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Journal of Research in Biology

An International Online Open Access
Original Research Paper
Publication group

Municipal Solid Waste (MSW) production and management in Kovilpatti
Municipality
Journal of Research in Biology

Authors: ABSTRACT:
Govindarajan B1
Senthilkumar P2 and
Prabakaran V3.

Studies were performed to appreciate the present status to municipal solid
waste (MSW) in Kovilpatti town. MSW is a complex waste. It is a classic example
Institution: where many different types of wastes aggregate from domestic, commercial and
1
Department of Zoology, industrial sources within a single waste stream. Solid waste problem is universal but
Manonmaniam Sundaranar recycling and utilization of wastes according to local conditions is an important task.
University, Thirunelveli- Kovilpatti is an important town for match box industries. The present study is aimed to
627012, Tamil Nadu, India. find out possible ways of utilization of MSW of Kovilpatti town.
2
Entomo Pathology Lab,
Institute of Forest Genetics
and Tree Breeding,
Coimbatore-02, TamilNadu,
India.
3
Department of Zoology,
Government Arts College,
Karur-639005, Tamil Nadu, Keywords:
India. Municipal Solid Waste (MSW), Kovilpatti municipality.

Corresponding author:
Govindarajan B.

Article Citation:
Govindarajan B Senthilkumar P and Prabakaran V.
Municipal Solid Waste (MSW) production and management in Kovilpatti Municipality.
Journal of research in Biology (2011) 5: 307-311.

Web Address: Dates:
http://jresearchbiology.com/ Received: 25 Aug 2011 /Accepted: 27 Aug 2011 /Published: 01 Sep 2011
Documents/RA0091.pdf.

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

307-311 | JRB | 2011 | Vol 1 | No 5
Journal of Research in biology
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An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Govindarajan et al.,2011

INTRODUCTION MATERIALS AND METHODS
India generates about 210 million tons of Collection and Physical characterization:
Municipal solid wastes (MSW) each day and the Physical characterization of municipal solid
quantum of wastes produced increases at a rate of waste components in various sanitary divisions of
1.5% per year (Abbasi and Ramasamy, 2001).The Kovilpatti was carried out according to the method
urban population in India is increasing explosively of Tchobanoglous et al (1977). To assess the
with proportionate increase in the solid waste, individual components present with in the mass of
causing great stress on environment. Improper the heterogeneous waste mix of the MSW, a truck
management of urban solid wastes causes pollution full of waste was collected from each sanitary
of ground and surface water, land and air covering division (1-6) and transported to a corner of the
biosphere problems. These problems are already dumping yard where the waste was segregated into
acute in cities, towns and villages as the disposal different components by hand sorting and listed.
facilities are not keep in pace with the quantum of
wastes being generated. RESULTS
Garbage is a heterogeneous bulky material The municipality town of Kovilpatti is
of solid wastes generated from human dwelling and divided into 36 wards and based on the
other city habitation, which includes both geographical location of these wards, grouped
biodegradable and non-biodegradable components. together to form 6 sanitary divisions. Number of
At all levels of development human beings produce population, streets, garbage bins, garbage collection
domestic, agricultural, industrial, hospital and per day and culverts in Kovilpatti (Table 1), wards
wastes at the public places. For designing the solid covered under each sanitary division and the
waste management system of an area first important population density are given in Table 2. Only 124
aspect is to be known how much solid waste is dust bins have been placed for the total area of 6.59
generated at a specific area. sq.km of Kovilpatti municipality. Slum area details
Solid waste problem is universal but are given in Table 3. The various kinds of waste
recycling and utilization of wastes according to composition in Kovilpatti municipality was debited
local conditions is a difficult task. Kovilpatti is an in Table 4. A sullage truck is used to collect the
important town for match box industries, and night soil from the public toilets when the tank is
vegetable market. It has a population of 87450 full. In addition to these tricycle is used to collect
(2001 census) in 36 wards with an extent of 6.59 and dump the bio-medical wastes from various
sq.km (www.municipality.tn.gov.in). The solid hospitals, dispensaries and clinics.
wastes produced by the city at present are disposed The profile of the current sanitary manpower
of by dumping the wastes in the dumping yard in of Kovilpatti municipality involved in solid waste
Alampatti Village, 3 km away from the town disposal is given in Table 5. The staffs grouped into
centre, which occupy an area of 3.81 acres. Since different categories, viz., general administrative
improper disposal of municipal wastes cause severe staff, revenue department, accounts section,
environmental pollution, the present study is aimed engineering division, town planning section, and
to find out possible ways of utilization of MSW of public health section. In all these sanitary divisions
Kovilpatti town. the organic materials such as food waste and the
The sanitary infrastructure of the plant residues dominated the other wastes. The
municipality, sanitary staff, population density, area percentage presence of glass, iron products, rubber
covered, type of area, total quantity of waste and leather were found to be negligible. At present
produced, vehicles used for collection, frequency of dumping site was not fully improved to carry out
collection, sources of waste and present disposal any composting activities. Therefore the municipal
method of municipality are the important aspects to authorities are trying to solve the problem.
be considered for planning effective solid waste
management. Hence efforts must be taken for DISCUSSION
proper scientific management of MSW in cities and Municipal solid waste management is a
towns. The present study was to assess the solid complex problem and it demands creative solutions
waste handling by Kovilpatti municipality. from many disciplines. Studies on solid waste
management have been carried out by many
workers in various cities and towns: Nanda et al
(2000 a, b) in Burla Town, Orissa, Goswami et al
308 Journal of Research in Biology (2011) 5: 307-311
Govindarajan et al.,2011

Table: 1 Municipal solid waste management at Kovilpatti town
Garbage
No. of No. of
Ward No. Population collection per Culverts
Streets Garbage bins
day (MT)
1. 2629 5 2 0.815 2
2. 1768 5 3 0.522 5
3. 3633 1 2 1.033 6
4. 1684 9 5 0.476 27
5. 3487 7 3 0.9522 9
6. 2633 8 4 0.790 9
7. 2460 5 2 0.678 7
8. 2964 5 3 0.942 19
9. 3321 3 4 1.016 12
10. 2104 4 3 0.662 16
11. 2883 11 5 0.855 22
12. 2940 9 2 0.862 7
13. 3006 6 2 0.917 12
14. 1812 8 5 0.562 10
15. 2190 9 4 0.702 17
16. 1530 8 3 0.514 6
17. 2380 5 4 0.722 7
18. 2272 6 5 0.739 10
19. 2219 4 4 0.678 4
20. 1480 11 4 0.467 5
21. 1845 4 6 0.551 10
22. 1366 5 2 0.473 2
23. 1059 5 4 0.896 5
24. 1845 4 6 0.551 10
25. 2348 6 2 0.854 10
26. 3067 6 5 0.714 5
27. 2880 8 3 1.104 7
28. 3047 7 3 0.923 6
29. 2958 19 2 0.889 7
30. 3158 5 4 0.878 13
31. 2225 5 3 0.741 10
32. 2047 4 4 0.613 8
33. 2751 11 3 0.862 16
34. 2543 5 3 0.760 11
35. 3061 4 4 0.641 3
36. 2106 7 4 0.606 10

(2001) in Gauhati, Assam,; Saxena & Joshi (2002) purposes. Munnoli and Bhosale (2002) also
in Hardwar, Uttranchal,; Daniel & Paul (2004 a); reported that the quantity of solid waste generation
Paul (2005) and Paul & Daniel (2007) in Dindigul, shoots up because of the floating population in Goa.
Tamil Nadu, India. The quantity of waste produced Similar type of results was observed by
in the developing countries like India is lesser than Cailas et al (1996) and Beede and Bloom (1995) in
that in the developed countries and is normally their studies on the rate of waste production with
observed to vary between 0.2 to 0.5 kg/head/d reference to the population size. The wards covered
(CPHERJ 1973; Ahsan 1999; Palnitkar 2000). under the thickly populated domestic area, i.e.,
The present study has shown that the ward no 10 is a slum area and hence the population
average per capita waste production of Kovilpatti is dense. The residential household waste
town is 27m.t/d. It is high because of largest match dominated the non-residential sector because of the
industry area. Villages around the town regularly large population, but the non-residential source
visit this area on all days for various commercial contained more quantity of organic fractions and

Journal of Research in Biology (2011) 5: 307-311 309
Govindarajan et al.,2011
Table: 2 Zone wise (Sanitary Division) Details
Sanitary Division Wards Covered No of individual Waste Generation
1. 1,2,3,4,23,24 12,144 3.64
2. 5,6,7,8,12,13,14 19006 5.81
3. 15,16,17,18,19 11,023 3.30
4. 20,21,22,25,26,27 12,291 3.68
5. 28,29,30,31,32,33 15,134 4.54
6. 9,10,11,34,35,36 21,108 6.33
Total 90,706 27.30

Table: 3 Slum Areas and Population
Name of the Slum Ward No. Population No. of Household
Bharathi Nagar 30 2225 556
Stalin Colony 31 3065 766
Natarajapuram 35 1633 408
Subramaniapuram 36 2107 536
Anna Nagar 29 2958 740
Valluvar Nagar 10 3428 851
Total 13661 3857

that was due to the presence of the match box in the whole Kovilpatti town with a 6.59 sq.km.
industries and vegetable wastes and large quantities area. Park (1997) has explained the advantage of
of leaves in the wastes, i.e., the wastes produced by house to house collection and it has resulted in a
the markets, hotels and wedding halls. Some of the simultaneous reduction in the number of public
waste produced by the wedding halls and the hotels bins. Plastics are the major compounds that pollute
are collected and used as dog feed by those who the environment, but comparatively its presence
rear them around the Kovilpatti municipality limits. was very less. The same type of observation has
Open and unregulated dumping is the been made by Munnoli & Bhosale (2002) in Goa,
predominant method of waste disposal in, and Reddy et al (2002) in Bangalore.
Kovilpatti. The waste piles are exposed to wind and The composition of the hospital wastes of
rain and also to rats, flies, insects, pigs and other Kovilpatti town showed only 0.2 mt was found
animals. In some places the people set fire to the highly infectious. The WHO (2002) also has
wastes. The rag pickers spend their days sorting observed the presence of 20% of hazardous
through the garbage for reusable and recyclable materials and 80% of benign materials in bio-
materials. The trashes were found to be medical wastes. For planning an effective solid
accumulated along the road sides, in vacant places, waste management by Kovilpatti municipality, a
particularly in the poorer sections of the town. newly modified profile recommended. Recycling is
Placing dust bins may lead to reduction in a major play in MSW reduction. Glass, aluminium
the problem of waste accumulation along the cans, plastics, newspapers and organic materials can
roadsides, but only 124 dust bins have been placed be recycled, the construction waste can be used for
Table: 4 Details of Solid Waste Composition landfills and the hospital waste can be incinerated.
It is essential to get public support for a sound
Waste Composition Quantity %
(MT) Generation
Table: 5 Man power involved in Kovilpatti
Hoseholds, Petty shops Municipality
21.00 77.7
and Establishments
Vegetable, Fruit, S.No Position Numbers
3.00 11.11 1 General Administration 19
Flower market
Meat, Fish, and 2 Revenue Department 12
0.5 1.8
Slaughter house 3 Account Section 1
Construction 2.5 9.39 4 Engineering Division 34
Hospital waste 0.200 Negligible 5 Town Planning Section 3
Total 27 100 6 Public health Department 26
310 Journal of Research in Biology (2011) 5: 307-311
Govindarajan et al.,2011

waste management programme. Creation of Nanda SN, Mishra B and Tiwari TN 2000-a.
awareness among the public on waste management Municipal solid wastes in Burla Town (Orissa): (1)
will improve sanitary conditions and the scenic Preliminary Survey. Indian J. Environ. and
beauty of Kovilpatti. Ecoplan., 3(3):643-646.

ACKNOWLEDGEMENT Nanda SN, Mishra B and Tiwari TN. 2000-b.
Authors sincerely thank kovilpatti municipality Municipal solid wastes in Burla town (Orissa): (II)
staffs and heartful thanks to Mr K. Naveenkumar, Physico-chemical characteristics. Indian J. Environ.
B.E., and Ecoplan., 3(3):647-652.

REFERENCES Park K. 1997. Environment and health. In:
Abbasi SA and Ramasamy E. 2001. Waste and Preventive and social medicine, 15th Edn. Pub:
wealth. In: Soild waste management with Banarsidas Bhanot, Jabalpur.468-541.
earthworms. Discovery Publishing house, New
Delhi. P. 1-7. Palnitkar S. 2000. Municipal solid wastes. In:
manual on municipal solid waste. Ed: Palinitkar S
Ahsan N. 1999. Solid waste management plan for Pub: All India Institute of local self-government. pp
Indian megacities Indian J. Environ. Protection. 19 9-19.
(2):90-95.
Paul JA, Karmagam N and Daniel T. 2005.
Beede DN and Blom DE. 1995. The economics of Effect of worm population on biomass of Eudrilus
municipal solid waste. The World Bank Observer. eugeniae in municipal solid waste and the status of
10(2):113-150. microorganisms. J. Ecobiol., 17(6):567-570.

Cailas MD, Kerzee RG, Bing-Canar J, Mehash Paul JA and Daniel T. 2007. Standardization of
EX, Croke KG and Swager RR 1996. An sampling method for physical characterization of
indicator of solid waste generation potential for municipal solid waste. Asian J. Water Environment
Illinois using principal components analysis and and Pollution.
geographic information systems of the air and waste
management association. 466:414-421. Saxena P and Joshi N 2002. Organic Waste
Decomposition by using Mucor sps. and
Daniel T and Paul JAJ. 2004. Solid waste Penicillium sps. in combination. Indian J. Environ
management options for Class-I Towns. In: and Ecoplan., 6(3):583-586.
Proceedings of the National conference on
“Environment awareness and pollution impacts”, Tchobanoglous G, Hillary Theisen and Roif
Centre for Energy and Environmental Science and Eliassen 1977. In: Solid Wastes: Engineering-
Technology, NIT, Trichy, Tamil Nadu. India. 157- Principles and Management Issues. McGraw Hill
165. International Students Edition, New York.

Goswami B, Kalita MC and Talukdar S. 2001 WHO 2002. Wastes from health care activities,
Bioconversion of municipal solid waste through World Health Organization Information, Fact
vermicomposting. Asian J. Microbiol. Biotech. Sheet No. 253.
Environ. Sci., 3:205-207.
www.municipality.tn.gov.in.
Kawata K. 1963. Environmental sanitation in
India. Christian Medical College, Ludhiana, Punjab.

Munnoli P and Bhosale S. 2002. Solid waste
disposal in Goa. Proceedings ofNational seminar on
Solid waste management-current status and
Strategies for future (Eds) R.K. somashekar and
M.A.R. Iyengar, Bangalore, India. 9-13.

Journal of Research in Biology (2011) 5: 307-311 311
Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Effect of prebiotic (GroBiotic®-A) on the growth performance and intestinal
microflora on rainbow trout (Oncorhynchus mykiss Walbaum)
Journal of Research in Biology

Authors: ABSTRACT:
Azari AH1, 2, Hashim R1,
Azari Takami G3, Farabi
SMV2, Darvish M4 and
Safari R5.
The use of prebiotics as feed supplements that improve efficiency of
intestinal bacteria is becoming de rigueur in animal husbandry in many regions
Institution: worldwide. We tested the effects of a commercial prebiotic (GroBiotic®-A) including
1
Laboratory of Feeds and yeast and dairy fractions in different levels on survival, growth, carcass composition
Feeding Management, and intestinal microflora on rainbow trout (Oncorhynchus mykiss Walbaum). In the
Aquaculture Research present study, we were able to detect high amounts of lactic acid bacteria (LAB) in the
Group, School of Biological intestine after 12 weeks of prebiotic supplementation, conducted. Ultimately, when
Sciences, Universiti Sains all supplementation diets are considered, GroBiotic®-A inclusion to diets for appearing
Malaysia, 11800 Penang, on increase on growth, survival, and carcass composition on O. mykiss.
Malaysia
2
Department of Aquaculture,
Caspian Sea Research
Institute of Ecology, Sari,
Iran
3
Department of Aquatics
Disease and Health, Faculty Keywords:
of Veterinary Science, Rainbow trout, prebiotics, GroBiotic®-A and lactic acid bacteria (LAB).
Tehran University, Tehran,
Iran
4
Department of Marin
Chemistry Science and
Technology, Islamic Azad
University, North Tehran
Branch, Tehran, Iran
5
Department of
Biotechnology, Caspian Sea
Research Institute of
Ecology, Sari, Iran.
Article Citation:
Azari AH, Hashim R, Azari Takami G, Farabi SMV, Darvish M and Safari R.
Corresponding author: Effect of prebiotic (GroBiotic®-A) on the growth performance and intestinal microflora
Abdolhamid Azari on rainbow trout (Oncorhynchus mykiss Walbaum).
Journal of research in Biology (2011) 5: 325-334

Dates:
Email: Received: 03 Jul 2011 /Accepted: 08 Jul 2011 /Published: 12 sep 2011
ahazaritakami@yahoo.com
© Ficus Publishers.
Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://
http://jresearchbiology.com/ creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
Documents/RA0055.pdf. commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

325-334 | JRB | 2011 | Vol 1 | No 5
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Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Azari et al.,2011

INTRODUCTION: 4.5g were obtained from private farm at Haraz,
The culture of salmonids particularly Niyak Ghezel. The fish were acclimated for one
Atlantic salmon, Salmo salar and rainbow trout week in four fiberglass tanks (4×4×1 m, 8 m3) to
(Oncorhynchus mykiss) is one of the most fish house conditions before the start of the trial.
important examples of commercially successful Fish were kept in fiberglass tanks in dimension of
intensive aquaculture in the world. Rainbow trout is 1.5×1.5×0.4 m, 1 m3 with an open system of supply
a large economic fish in Iran and artificial breeding located in the north of Iran. Fishes randomly were
farms of this fish species are increasing apparently. divided into 7 groups which were placed in
It is also the second cultured fish species in Iran fiberglass tanks as described in above and in fresh
(FAO, 2006). The increasing economic and social water-flow at 9-12oC for 84 days prior to use in
concerns by decreasing the use of antibiotics and experiments.
other chemicals used in fish farming have Experimental Procedure
encouraged more environment friendly approaches The present work was performed at the
for increasing growth (Verschuere et al., 2000). Department of Aquaculture, Ecological Academy of
Prebiotic, unlike probiotic, is not an organism and Caspian Sea, Sari, Iran. The trial was performed for
has less influence in natural environment. These so- 12-weeks using fish in 3 replicates per treatment.
called prebiotic carbohydrates are defined as Before the start of the feeding trial, three fish were
„„nondigestible food ingredient(s) that beneficially randomly sacrificed with an overdose of clove oil,
affect host health by selectively stimulating the and triplicate pooled samples were taken for the
growth and/or activity of one or a limited number of determination of initial whole body composition. At
bacteria in the colon‟‟ (Gibson and Roberfroid the end of the trial, all fish were weighed and
1995). Therefore, the need for alternative counted and three fishes from each fiberglass tank
techniques is increasing, and the contribution of were collected for resolution of whole carcass
prebiotics such as xylooligosaccharides (XOS) may composition.
be considerable. XOS, fructooligosaccharides Experimental diets
(FOS), inulin, and other related carbohydrates have The fish were fed with a standard
received considerable attention because of the commercial food (Chine Co.) at a rate of 2-5 % of
health benefits they are believed to confer on the the biomass per day. Commercial dry food served
host (Mussatto and Mancilha 2007; Cerezuela et al., as a basis in which the various concentrations of
2008). However, it is evident that they play an prebiotic, GroBiotic®-A were added, in 20 ml of
important role in generating a colonic microflora sunflower oil, to commercial dry feed followed by
that comprises predominantly bifidobacteria in mixing with a blender or by handy. Six
young mammals (Houdijk et al., 1998; Erney et al. experimental diets were formulated with increasing
2000; Costalos et al., 2008). levels of GroBiotic®-A (1, 1.5, 2, 2.5 and 3% of
A „functional nutrient‟ can be further basal diet) and non-supplemented diet (control): as
defined as a dietary ingredient that employs diet 1, 2, 3, 4, 5, 6 and 7 containing 43.30 ± 0.07%
possible positive effects on health in addition to its and 40.56 ± 0.06% protein, 13.57 ± 0.15% and
direct role as a nutrient. Therefore, the present study 15.19 ± 0.04% lipid and an estimated gross energy
was conducted to explore growth performance, level of 20.31 ± 0.03 and 20.61 ± 0.04 kj.g-1 (FFT,
survival and microflora into intestine of juvenile Fingerling Feed Rainbow Trout and GFT1, Growth
rainbow trout, that feed the variety levels of dietary Feed Rainbow Trout), respectively.
prebiotic, GroBiotic®-A which is a commercial Analytical methods
mixture of partially autolyzed brewer‟s yeast, dairy Chemical analyses
ingredient components and dried fermentation The food, experimental diets and fish
products (with 5, 10, 15, 20, 25 and 30g kg-1 carcass were analysed for proximate composition of
GroBiotic®-A). dry matter, crude protein, crude lipid, fibre and ash
using standard AOAC methods (1997). Briefly, dry
MATERIAL AND METHODS: matter was determined by drying at 100 0C to
Source and maintenance of fish constant weight; crude protein was determined by
The experimental system was used here for the Kjeldahl procedure (Nitrogen×6.25); crude fat
an experiment to optimize the feeding regime of by chloroform methanol extraction (2:1, v/v); crude
rainbow trout, Oncorhynchus mykiss. Apparently ash content by determining the residue after heating
healthy O. mykiss with an average body weight of in a muffle furnace at 550 0C for 5 h and crude fibre
326 Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011

by loss on ignition of dried residue after successive All data are expressed as mean ±SD, where n
digestion with 5% H2SO4. Nitrogen free extract represents the number of fish. The differences in
(NFE) was calculated by subtracting the sum of parameters were tested for significance using a one-
crude protein, crude fat, ash, and crude fibre from way analysis of variance (ANOVA) which was
the total dry matter content. Proximate analyses of performed using SPSS.V11.5 Subsequent
the trial diets are described in Table 1. significance between groups was delineated by
Bacterial analysis Duncan‟s test. A value of P<0.05 were taken for
The microbial analyses were done twice; significance in all statistical tests.
before the trial and at the end of the fourth week.
Ten fish from each group were sacrificed by RESULTS:
immersion in a tank containing MS-222 at a Growth, Survival and Feed Performance
concentration of 150 mg L-1 of water for 15 min, No external clinical sign occurred in any
and they were eviscerated aseptically and the whole treatment during the period of this experiment. The
intestine was removed. The intestine was dissected statistical analysis of different growth parameters of
and the contents were collected by carefully Oncorhynchus mykiss at the end of experimental
scraping with a rubber spatula and weighed. The period (Table 2) indicated a significant increase in
microbial analyses were performed by spreading body weight gain percent (WG %) between the six
appropriate dilutions in PBS from 101 to 106 on different groups and control diet (P<0.05). O.
tryptic soy agar (TSA, Merck, Darmstadt, mykiss in group G-A5 kept on diet supplemented
Germany), a general media for facultative aerobic with (GroBiotic®-A2.5%) were the fast grower
bacteria. The plates were incubated aerobically at followed by the O. mykiss in the groups G-A4 and
30°C for 2 days. For determination of anaerobic G-A6 received diet supplemented with (GroBiotic®-
bacteria was used from anaerobic jars with A 2 and 3% respectively) in comparison to other
disposable Anaerocult A bags (Merck, Darmstadt, groups. The specific growth rate (SGR) takes
Germany) for the generation of an anaerobic almost the same pattern of WG% in which O.
medium. For lactic acid bacteria (LAB) mykiss in the group G-A5 (2.5%) have the highest
determination, diluted samples were plated on SGR followed by O. mykiss in groups G-A4 (2%)
deMan, Rogosa, and Sharpe (MRS) agar (Merck, and G-A6 (3%) in comparison to other groups.
Darmstadt, Germany) and incubated at 30°C for 2– Average daily growth (ADG) of fish under
3 days in an anaerobic jars with disposable G-A (2, 2.5 and 3%) feeding were significantly
Anaerocult C bags (Merck, Darmstadt, Germany) greater (P<0.05) than the basal diet and other
(Jones et al., 2008). groups.
Calculation and Statistical Analysis Survival also was affected significantly
The following formulae were applied to the data: (P<0.05) by dietary treatments as the fish fed
control diet had lower survival 93.33 ±
Feed intake (FI) = [total feed intake / number of fish (g)] 2.31compared to an overall survival rate of 98.67 ±
2.31 among G-A5 and G-A6 fed the remaining diets
Specific growth rate (SGR %) = [((ln Wf – ln Wi) / T) × 100] (Table 2).
Feed efficiency (FE) = [total wet weight gain (g) / Apparent Protein Utilization
Protein utilization efficiency, measured in
total feed intake (g)]
term of protein efficiency ratio (PER) and net
Protein Efficiency Ratio (PER) = [wet weight gain protein utilization (NPU) is summarized in Table 3.
(g) / total protein intake] These were the same for protein efficiency ratio
(PER) in which the O. mykiss in the groups treated
Average Daily Growth (ADG = [((wf– wi) / wi) × (Tf-Ti)] with prebiotic 2, 2.5 and 3% supplemented diets
exceeded the value of other groups and even
Survival rate (%) = [(Number of fish which survived / control. The feed efficiency (FE) of O. mykiss takes
initial number of fish) × 100] almost the same patterns of PER in which in the
group G-A5 (2.5%) have the highest FE followed
Where Wf refers to the mean final weight, Wi is the by O. mykiss in group G-A2 and G-A3% in
mean initial weight of fish, T is the feeding trial comparison to other groups and basal diet.
period in days. Apparent net protein utilization (NPU) the groups

Journal of Research in Biology (2011) 5: 325-334 327
328
Table 1 Proximate analyses of the diets used in varying levels -response experiments in rainbow trout (Oncorhynchus mykiss) during 84 days
trial1.
Proximate composition

Treatments Experiment Moisture (%) Protein (%) Lipid (%) Fiber (%) Ash (%) NFE9 (%) GE10 (kj.g-1)
Diets
Control FFT1 5.83 ± 0.14b 43.30 ± 0.07a 13.57 ± 0.15b 6.57 ± 0.09b 9.12 ± 0.03a 27.44 ± 0.13b 20.31 ± 0.03g
GFT12 6.85 ± 0.15a 40.56 ± 0.06b 15.19 ± 0.04a 6.59 ± 0.07a 9.22 ± 0.02b 28.44 ± 0.07a 20.61 ± 0.04b
G-A13 FFT+G1 5.79 ± 0.12b 43.19 ± 0.11a 13.70 ± 0.15b 6.52 ± 0.11b 9.12 ± 0.17a 27.47 ± 0.44b 20.34 ± 0.06fg
GFT1+G1 6.91 ± 0.07a 40.42 ± 0.10b 15.15 ± 0.06a 7.18 ± 0.06a 8.58 ± 0.12b 28.67 ± 0.13a 20.44 ± 0.01de
G-A24 FFT+G2 5.89 ± 0.05b 43.29 ± 0.21a 13.60 ± 0.21b 6.48 ± 0.15b 9.14 ± 0.04b 27.43 ± 0.22b 20.38 ± 0.07efg
GFT1+G2 6.91 ± 0.03a 40.35 ± 0.06b 15.15 ± 0.03a 7.22 ± 0.09a 8.57 ± 0.18a 28.70 ± 0.25a 20.41 ± 0.02def
G-A35 FFT+G3 5.85 ± 0.11b 43.18 ± 0.04a 13.65 ± 0.30b 6.49 ± 0.20b 9.15 ± 0.06b 27.53 ± 0.42b 20.43 ± 0.06de
GFT1+G3 6.92 ± 0.06a 40.58 ± 0.28b 15.17 ± 0.11a 7.31 ± 0.05a 8.50 ± 0.17a 28.44 ± 0.30a 20.40 ± 0.01def
G-A46 FFT+G4 5.76 ± 0.11b 43.19 ± 0.11a 13.69 ± 0.12b 6.51 ± 0.07b 9.13 ± 0.03b 27.47 ± 0.19b 20.49 ± 0.10cd
GFT1+G4 6.83 ± 0.05a 40.38 ± 0.06b 15.18 ± 0.15a 7.33 ± 0.10a 8.51 ± 0.04a 28.59 ± 0.09a 20.76 ± 0.04a
G-A57 FFT+G5 5.92 ± 0.10b 43.21 ± 0.20a 13.68 ± 0.06b 6.47 ± 0.17b 9.20 ± 0.10b 27.43 ± 0.10b 20.67 ± 0.03b
GFT1+G5 6.80 ± 0.10a 40.37 ± 0.05b 15.19 ± 0.15a 7.20 ± 0.11a 8.51 ± 0.08a 28.72 ± 0.26a 20.49 ± 0.05cd
G-A68 FFT+G6 5.73 ± 0.16b 43.30 ± 0.13a 13.68 ± 0.03b 6.56 ± 0.06b 9.14 ± 0.09b 27.31 ± 0.27b 20.53 ± 0.03c
GFT1+G6 6.74 ± 0.01a 40.36 ± 0.17b 15.16 ± 0.05a 7.24 ± 0.08a 8.52 ± 0.09a 28.73 ± 0.22a 20.48 ± 0.03cd
1
Fingerling Feed Rainbow Trout (commercial Rainbow Trout food, Chinne)
2
Growth Feed Rainbow Trout (commercial Rainbow Trout food, Chinne)
3
GroBiotic®-A (A commercial prebiotic) 0.5% of diet
4
GroBiotic®-A (A commercial prebiotic) 1% of diet
5
GroBiotic®-A (A commercial prebiotic) 1.5% of diet
6
GroBiotic®-A (A commercial prebiotic) 2% of diet
7
GroBiotic®-A (A commercial prebiotic) 2.5% of diet
8
GroBiotic®-A (A commercial prebiotic) 3% of diet
9
Nitrogen free extract {100 – (protein + lipid + ash + fiber)}
10
Gross energy content (Brafield 1985).
Azari et al.,2011

Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011

which receiving G-A feeding were vary
Table 2 Initial weight, final weight, percentage weight gain, specific growth rate, average daily growth and survival of rainbow trout(Oncorhynchus significantly (P < 0.05) with these supplements

724.04 ± 41.60c
level in the diets, similarly to PER. FI (feed intake)

93.33 ± 2.31b
36.76 ± 2.14c

2.51 ± 0.06c
38.45 ± 2.50c
4.46 ± 0.05
Control
for G-A feeding tended to have better values than
control diet with increasing supplements level and
also feeding to G-A produced a better productive.
The protein productive values (PPV) of the
fingerlings fed with different experimental diets are

Survival rate (%) = (Final fish number / Initial fish number) × 100
presented in Table 3. Fish fed 25 g G-A kg- 1 diet (G

97.33 ± 2.31ab
Values are mean ± SD (n=3). Mean values within columns not sharing the same superscript are significantly different (P<0.05)
871.51 ± 29.04b
43.24 ± 1.13ab

46.18 ± 1.36ab
2.71 ± 0.04b
-A5) had maximum PPV and there was no
4.45 ± 0.05
G-A 67

improvement (P>0.05) in PPV beyond this level in
comparison to basal diet.
Body Composition
In order to determine the nutritional effects
GroBiotic®-A (A commercial prebiotic) 3% of diet
GroBiotic®-A (A commercial prebiotic) 2% of diet
GroBiotic®-A (A commercial prebiotic) 1% of diet

of administered prebiotic on rainbow trout fry, the
SGR% = {(LnWf – LnWi) / Total days} × 100
biochemical composition of carcass was analyzed.
933.09 ± 14.86a
45.21 ± 1.46a

2.78 ± 0.02a
48.60 ± 1.62a
98.67 ± 2.31a
4.40 ± 0.11

The results are presented in Table 4. At the end of
G-A 56

the experiment, in comparison to initial values, all
the experimental groups within control showed
higher percentage of protein and ash but a lower
mykiss) fed diets containing varying Concentrations of GroBiotic ®-A and control for 84 days1.

percentage lipid and moisture. Protein values of
carcass in all treatments were significantly higher
847.08 ± 18.41b

(P<0.05) than controls. The best result was obtained
41.93 ± 1.12b

2.68 ± 0.02b
44.64 ± 1.29b
98.67 ± 2.31a
4.43 ± 0.03
G-A 45

from G-A5 (17.06 ± 0.06%). Significantly (P<0.05)
different lipid values of carcass in prebiotic groups,
Wf = Final weight

compared to the controls, were indicated.
GroBiotic®-A feeding resulted in a decrease in body
lipid (fat) with increase in dietary supplements.
Moisture values of G-A2, G-A3, G-A4, G-A5 and
770.08 ± 33.11c

97.33 ± 2.31ab
38.24 ± 1.17c

2.57 ± 0.05c
40.29 ± 1.42c
4.40 ± 0.06

G-A6 indicated a significant (P<0.05) difference as
G-A 34

well (Table 4). Fish fed the diet (commercial
11
9

13
7
3
5

rainbow trout food, CHINE) under feeding received
G-A 2, 2.5 and 3% showed significant (P<0.05)
difference that had higher body ash than fish from
GroBiotic -A (A commercial prebiotic) 1.5% of diet
GroBiotic -A (A commercial prebiotic) 2.5% of diet
GroBiotic®-A (A commercial prebiotic) 0.5% of diet

the control and other treatments (Table 4).
768.86 ± 39.36c

94.67 ± 2.31ab
38.92 ± 1.84c

2.57 ± 0.05c
41.00 ± 2.17c
4.48 ± 0.04

Bacterial Analysis
G-A 23

In all treatments, lactic acid bacteria (LAB)
ADG (%) = {(Wf – Wi) / Total days} × 100

successfully colonized the O. mykiss digestive tract
on the end of trials (12-weeks). At the beginning of
the study, we observed that all the fish were not
significantly different on the aerobic, anaerobic and
774.09 ± 18.14c

97.33 ± 2.31ab
38.98 ± 0.74c

2.58 ± 0.02c
41.10 ± 0.86c
4.46 ± 0.07

LAB media count (P>0.05; Table 5). Total bacterial
Wg = {(Wf – Wi)/Wi} × 100
Treatments
G-A 12

counts among prebiotic supplemented groups were
significantly different from total bacterial counts in
controls in digestive tract of fish (Table 5; P<0.05).
Wi = Initial weight,

The mean of total bacterial counts of intestine with
control diet in aerobic and anaerobic condition in
Survival(%)13

®
®
Parameters

TSA media were 4.46 ± 0.01 CFU/g and 4.80 ±
ADG (%)12
SGR (%)11
Wg (%)10

0.01 CFU/g, respectively increased exponentially
Wf (g)9
Wi (g)8

from the experimental groups in intestine (P<0.05).
On the other hand, LAB colonization was detected
10
12
1
2
4
6
8

artificially dominant in experimental groups,

Journal of Research in Biology (2011) 5: 325-334 329
Azari et al.,2011

Table 3 Feed intake, feed efficiency, protein efficiency ratio, Nett protein utilization and productive protein
value of rainbow trout (Oncorhynchus mykiss) fed diets containing varying Concentrations of GroBiotic ®-A and
control diet for 84 days1.
Parameters
Treatments Feed intake FE8 PER9 NPU (%)10 PPV11
G-A 12 40.24 ± 1.78bc 0.86 ± 0.03cd 1.99 ± 0.06cd 4.77 ± 2.61cd 89.50 ± 6.14a
G-A 23 41.20 ± 0.23ab 0.84 ± 0.04de 1.97 ± 0.09d 2.40 ± 0.01d 86.21 ± 0.48a
G-A 34 41.77 ± 1.07ab 0.81 ± 0.01e 1.89 ± 0.03d 7.46 ± 0.92bc 88.46 ± 1.61a
5 ab b bc b
G-A 4 41.55 ± 0.85 0.90 ± 0.01 2.08 ± 0.02 8.27 ± 1.28 89.88 ± 2.73a
G-A 56 42.99 ± 0.65a 0.95 ± 0.02a 2.19 ± 0.04a 13.84 ± 1.29a 91.44 ± 1.17a
G-A 67 41.36 ± 1.16ab 0.94 ± 0.00ab 2.16 ± 0.01ab 9.97 ± 2.79b 90.33 ± 2.73a
Control 39.00 ± 0.86c 0.83 ± 0.04de 1.89 ± 0.08d 2.24 ± 0.61d 88.23 ± 1.69a
1
Values are mean ± SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic®-A (A commercial prebiotic) 0.5% of diet 3
GroBiotic®-A (A commercial prebiotic) 1% of diet
4 ® 5
GroBiotic -A (A commercial prebiotic) 1.5% of diet GroBiotic®-A (A commercial prebiotic) 2% of diet
6 ® 7
GroBiotic -A (A commercial prebiotic) 2.5% of diet GroBiotic®-A (A commercial prebiotic) 3% of diet
8
Feed efficiency = weight gain (g) / food intake (g)
9
Protein efficiency ratio = weight gain (g) / protein intake (g)
10
Nett Protein Utilization (NPU=Wf × Protein Muscle Final – Wi × Protein Muscle Initial/Protein Consumed)
11
Productive protein value = protein gain (g) × 100 / protein intake (g).

however, the opposite pattern was observed for the selectively stimulating the growth and/or activity of
digestive tracts of O. mykiss, in which the mean of one or a limited number of bacteria in the colon,
total LAB counts among prebiotics administered and thus improves host health. Besides FAO
groups were more than control (P<0.05). The experts “A prebiotic is a non-viable food
results of identification according to media on to component that presents a health benefit on the host
aerobic, anaerobic and lactic acid bacteria a s s oc ia t ed w it h mo du la t i o n o f t h e
condition, the highest count revealed the microbiota” (FAO 2007). The data of prebiotic
experiment that was under G-A5 (4.92 ± 0.01, 7.21 research is still young, yet the progress made in
± 0.02 and 6.60 ± 0.01 CFU/g, respectively; Table elucidating the useful health effects of specific
5). prebiotics is significant not only for humans and
animals, but for fish species also as well. In the
DISCUSSION: present study, growth performance was increased
Growth Performance and Protein utilization about 23 % in fish fed prebiotic (GroBiotic®-A) diet
Fish growth performance is affected by compared to fish fed control diet; growth
different factors including water quality, stresses performance was significantly higher (P<0.05)
and diseases, diet quality and quantity etc. Fish exhibited in O. mykiss juvenile maintained on the G
under intensive culture conditions will be badly -A diet supplemented with (prebiotic diet). The
affected and often fall target to different microbial specific growth rate (SGR) illustrated almost the
pathogens that have been conducted with same pattern of W.G% in which O. mykiss in the
chemotherapeutic substances of which antibiotics group that received G-A5 have the highest SGR
were used. The use of biological products namely followed by O. mykiss in group G-A4 and G-A6
the probiotic either alone or in combination with content in comparison to control group, G-A1, G-
prebiotics is recently the aim of the disease A2 and G-A3. These were also true for protein
biocontrol program in aquaculture as they improve efficiency ratio (PER), NPU (protein utilization)
the fish health and alter the fish associated and survival in which the O. mykiss in groups
microbial population (Gibson and Roberfroid, treated with prebiotic supplemented diets was more
1995). While Gibson and Roberfroid (1995) than the amount of control group. In our study,
defined, prebiotic term as “A nondigestible foods survival of fish in the treatment which produced the
ingredient (s) that beneficially affects the host by highest growth performance was 5.7 % than the
330 Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011

lowest survival value. Since the feed efficiency (P<0.01) and mortality decreased from 28.33 to
(FE) of O. mykiss kept on a basal diet (control) was 16.67% (P<0.01). These data supported the findings
lower than the diets supplemented with prebiotics, of Hanley et al., (1995) who also have shown that
while the highest FE was belonged to group which hybrid red tilapia juveniles, fed 0.6% prebiotic
had G-A 2.5 % and a 14.5 % increased on feed (Aqua-Mos™, Alltech Inc., KY, USA) in their
efficiency, corroborating results of other previous hatchery diets, that have had a 22.5% improved
studies. The PER results indicated that survival with 27.2% increase in weight gain. The
supplementing diets with prebiotics significantly results revealed that both groups received prebiotic-
improved protein utilization in rainbow trout. supplemented diets showed higher growth rate than
Similar results have been reported by Li et al., those kept on a basal diet, suggesting that the
(2005) who observed increased weight gain and addition of prebiotics enhanced the growth have
feed efficiency which were generally observed in performance and feed utilization.
hybrid striped bass fed diets supplemented with In our study observed proximate analysis at
partially autolyzed brewer‟s yeast and GroBiotic®- the end of the experiment indicated significant
A at each sampling time (4, 8, 12 and 16 weeks). (P<0.05) differences in muscle protein, lipid, ash
Staykov et al., (2007) have conducted rainbow trout and moisture contents of all the treatments.
(O. mykiss) with supplementation of prebiotic Bio Although the diets included containing different
Mos® (0.2%) in standard commercial extruded levels of G-A, had increased effect on total muscle
feeds which were raised in net cages and raceways, protein and ash (13.3% and 20.6%), respectively
at the end of the six weeks trial period, the mean however decreased influence on muscle lipid and
body weight of fish receiving prebiotic was 13.7% moisture (20.6% and 1.3%), respectively content of
and 9.97%, respectively higher, compared to the the fish. This study has found that supplementation
control groups (P<0.01). The supplementation of with G-A may help to positively change body
prebiotic in the diet significantly decreased FCR composition by increasing lean body mass (which
(P<0.05) and mortality (P<0.01). This result was in includes muscle mass) and decreasing fat mass.
agreement with the results expressed by Bogut et The fillets (MR%) of the groups of rainbow
al., (2006) which demonstrated improvements in trout differed with regard to the received of G-A
growth with the use of prebiotic MOS (Bio-Mos®) and non- G-A; the groups that fed G-A
to the diet of other freshwater species, European supplemented diets exhibited higher values than of
catfish (Silurus glanis) juveniles from 22 to 76 g in basal diet (P< 0.05). Results demonstrated that
the control groups and 83 g in the Bio-Mos® prebiotic G-A can be an influence device for the
groups, a 9.7% higher average body weight enhancement of growth of performance, health
(P<0.01). Here the FCR was also lower to 11.6% status and feed efficiency of rainbow trout grown in
Table 4 Carcass proximate compositions of rainbow trout (Oncorhynchus mykiss) fed control and varying
Concentrations of GroBiotic®-A and control diet for 84 days1.

Protein (%) Lipid (%) Ash (%) Moisture (%)
At the start 14.68 ± 0.13 9.40 ± 0.05 1.87 ± 0.03 75.10 ± 0.36
2 d a b
At the end G-A 1 15.50 ± 0.43 8.23 ± 0.06 1.22 ± 0.02 75.01 ± 0.02a
G-A 23 15.10 ± 0.00e 7.59 ± 0.45b 1.21 ± 0.06b 74.93 ± 0.05ab
G-A 34 15.85 ± 0.16c 7.44 ± 0.12b 1.23 ± 0.23b 74.89 ± 0.63ab
G-A 45 16.17 ± 0.21bc 7.44 ± 0.03b 1.38 ± 0.11ab 74.49 ± 0.10bc
G-A 56 17.06 ± 0.06a 7.05 ± 0.04c 1.46 ± 0.05a 74.24 ± 0.09c
G-A 67 16.23 ± 0.06b 7.09 ± 0.05c 1.44 ± 0.05a 74.51 ± 0.18bc
Control 15.06 ± 0.11e 8.02 ± 0.07a 1.21 ± 0.02b 75.16 ± 0.13a
1
Values are mean ± SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic®-A (A commercial prebiotic) 0.5% of diet 3
GroBiotic®-A (A commercial prebiotic) 1% of diet
4 ® 5
GroBiotic -A (A commercial prebiotic) 1.5% of diet GroBiotic®-A (A commercial prebiotic) 2% of diet
6 ® 7
GroBiotic -A (A commercial prebiotic) 2.5% of diet GroBiotic®-A (A commercial prebiotic) 3% of diet

Journal of Research in Biology (2011) 5: 325-334 331
Azari et al.,2011

Table 5 Total bacterial levels (log CFU/mg intestinal contents) in TSA medium (Aerobic, Anaerobic) and LAB
(Lactic Acid Bacteria) condition in the juvenile O.mykiss fed with test diets for 12 weeks1.

Initial Final
Anaerobic Anaerobic
Source Aerobic (TSA) LAB Aerobic (TSA) LAB
(TSA) (TSA)
G-A12 4.49 ± 0.02 4.80 ± 0.01 0.00 ± 0.00 4.49 ± 0.02e 4.81 ± 0.01e 3.63 ± 0.01f
G-A23 4.49 ± 0.02 4.80 ± 0.01 0.00 ± 0.00 4.65 ± 0.02d 5.22 ± 0.01d 3.90 ± 0.03e
G-A34 4.48 ± 0.02 4.80 ± 0.01 0.00 ± 0.00 4.49 ± 0.02c 6.82 ± 0.01c 6.06 ± 0.05d
G-A45 4.48 ± 0.03 4.81 ± 0.01 0.00 ± 0.00 4.86 ± 0.02b 7.20 ± 0.02a 6.46 ± 0.01c
G-A56 4.46 ± 0.02 4.80 ± 0.01 0.00 ± 0.00 4.92 ± 0.01a 7.21 ± 0.02a 6.60 ± 0.01a
G-A67 4.47 ± 0.01 4.81 ± 0.02 0.00 ± 0.00 4.87 ± 0.01b 7.15 ± 0.01b 6.55 ± 0.03b
Control 4.46 ± 0.02 4.80 ± 0.00 0.00 ± 0.00 4.46 ± 0.01e 4.80 ± 0.01e 0.00 ± 0.00j
1
Values are mean ± SD (n=3). Mean values within columns not sharing the same superscript are significantly
different (P<0.05)
2
GroBiotic®-A (A commercial prebiotic) 0.5% of diet 3 GroBiotic®-A (A commercial prebiotic) 1% of diet
4
GroBiotic®-A (A commercial prebiotic) 1.5% of diet 5 GroBiotic®-A (A commercial prebiotic) 2% of diet
6
GroBiotic®-A (A commercial prebiotic) 2.5% of diet 7 GroBiotic®-A (A commercial prebiotic) 3% of diet.

a variety of production systems. On other hand, the supplemented diets and basal group, isolated from
beneficial influence of G-A supplemented on the inner digestive tract bacteria of rainbow trout,
growth may be due to a change of the intestinal have a big capacity to adhere to and survive in the
microflora by mannanoligofructose, lactose or other intestinal tract. LAB supplementation, however,
carbohydrates from the dairy ingredient, partially demonstrated an ability to challenge the resident
autolyzed yeast and/or dried fermentation products. microbiota, since the increase in LAB observed was
In the present study, it is given the evidence of possibly the result of a fall in intestinal pH motive
improvement in the health and growth performance by lactic acid or other fermented products produced
of fish in spite of the differences in methods and by LAB strains. The previous studies (Ringø et al.,
species used. 1998; Ringø and Olsen 1999) indicated that dietary
Intestinal microflora fatty acids and carbohydrates changed the bacterial
At the end of trial, it was noticed that the flora of the gastrointestinal tract of fish. Similar
prebiotic supplementation into diet (extruded pellet findings were pointed out by Li and Gatlin (2004,
food) in these studies resulted significant (P<0.05) 2005), who investigated the effect of commercial
increases in total bacterial counts among prebiotic prebiotics GrobioticTM AE and GrobioticTM
supplemented groups, which were significantly supplemented in diets of hybrid striped bass
different in compared to total bacterial counts in (Morone chrysops × M. saxatilis) growth and
controls at the digestive tract of O. mykiss (P<0.05). exhibited that the prebiotic promoted the growth
In the present study, we were able to detect high performance. The results of this present study
amounts of lactic acid bacteria (LAB) in the clearly indicated that diet supplemented with G-A
intestine after 12 weeks of prebiotic at a suitable concentration (20, 25 and 30 g kg-1)
supplementation. However the values noted on could improve the total bacterial counts and LAB of
different media such as: (Aerobic; 4.92 vs. 4.46 all intestine O. mykiss (Table 5). Lactic acid
CFU/g), (Anaerobic; 7.21 vs. 4.80 CFU/g) and bacteria had been considered beneficial residents of
(LAB; 6.60 vs. 0 CFU/g) G-A group treatments and the fish intestinal function because of producing
basal which these present data the beneficial bacteriocins and hence positively affecting the
influence of prebiotic on growth was perhaps due to host‟s microflora (Ringø et al., 1998; Irianto and
an alteration of the intestinal microflora. After 12 Austin 2002; Ringø et al., 2006). Prebiotic
weeks of G-A-feeding in the present study, we were oligosaccharides such as inulin and oligofructose
able to detect high amounts of LAB and total are fermented in the colon where they promote the
bacterial counts on aerobic and anaerobic media in growth of bacterial populations associated with a
the intestine, which demonstrates that G-A healthy, well functioning colon. This selective
332 Journal of Research in Biology (2011) 5: 325-334
Azari et al.,2011

stimulation occurs because oligosaccharides are human milk neutral oligosaccharides in a diverse
readily fermented by beneficial types of colonic population. J. Pediatr, Gastroenterol Nutr., 30:131-
bacteria and are not used effectively by potentially 133.
pathogenic bacteria species (Flickinger et al.,
2003). Our results have demonstrated that G-A, as FAO. 2006. The state of world fisheries and
well as other prebiotic ingredients, may promote the aquaculture 2004. Rome, Italy. pp 14-17.
maintenance of lactic acid-production as some
reports have also supported the point (Mussatto and FAO. 2007. Technical Meeting on Prebiotics.
Mancilha 2007; Moura et al., 2007). Based on our September 15-16, FAO, Rome, Italy. 11.
data, the beneficial influence of prebiotic on growth
was possible due to an alteration of the intestinal Flickinger EA, Van Loo J, Fahey GC. 2003.
microflora. Nutritional responses to the presence of inulin and
oligofructose in the diets of domesticated animals, a
ACKNOWLEDGMENTS review. Crit. Rev. Food Sci. Nutr., 43:19-60.
We express our best sincere gratitude to
Ghezelalaparvar Trout farming (private fish Gibson GR and Roberfroid MB. 1995. Dietary
farming), Caspian Sea Research Institute of modulation of the human colonic microbiota
Ecology of Iran and IIC, International Ingredient introducing the concept of prebiotics. J, nutr., 125:
Corporation of USA for providing the fry and 1401-12.
facilities for the study.
Hanley F, Brown H and Carberry J. 1995. First
REFERENCE: observations on the effects of mannan
AOAC. 1997. Official Methods of Analysis of oligosaccharide added to the hatchery diets for
AOAC International, 16th edn, . Association of warmwater Hybrid Red Tilapia. In: Nutritional
Official Analytical Chemists, Arlington, VA, USA. Biotechnology in the Feed and Food Industries:
1(4):1–3 Proceedings of Alltech‟s 11th Annual Symposium
(Suppl. 1, Abstracts of Posters presented), May 20-
Bogut I, Milakovic Z, Pavlicevic J, Petrovic D. 23, Lexington, KY, USA. 11.
2006. Effect of Bio-Mos® on performance and
health of European catfish (Silirus glanis). In: Houdijk JGM, Bosch MW, Verstegen MWA.
Nutritional Biotechnology in the Feed and Food and Berenpas HJ. 1998. Effects of dietary
Industries: Proceedings of Alltech‟s 22nd Annual oligosaccharides on the growth performance and
Symposium (Suppl., Abstracts of Posters faecal characteristics of young growing pigs. Anim,
presented), April 23-26, Lexington, KY, USA. 90. Feed Sci, Technol., 71, 5-48.

Brafield AE. 1985. Laboratories studies on energy Irianto A and Austin B. 2002. Probiotics in
budgets. In: Fish Energetics. New Perspective aquaculture. J. Fish Dis 25, 633–642.
(Tytler, P. & Calow, P. eds), Croom Helm, London
and sydney. 257-281. Jones R, Hussein HM and Zagorec M. 2008.
Food Microbiology 25:228-234.
Cerezuela R, Cuesta A, Meseguer J, Esteban M.
A. 2008. Effects of inulin on gilthead seabream Li P and Gatlin DM. 2004. Dietary brewer‟s yeast
(Sparus aurata L.) innate immune parameters. Fish and the prebiotic Grobiotic® AE influence growth
Shellfish Immunol., 24:663-668. performance, immune responses and resistance of
hybrid striped bass (Morone chrysops ×M.
Costalos C, Kapiki A, Apostolou M, Papathoma saxatilis) to Streptococcus iniae infection.
E. 2008. The effect of a prebiotic supplemented Aquaculture 231:445-456.
formula on growth and stool microbiology of term
infants. Early Hum Dev., 84:45-49. Li P, Gatlin DM. 2005. Evaluation of the prebiotic
Grobiotic® AE and brewer‟s yeast as dietary
Erney RM, Malone WT, Skelding MB, Marcon supplements for sub-adult hybrid striped bass
AA, Kleman-Leyer KM. 2000. Variability of (Morone chrysops × M. saxatilis) challenged in situ

Journal of Research in Biology (2011) 5: 325-334 333
Azari et al.,2011

with Mycobacterium marinum. Aquaculture 248:
197-205.

Moura P, Marques S, Alves L, Freire JPB,
Cunha LF, Esteves MP. 2007. Effect of xylo
oligosaccharides from corn cobs autohydrolysis on
the intestinal microbiota of piglets after weaning.
Livest. Sci., 108:244-248.

Mussatto SI, Mancilha IM. 2007. Non-digestible
oligosaccharides: a review. Carbohydr Polym.,
68:587-597.

Ringø E and Gatesoupe FJ. 1998. Lactic acid
bacteria in fish: a review. Aquaculture 160:177-
203.

Ringø E and Olsen RE. 1999. The effect of diet on
aerobic bacterial flora associated with intestine of
Arctic charr, Salvelinus alpinus L. J. Appl.
Microbiol., 86:22-28.

Ringø E, Sperstad S, Myklebust R, Refstie S and
Krogdahl A. 2006. Characterisation of the
microbiota associated with intestine of Atlantic cod
(Gadus morhua L.). Aquaculture 261:829-841.

Staykov Y, Spring P, Denev SA and Sweetman J.
2007. Effect of a mannan oligosaccharide on the
growth performance and immune status of rainbow
trout (Oncorhynchus mykiss), Aquacul. Int., 2:153-
161.

Verschuere L, Rombaut G, Sorgeloos P and
Verstraete W. 2000. Probiotic bacteria as
biological control agents in aquaculture, Microbiol,
Mol, Biol, Rev, 64:655-671.

334 Journal of Research in Biology (2011) 5: 325-334
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Isolation, screening and induction of mutation in strain for extra cellular
lignin peroxidase producing bacteria from soil and its partial purification
Journal of Research in Biology

Authors: ABSTRACT:
Renugadevi R,
Ayyappadas MP,
Preethy PH and Savetha S.
The Lignin peroxidase enzyme production was carried out through bacteria
isolated from sample soil collected around decayed wood at Sulur, Coimbatore. In this
Institution:
Assistant Professors, report, a mutant of Bacillus subtilis LPTK was obtained after chemical mutagenesis
Department of with EMS and screened for better Lignin peroxidase production. In the production
Biotechnology optimization studies, the bacterial strain needs temperature around 37OC, pH.6,
RVS College of Arts lactose as a carbon source, peptone as nitrogen source and incubation time for 36
and Science, Sulur. hours for its higher enzyme productivity. The partial purification was also done.

Corresponding author:
Renugadevi R

Email: Keywords:
renugadevi@rvsgroup.com Lignin, LP, Bacillus subtilis and enzyme activity.

Web Address: Article Citation:
http://jresearchbiology.com/ Renugadevi R, Ayyappadas MP, Preethy PH and Savetha S.
Documents/RA0085.pdf.
Isolation, screening and induction of mutation in strain for extra cellular lignin
peroxidase producing bacteria from soil and its partial purification
Journal of research in Biology (2011) 4: 312-318

Dates:
Received: 17 Aug 2011 /Accepted: 27 Aug 2011 /Published: 12 Sep 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

312-318 | JRB | 2011 | Vol 1 | No 5
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Renugadevi et al.,2011

Introduction up to 107 dilutions using sterile saline as a blank and
Lignin is the most abundant renewable the diluted samples were plated into the sterile
carbon source on earth (Bo Zhang et al., 2008). nutrient agar using spread plate method. The
When plants die, it will rot and degrade in soil. nutrient agar plates were incubated at 37°C for 24
Researches are widely done on fungi which degrade hours. The isolated colonies were further purified
lignin. While bacteria grow and multiply faster, it is by streak plate method using sterile media plates.
anticipated to be better in production of lignin The pure cultures were inoculated into sterile
degrading enzymes. There are many types of lignin nutrient agar slants and nutrient broth for further
degrading enzymes such as lignin peroxidase, use.
manganase peroxidase, laccase and glyoxal oxidase Screening for the Enzyme Production
are produced by fungi as well as bacteria such as The isolated pure strains were screened for
lignin peroxidase by the bacteria Streptomyces the production of extra cellular lignin peroxidase
viridosporus (Ramachandra et al., 1987). using screening medium which contains lignin as a
Lignin peroxidase is an enzyme that is used substrate (Yeoh Teik Loon et al., 2006). The pure
to degrade lignin. It was first discovered in 1983. cultures were streaked at the lignin agar plates and
Lignin peroxidases are produced by many wood the plates were incubated at 37°C for 24 hours. The
degrading fungi as a family of isoenzymes (Kirk observation was made to see the substrate utilized
and Farrell, 1987). Recent researches also showed zone around the colony. Only the strain showing
that it can be produced from bacteria such as positive and better zone was taken for further study.
Streptomyces viridosporus. These hemeproteins are Strain Improvement by Mutation
similar to the more familiar plant peroxidases in The chemical mutagenesis was carried out
structure and mechanism, and utilize hydrogen according to Kotchoni and D. Bartels (2003). Ethyl
peroxide and organic peroxides to oxidize a variety methanesulphonate (EMS) was used as the
of substrates (Tien et al., 1985, 1986). Some of the mutagenic agent. The better zone formed strain was
most important features distinguishing these grown to mid logarithmic phase of growth in LB
enzymes from other oxidoreductases (such as broth and from that cells were centrifuged at room
horseradish peroxidase), for example, are their very temperature. The pellet was re-suspended in normal
low pH optima and much higher redox potentials. saline and a suspension of (v/v) of EMS was made.
The substrates of lignin peroxidase include Appropriate dilutions were made and incubated
both phenolic and non-phenolic aromatic overnight at room temperature to allow mutational
compounds; the phenolic substrates are oxidized to segregation. The culture was then spread on agar
yield products similar to those produced by media and incubated overnight at 37°C. Single
classical peroxidases, while the oxidation of the non mutant clones were further grown in new plates till
-phenolic methoxybenzenes are unique to the lignin the third generation. The third generations (M3) of
peroxidases (Korsten L and Cook N, 1996), the the mutants were replica-plated in nutrient agar
oxidation of these substrates yield aryl cation plates. The wild and mutant strain was screened
radicals. Lignin peroxidase can catalyze the again using lignin screening medium. The selection
oxidation of substrates with a reduction potential of mutants was based on the diameter of the clear
greater than 1.3 volts. The enzyme is capable to zone surrounding the colonies.
oxidizing lignin monomers, dimers and trimers as Enzyme Production
well as polycyclic aromatic compounds such as Preparation of Inoculum
benzopyrene (Haemmerli et al., 1996). The The inoculum for further production of
powerful and relatively non-specific nature of this enzyme and other studies was prepared using Luria
enzyme has lead to the investigation of its potential broth (Zolta´n Pra´Gai, 2002). The pure culture was
use in diverse fields of biodegradation of toxic inoculated into sterile inoculum broth and was
chemicals, pulp, paper processing, and in the textile incubated at 37°C in a rotary shaker for overnight.
industry. The fresh over night culture was used as inoculum
for the production of enzyme.
METHODS AND MATERIALS Production
Sample Collection and Isolation of Bacteria The enzyme production was carried out by
The sample soil was collected around shake flask fermentation using production medium
decayed wood at Sulur, Coimbatore in a sterile with pH 8. The inoculated medium was incubated
container. The collected sample was serially diluted at 37°C for 48 hours. The medium was agitated at
313 Journal of Research in Biology (2011) 5: 312-318
Renugadevi et al.,2011

200 rpm for better aeration and growth of the medium was incubated at 37°C temperature with
organism. shaking around 150 rpm. After incubation, around
Lignin peroxidase Assay 20 ml of culture was aseptically withdrawn
Bacterial crude culture prepration periodically at 6 hours intervals up to 48 hours. The
Aliquots of 10ml of the culture suspension culture filtrate was tested for the total protein
were centrifuged at 5000 rpm for 15 minutes and content and lignin peroxidase activity (Tuncer et al,
cell free extract was subjected to enzyme assay. The 2004).
extract was stored at 4°C for further analysis. Temperature
Plate Assay Hundred ml of sterile production medium
The plate assay was performed using agar for bacteria was prepared in different conical flask
plates amended with Lignin. The agar plates were at pH 8.0 and inoculated with 5% inoculum. Each
prepared by mixing of 1% Lignin with 1.7% agar. flask was incubated at different temperatures such
After solidification of agar, around 10 mm as 28°C, 32°C, 37°C, 42°C, 47°C, and 52°C for 36
diameters of wells were cut out aseptically with the hours. The protein estimation and enzyme activity
help of cork-borer. The well was filled with the were estimated followed by Tuncer et al (2004).
culture filtrate and incubated at 37°C for overnight. pH
The observation was made to see the hydrolytic Hundred ml of sterile production medium
zone around the wells. Two wells were filled with was prepared for bacterial strain in different
distilled water and production medium without conical flasks and each flask was adjusted to
culture acts as a control (Yeoh Teik Loon et al., different pH such as 4, 5, 6, 7, 8 ,9 using 0.1N
2006) NaOH and 0.1N HCl. After sterilization, flasks
Chemical Assay were inoculated with 5% inoculum. The flasks were
Lignin peroxidase activity was determined incubated at 37ºC for 36 hours. The protein
by estimating the reducing sugar produced during estimation and enzyme activity were estimated
enzymatic reaction by dinitro salicylic acid method. (Tuncer et al, 2004).
Assay for lignin peroxidase were performed using Carbon Sources
substrate composed of alkaline lignin dissolved in Hundred ml of sterile production medium
0.1M phosphate buffer with pH of 7.0. After (pH.6) for bacteria was prepared in different conical
incubation at 33°C for 24 hours, the broth is flasks. Each flask was amended with different
transferred for centrifugation. Centrifugation was carbon sources such as Glucose, Fructose, Maltose,
done for 5 minutes. The supernatant obtained was Lactose, Sucrose and Mannitol. The flasks were
used as a crude enzyme. The crude enzyme was inoculated with 5% inoculum and incubated at 37°C
incubated with substrate at 33°C. The reaction for 36 hours. The culture filtrate was collected for
mixtures were tested for its simple sugar using DNS protein estimation and enzyme activity (Tuncer et
method. al, 2004).
Estimation of Total Protein Nitrogen source
The chemical assay for the total protein Hundred ml of sterile production media for
content from the sample was determined using bacteria (pH 6) was prepared in different conical
Bradford method (Bradford, 1976). To the culture flasks. Each flask was amended with different
filtrate Bradford reagent was added. The tube was nitrogen sources such as Beef extract, Casein,
gently tilted once for mixing and the absorbency Peptone, Gelatin, Ammonium chloride, Ammonium
was taken at 595nm in UV- VIS spectrophotometer. sulphate and Potassium nitrate. The flasks
The blank was prepared by mixing distilled water containing bacterial medium were inoculated with
reagent. The protein concentration was determined 5% inoculum and incubated at 37ºC for 36 hours.
by comparing the value with standard graph The culture filtrate was collected for Protein
prepared using Bovine serum albumin. estimation and Enzyme activity (Tuncer et al,
PARAMETER OPTIMISATION STUDIES 2004).
Effect of incubation time on lignin peroxidase Partial Purification
production Ammonium Sulphate Fractionation of Proteins
Around 500ml of sterile production medium The enzyme separation from the exhausted
for bacterial were prepared and 5% bacterial medium was done by 70% Ammonium sulphate
inoculum was added aseptically. The inoculated saturation (Elba et al, 1999). The mixture was then

Journal of Research in Biology (2011) 5: 312-318 314
Renugadevi et al.,2011

stored in a cold room for 24 hours to precipitate all culture was withdrawn for enzyme activity. The
the proteins and the precipitation was separated by bacterial cultures were withdrawn once in every six
centrifugation for 10 minutes. The supernatant was hours. The results revealed that there is gradual
discarded and the remaining precipitate was increasing of production has occurred from 18 th
dissolved with 5 ml of 1M-citrate phosphate buffer hour to 36th hours and higher production has
(pH.8). Then the mixture was subjected to dialysis. occurred at 36th hours (Table.1). This shows that
Dialysis bacterial isolate should have maintained its log
The purpose of dialysis is to remove phase from around 18th hour to 36th hour. Besides, it
undesired small molecular weight molecules from a is believed that the higher production of lignin
mixture in which the desired species of molecules peroxidase has occurred in extreme log phase
are too large to travel across the membrane. because even though the log phase was maintained
Ordinarily this process is utilized during protein around 18th to 36 hours, the followed drop of
purification in which salting out procedure has been production has indicated that the organism should
employed as the initial step with ammonium have entered into stationary phase of growth (Fig
sulphate. After the protein is precipitated from the 1).
initial source, it is re-dissolved in buffer and then Effect of Temperature
poured into a dialysis bag. The environmental parameters are showing
great influence in the growth of the organisms and
RESULTS the production of enzymes. The optimum
Naturally occurring microorganisms are temperature for the better production was made in
having the ability to produce various enzymes. Now various temperatures. It was found that like other
a day’s most of the enzymes are important for mesophilic organisms, the higher lignin peroxidase
human welfare and industry. In this study, the activity was found (41U/ml) at 37ºC from bacteria
bacterial strains were isolated from the sample soil
at Sulur area, Coimbatore because most of the
natural wastes are degraded by the native microbes
that are growing over that waste. In such a way, it is
fact that the microbes which are isolated from
decayed wood soil may have ability to produce
lignin peroxidase. From the samples, around 15
bacterial strains were isolated and screened; it was
found that four bacterial strains showed positive
results on lignin peroxidase production. The better
zone formed bacterial strain was considered for
further strain improvement study.
Strain improvement was done by Kotchoni
and Shonukan (2002) method. In this experiment
Figure 1. Enzyme production and total protein
the better positive strain for lignin peroxidase was content of bacterial isolate at different incubation time
treated with Ethyl methanesulphonate and the M3
generation mutant strain produced much better (Table.2). These indicate that the optimum
yellow zone then compared to wild strain. This temperatures for better production of bacterial
strain was used for further studies. isolates are 37ºC (Fig.2). The temperature
In plate assay the lignin peroxidase activity requirement of the organism is based on the nature
was identified by a clear zone when compared with of organisms. Many thermophilic bacteria like
the control and the chemical assay determined by Clostridium sp. needs 55ºC for better production of
DNSA (3, 5- dinitro salicylic acid) method using lignin peroxidase (Tuncer et al, 2004).
starch as a substrate.
Effect of Time Effect of pH
The growth study of the organism is The pH is the important parameter which
essential for the production of enzyme because determines the growth of the organism and lignin
most of the extra cellular enzymes are produced peroxidase production. The study results showed
during log phase of the organisms. Generally during that the optimum pH around six is better for
growth, the bio mass of the cells was estimated. The bacterial isolate (Table. 3 & Fig.3). Like
315 Journal of Research in Biology (2011) 5: 312-318
Renugadevi et al.,2011

temperature, different organisms need different pH Table.3 Effect of pH on total protein and enzyme
ranges for its lignin peroxidase production. The production of bacterial isolate
Thermophilic bacterium has produced high amount Enzyme
of lignin peroxidase between pH 4-6 (Tuncer et al, S. pH
Total protein content
activity
2004). NO (μg/ ml)
(U/ml)
1 4 9 4
Effect of Carbon source 2 5 31 21
Table.2 Effect of Temperature on total protein and 3 6 73 41
enzyme production by bacterial isolate 4 7 92 33
5 8 70 15
Total protein Enzyme
S. Temperatur 6 9 62 12
content activity
NO e (ºC)
(μg/ml) (U/ml)
1 28 49 15
2 32 50 20
3 37 52 41
4 42 40 26
5 47 31 12
6 52 28 4

Figure.3 Effect of pH on total protein and enzyme
production of bacterial isolate

nitrogen sources are of secondary energy sources
for the organisms, which play an important role in
the growth of the organism and the production
(Table.5 & Fig.5).

Figure 2. Effect of Temperature on total protein and DISCUSSION
enzyme production by bacterial isolate The Lignin peroxidase enzyme production
Table. 4 Enzyme production and total protein
The carbohydrates are soul energy source for oduction on different carbon sources
most of the heterotrophic organisms. These shows
great influence on the production of many enzymes. Bacteria
Carbon Total protein Enzyme
In this study, lactose was found to be a right carbon S.No
sources content activity
source for bacterial strain for higher production of (μg/ ml) (U/ml)
lignin peroxidase (Table. 4 & Fig.4). Lignin 1 Glucose 48 6
peroxidase production will be low in the medium 2 Fructose 57 18
amended with glucose, fructose, maltose,sucrose 3 Maltose 38 9
and mannitol when compare to lactose (Tuncer et 4 Lactose 86 31
al, 2004). 5 Sucrose 51 15
Effect of Nitrogen source 6 Mannitol 47 10
The nitrogen sources are of secondary
energy sources for the organisms, which play an was carried out through bacteria isolated from
important role in the growth of the organism and sample soil collected around decayed wood at
the production. The nature of the compound and the Sulur. Trichoderma harzianum isolates (D1/4)
concentration that we are using may stimulate or proved to be the highest producer for the two
down modulate the production of enzymes. The enzymes such as 160% and 186% of CMCase and β

Journal of Research in Biology (2011) 5: 312-318 316
Renugadevi et al.,2011

Figure.4 Enzyme production and total protein
production on different carbon sources
-glucosidase respectively, more than the original
strain (Ahmed M. El-Bondkly et al., 2010). In this
report, a mutant of Bacillus subtilis LPTK was Figure. 5 Enzyme and total protein production on
obtained after chemical mutagenesis with EMS and different nitrogen sources
screened for better Lignin peroxidase production.
The effect of carbon and nitrogen sources on lignin Radwan and Dora SA. 2010. Successive
peroxidase (LiP) activity of Aspergillus sp., showed Construction of β-Glucosidase Hyperproducers of
that glucose (1%) and ammonium sulphate were the Trichoderma Harzianum Using Microbial
best carbon and nitrogen sources (Ahammed S and Biotechnology Techniques, J. Microbial &
Prema P,2002). In the production optimization Biomedical Technology 2:070-073.
studies, the bacterial strain needs temperature
around 37OC, pH.6, lactose as a carbon source, Bradford MM. 1976. A rapid and sensitive for the
peptone as nitrogen source and incubation time for quantization of microgram quantities of protein
36 hours for its higher enzyme productivity. utilizing the principle of protein-dye binding.
Analytical Biochemistry 72:248-254.
REFERENCES
Ahammed S and Prema P. 2002. Influence of Bo Zhang, Hua-Jiang Huang and Shri
media nutrients on synthesis of lignin peroxidase Ramaswamy. 2008. Reaction Kinetics of the
from Aspergillus sp., Appl Biochem Biotechnol, Hydrothermal Treatment of Lignin, J. Applied
102-103(1-6):327-36. Biochemistry and Biotechnology 147:119-131.

Ahmed M. El-Bondkly AAM, Aboshosha NH. Elba P S. Bon, Hilton J Nasecimento, jacyara M,
B Macedo and Jose G, silva Jr. 1999. Lignin
Table.5 Enzyme and total protein production on peroxidase isofor ms from streptomyces
different nitrogen sources viridosporus T7A: are they a monomer based
Bacteria structure, Biotechnology Techniques 13:289-293.
Nitrogen Total protein Enzyme
S.No
sources content activity Haemmerli SD, Leilosa MSA, Sanglard D and
(μg/ ml) (U/ ml) Fiechter A. 1996. Oxidation of benzo(a) pyrene by
1 Beef extract 46 24
extracellular ligninases of phanerochaete
2 Casein 98 32
chrysossporium: veratyl alcohol and stability of
3 Peptone 90 41
ligninases, J. Biochem, 261:6900-6903.
4. Gelatin 94 34
Ammonium
5 38 24 Kirk TK and Farrell. 1987. Enzymatic
chloride
Ammonium combustion: the microbial degradation of lignin,
6 36 22 ann, ERv, Microbiol, 41:465-505.
sulphate
Potassium
7 31 23
nitrate Korsten L and Cook N. 1996. Optimizing
317 Journal of Research in Biology (2011) 5: 312-318
Renugadevi et al.,2011

culturing conditions for bacillus subtilis, South
African Avocado Growers Association Yearbook,
19:54-58.

Kotchoni SO and Shonukan OO. 2002.
Regualtory mutations affecting the synthesis of
cellulase, World J Microbiol Biotechnol., 18:487-
491.

Ramachandra M, Crawford DL and Hertel G.
1988. Characterization of an extracellular lignin
peroxidase of the lignocellulolytic actinomycete,
streptomyces and viridosporus, App. Environ,
Microbiol., 54:3057-3064.

Tien M, Kersten PJ, Kalyanaraman B and Kirk
TK. 1985. The ligninase of Phanerochaete
chrysosporium generates cation redicals from
methoxybenzenes. J Biol Chem, 10:260(5):2609-
2612.

Tien M, Kirk TK, Bull C and Fee JA. 1986.
Steady state and transient state kinetics on the
oxidation of 3, 4-dimethoxybenzyl alcohol
catatyzed by the ligninase of phanerocheate
chrysosporium burds, J. Biol Chem, 261(4):1687-
1693.

Tuncer M, Kurul A, Isikli M and elenk FGC.
2004. Optimization of extracellular endoxylanase,
endoglucanase and peroxidase production by
strptomyces sp. F2621 isolated in turkey, J. Applied
Microbiology 97:783-791.

Yeoh teik loon. 2006. Screening of lignin degrader
from soil, thesis. Submitted for the degree of
bachelor of chemical engineering.

Zolta’n pragai and Colin R Harwood. 2002.
Regulatory interactions between the pho and rB-
dependent general stress regulations of bacillus
subtilis 148:1593-1602.

Journal of Research in Biology (2011) 5: 312-318 318
Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Relationship between Anemia and Parasitic Infections in Shekhan District,
Iraq
Journal of Research in Biology

Authors: ABSTRACT:
Bushra H. Al-Naemi, A total of 431 of stool samples were collected from patients consulting the
Zohair IF. Rahemo*, governmental health center in Al-Shakhan district (Mosul, Iraq) , their ages range from
Sundus N. Al-Kallak. 1-50 years, the period of collection lie between January 2009-October 2009. The
highest percentage of the occurrence of cysts was those of Entamoeba histolytica
(35.8.0%) followed by eggs of Enterbius vermicularis (32.60%), then cysts of Giardia
lamblia (26.81%), then the eggs of Ancylostoma dudenale (2.17%), then eggs of
Ascaris lumbricoides (1.44 %) and the lowest was eggs of Trichuris trichura (1.08%).
The percentage of anemia is higher in infected persons compared with uninfected as
infection with E. histolytica (38.98% and 35% respectively), similarly G. lamblia
Institution: (28.18% and 26.2% respectively) , A. duedenale ( 8.47% and 0.4% respectively)T.
Department of Basic trichura (5.08% and 0.0 respectively, and in A. lumbricoides (1.69% and 1.3%
Medical Sciences, College of respectively), while infection with E. vermicularis the percentage of infection is higher
Nursing, Mosul University, in unanemic persons(36.8% and 16.9% respectively). F u r t h e r m o r e , the
Iraq highestpercentage of infection by E. histolytica occurs in the age 11-20(40%) while the
*Department of Biology, lowest was in the age 41-50 and more(21.4 %). As regard infection of G. lamblia the
College of Science, highest infection was in the age 1-10 (39.5%) while the lowest was in the age 31-40
University of Mosul, Mosul, (11.4%). The infection with E. vermicularis , the highest was in the age 11-20(43.4%)
Iraq. and the lowest in the age 1-10(19.7%). The highest infection with A. duedenale was in
the age 11-20(3%) while the lowest in the age 1-10(2 %). As concern infection with A.
lumbricoides the highest infection rate (3.1%) was in the age 1-10 while the lowest
was in the age 11-20(1%). Infection with T. trichuris the highest infection rate (2%) was
in the age 1-10 while the lowest ( 1%) in the age 11-20. As regards haemoglobin
concentration and the type of the parasite, the highest concentration was in case of
infection by G. lamblia (11%), then E. histolytica(10.3%), A. lumbricoides(10%) then E.
Corresponding author: vermicularis (9.5%) then in case of T. trichura( 9 ), then the lowest was in A. duodenale
Zohair IF. Rahemo (8%).while concentration of hemoglobin in different age group: the highest
concentration was in age group41-50(10.5 g/dl) and then the 31-40(10 g/dl) then the
age group 1-10(9.7% g/dl) then in age group 11-20( 9.2 g/dl) and the lowest was in the
age group 21-30(8.1% g/dl).

Article Citation:
Bushra H. Al-Naemi, Zohair IF. Rahemo, Sundus N. Al-Kallak.
Relationship between Anemia and Parasitic Infections in Shekhan District, Iraq
Journal of research in Biology (2011) 5: 319-324
Web Address:
http://jresearchbiology.com/
Documents/RA0087.pdf. Dates:
Received: 17 Aug 2011 /Accepted: 27 Aug 2011 /Published: 12 Sep 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

319-324 | JRB | 2011 | Vol 1 | No 5
Journal of Research in biology
Submit Your Manuscript
An International Open Access Online
Research Journal www.ficuspublishers.com www.jresearchbiology.com
Al-Naemi et al.,2011

INTRODUCTION MATERIALS AND METHODS
Anemia is a common disease observed in A total of 431 of stool samples were
tropics. Iron deficiency anemia affects about 1.3 collected from patients consulting the governmental
billion people with the highest prevalence and Health Center in Al-Shekhan district. Al –Shekhan
morbidity in young children and pregnant women district situated about 50 Km northern of mosul
(Gillespie et al.1991).The only study investigated city, it is a hily area with continental climate similar
the relationship between intestinal parasites and to mosul climate especially in spring and autumn,
anemia was done persons in Nineveh Governorate while in winter temperature range for -5 to 8c and
(Al-Heali,2009), she examined 499 stool samples in summer 40-50 c. Its population can be estimation
and found six species of parasites and she to be more than 70.000 person including the center
concluded that the haemoglobin decreased in the out its suburbs. The water supply come from wells
blood of infected persons with 22% . However situated in the hilly area. Most of Shekhan people
anemia is considered a serious public health and are working in agriculture especially wheat and
Schistosoma mansoni is considered a risk factor in barley in addition to animal breeding such as sheep,
Egypt (Curtale et al. 1998).Food and drinks are the goats, and poultry. Their ages range between 1-50
main routes in which intestinal parasites can inter year, the period of collection lie between January
the body and to certain extent other routes such as 2009- October 2009.Patients attending this
flies and mosquitoes (Greets and Gryseels, 2000). governmental outpatient clinic are usually suffering
Furthermore, bad hygiene and social behavior in from common cold, diarrhea, renal colic,
addition to general health can increase the dermatological problems, diabetes, heart pressure,
possibility of anemia occurrence(El Kichaoi and breast cancer, gynecological diseases, and sterility.
Abd Rabou, 2004). Intestinal helminthes such as The patient who suffer from diarrhea, and
Ancylostoma duedenale infect 9000 million people abdominal pain, and vomiting were selected for this
in the world, while Ascaris lumbricoides and study. Stool specimens were preserved in a 15 cc
Trichuris trichura infect 800 million, and vial containing 10% formaline as a fixative with
Enterobius vermicularis infect 42 million in fitted cover and the age of the patient was recorded
developed countries(Lobo et al. 1998). Most of for each sample, and stored for two –three days.
persons infected with parasitic infection suffering Two to three slides from each vial were prepared, a
from nutrition, nausea, weakness, diarrhea, anemia drop of Lugols iodine was added, then examined
(Pothinpak et al.2005). Patients with heavy under the microscope by direct method according
infection may lose up to 200 ml of blood per day, to Neimeister et al. (1990). Haemoglobine was
but around 40% of iron may be reabsorbed before it estimated using Sahli method (Lewis et al., 2001).
leaves the intestine. Nevertheless, a moderate The normal haemoglobine levels:
Ancylostoma doudenale infection will gradually Age Hemoglobin levels
produce an iron-deficiency anemia as body reserves Less than 7 days 17 to 22 h/dl
of iron are used. Severity of anemia depend on 1 Week 15 to 20 g/dl
worm load and dietary iron intake of patient 1 Month 11 to 15 g/dl
(Roberts and Janovy, 2005). Children 11 to 13 g/dl
Anemia is the most common disease among Adult males 14 to 18 g/dl
malnutrition and among the main causes is Elderly males 12.4 to 14.9 g/dl
insufficient protein , folic acid , B6 or B12 and Adult females 12 to 16 g/dl
vitamine C, while the other causes of anemia are Elderly females 11.7 to 13.8 g/dl
intestinal helminthes, amoebiasis and malaria
(Koukounari et al. 2008). The most common RESULTS
symptoms are bale blood, eye white, feeling of tire, As indicated in Table (1), the highest
nausea, lips fractures(Shubair et al.2005). percentage of the occurrence of cysts was those of
The aim of the present study is to investigate Entamoeba histolytica (35.8.0%) followed by eggs
the relationship between the anemic persons and the of Enterbius vermicularis (32.60%), then cysts of
presence of intestinal parasites in different age Giardia lamblia (26.81%), then the eggs of
group in a sample taken from Shekhan district, Ancylostoma dudenale (2.17%), then eggs of
Mosul, Iraq. Ascaris lumbricoides (1.44 %) and the lowest was
eggs of Trichuris trichura (1.08%).

320 Journal of Research in Biology (2011) 5: 319-324
Al-Naemi et al.,2011

Table (1): the different intestinal parasites and their lowest in the age 1-10(2 %). As concern infection
percentages. with A. lumbricoides the highest infection rate
Total number Percentage of (3.1%) was in the age 1-10 while the lowest was in
Parasite
of infection infection the age 11-20(1%). Infection with T. trichuris the
Entamoeba highest infection rate (2%) was in the age 1-10
99 35.8
histolytica while the lowest ( 1%) in the age 11-20.
Giardia lamblia 74 26.81 As indicated in Table (4), showing
Enterobius haemoglobin concentration and the type of the
90 32.60
vermicularis parasite, the highest concentration was in case of
Ancylostoma
6 2.17 infection by G. lamblia (11%), then E. histolytica
duodenale
Ascaris
(10.3%), A. lumbricoides(10%) then E.
4 1.44 vermicularis (9.5%) then in case of T. trichura( 9 ),
lumbricoides
Trichuris then the lowest was in A. duodenale(8%).
3 1.08 Table(5), showing the concentration of
trichura
total 276 100.0 hemoglobin in different age group. The highest
concentration was in age group41-50(10.5 g/dl) and
As shown in Table(2), the percentage of then the 31-40(10 g/dl) then the age group 1-10
anemia is higher in infected persons compared with (9.7% g/dl) then in age group 11-20( 9.2 g/dl) and
uninfected as infection with E. histolytica (38.98% the lowest was in the age group 21-30(8.1% g/dl).
and 35% respectively), similarly G. lamblia
(28.18% and 26.2% respectively), A. duedenale DISCUSSION
( 8.47% and 0.4% respectively)T. trichura (5.08% As seen in Table(1), six parasites have been
and 0.0 respectively, and in A. lumbricoides (1.69% encountered in persons consulting Shekhan district
and 1.3% respectively), while infection with E. health center including two protozoans( E.
vermicularis the percentage of infection is higher in histolytica and G. lamblia) and four nematodes
unanemic persons(36.8% and 16.9% respectively). namely E. vermicularis, A. duodenale , A.
Table(3), indicated the revealed parasites lumbricoides, and T. trachura. In the present study
listed with their age group . The highest percentage E. histolytica was the most abundant parasite
of infection by E. histolytica occurs in the age 11- (35.8%) while that with lowest infection was T.
20(40%) while the lowest was in the age 41-50 and trichura(1%).
more(21.4 %). As regard infection of G. lamblia the Similar parasites were previously reported
highest infection was in the age 1-10 (39.5%) while from the school pupils and food handler by Al-
the lowest was in the age 31-40(11.4%). Daoody(1998) the most abundant one was E. coli
The infection with E. vermicularis, the 24.7% Al- Shirifi (2000) found that the most
highest was in the age 11-20(43.4%) and the lowest abundant parasites was G. lamblia (15.69%) while
in the age 1-10(19.7%). The highest infection with E. histolytica was 8.45% . Al-Abbady (2001) found
A. duedenale was in the age 11-20(3%) while the the abundant parasites was E. coli while E.
Table (2): intestinal parasite in patients with or histolytica was only 11.07%. Hama(2007)found
without anemia that E. vermicularis was the predominant
Parasite With anemia Without anemia helminthes with high rate of infection (29.8%)
Entamoeba while G. lamblia was the most abundant among
23 38.9 76 35.0 protozoa in schoolchildren in Erbil province
histolytica
Giardia (37.44%). Anyhow the abundance of E. histolytica
17 28.18 57 26.2
lamblia is likely because its life cycle is simple as there is
Enterobius no intermediate host, and the infection comes
10 16.95 80 36.8
vermicularis usually through polluted food and drinks and the
Ancylostoma house flies also is very known mechanical
5 8.47 1 0.4
duodenale transmitter in such cases, similar was drawn was
Ascaris conclusion by Niazi et al.1976.The lowest infection
1 1.69 3 1.3
lumbricoides
by T. trichura is expected as the worm is one of the
Trichuris
3 5.08 0 0.00 geohelminths which need some period about 10
trichura
days in the soil to be developed to infected stage
total 59 100.00 217 99.7

Journal of Research in Biology (2011) 5: 319-324 321
Al-Naemi et al.,2011

Table (3): prevalence of intestinal parasites by age in 276 patients
Age (year)
parasite 1-10 11-20 21-30 31-40 41-50
n % n % n % n % n %
E. histolytica 35 35.4 40 36.7 10 32.3 11 40.7 3 30.0
G. lamblia 38 38.4 21 19.3 9 29.0 4 14.8 2 20.0
E.vermicularis 19 19.2 43 39.4 12 38.7 11 40.7 5 50.0
A. duodenale 2 2.0 3 2.8 --- --- 1 3.7 --- 0.0
A.lumbricoides 3 3.0 1 0.9 --- --- --- 0.0 --- 0.0
T. trichura 2 2.0 1 0.9 --- ---- --- 0.0 --- 0.0
total 99 100.0 109 100.0 31 100.0 27 100.0 10 100.0

(Markell et al.1999) such soil may be not available, polyparasite infection profiles.
in other hand using chemical fertilizer instead of In Mexico, Brenllinger et al.(2003) after a
stool in farming may lead to reduction of infection. comparative study of infection and anemia in adult
As indicated in Table(2), the percentages of women found haemoglobin levels in hookworm-
anemia were more in infected persons with E. infected women - were significantly lower than
histolytica, G. lamblia, A. duodenale and T. uninfected woman anyhow anemia even if it is
trichuris, A. lubmricoides. The presence of the evident in the present population in Shekhan district
parasite which accompanied by anemia is likely due it is not clear whether it is due to falate and vitamin
to the effect of parasitism, or the person is in low B12 deficiencies and haemoglobinopathies like
nutritional level. On the other hand the presence of sickle-cell anemia and thalassemia. The cause of
parasite and the absence of anemia may be because anemia in our sample is more likely due to mild
the parasitic infection is in its early stage. Such iron deficiency which does not display microcytosis
finding is similar to the results found by Le et al. or other nutritional deficiencies similar to the
(2007) as she found more prevalence of anemia in results of Tysuyuoka et al. (1999) in Aracaju
schoolchildren with intestinal parasite infection in sergipe Brazil.
rural Vietnam especially in case of Trichuris As seen in Table(3), the highest percentages
infection(76%). In other study conducted in Kenya , of infection was in the age 11-20 and 1-10 by E.
Koukounari et al.(2008) found the children heavily histolytica , G. lamblia , E. vermicularis, A.
infected with S. mansoni were more likely to be dudenale, T. trichura. The highest percentage was
anemic compared with uninfected children. in this age means that the highest infection was in
However, in a group study of youngster in the childhood and adolescent group which mean the
northern Brazil, Ferrerria et al.(1998) found no persons are highly active and do not care about their
statistical difference when the association was made hygiene as persons are in close contact with
between each parasite (A. duodenale, T. trichuris, pollutant with soil as they play on the ground and
A. lumbricoides) and anemia. similar study was less care about their hygiene. Similar results of
conducted in Paroguoy( Labiano abello et al.1999). children infection especially by S. mansoni was
However, in a study conducted by observed by Koukounari et al.(2008) in Kenyan
Mupfasoni et al. (2009) in northern Rwanda schoolchildren similar conclusion was drawn by
concerning polyparasite in helminth infections and Erosie etal. (2001), (see Le et al.2007) when studies
their association to anemia nor the odds of stunting the relation between hookworm infection and
were found to be significantly different in the three haemoglobin status in rural elementary school

Table (4): concentration rate of hemoglobin according Table (5): concentration rate of hemoglobin according
to parasites species to age group
parasite n Haemoglobin g/dl Age group n Haemoglobin g/dl
E histolytica 23 10.3
1-10 19 9.7
G. lamblia 17 11
11-20 17 9.2
E. vermicularis 10 9.5
21-30 10 8.1
A. dudenale 5 8
31-40 8 10
A. lumbriciodes 1 10
T. trichura 3 9 41-50 5 10.5
322 Journal of Research in Biology (2011) 5: 319-324
Al-Naemi et al.,2011

children in southern Ethiopia as they claims that
farming and playing in moist soil resulting in higher Al Daoody AA. 1998. Epidemiology of intestinal
exposure to infective egg and filariform larvae in Parasites among pupils of a number of primary
the soil. Scholchildren in rural Vietnam was also schools and foodhandlers in Ninevah governorate.
highly infected with the highest prevalence for T. M.Sc. thesis, Mosul. 117.
trichura , A. lumbricoides as concluded by Le et al.
(2007). while Table(4),showing the highest Al Heali FM. 2009. The roles of parasites as
concentration of hemoglobin was in case of G. caused of anemia for elderly persons in Nineveh
lamblia.These results are puzzling as it is well governorate. Proceeding of the 6 th conference of
known that G. lamblia cause fatty stool with no Nursing College. 119-125.
blood (Roberts and Janovy,2005). Anyhow possibly
a virulent strain ( see Roberts and Janovy, 2005) Al Shirifi HMH. 2000. Prevalence of intestinal
may exist in Shekhan and caused such anemia . parasites among pupils of primary schools and
On the other hand in the present results A. foodhandlers in Al-Taamem province –iraq. A
duodenale seem to cause less anemia compared to thesis submitted by college Education , Mosul
G. lamblia. However, it was also known that blood university .
loss per worm is about 0.03 ml per day in N.
americanus and 0.26 ml per day for A. duodenale Brenitlinger PE, Capps L, Denson M. 2003.
(Roberts and Janovy, 2005) Possibly these Hookworm infection and anemia in adult women in
fluctuations of anemia depends on nutritional status rural chipas, Mexico(a), Salud Publica Mex.
of individuals which were not estimated in the 45:1179-1189.
present investigation, as nutritional status is an
important criterion in determination of anemic Curtale F, Nabil M, ElWakeel A, Shamy MY,
persons. Beheral Survey team. 1998. Anemia and intestinal
As regard Table (5), the anemia in older Parasitic Infections among school age children in
group is expected as immunity usually decrease and Behera Governorate. Egypt.J.Trop. Pediatr.44(6):
intestinal parasites with more percentage while the 323-328.
lowest anemia was in the age 21-30 which is the
climax of young stage . It puzzling that the highest El Kichaoi AY , Abd Rabou AFN. 2004.
concentration of hemoglobin in age 41-50 while Changing trend in frequency of intestinal parasites
lower in age 21-30 . These seem to contradict with in Gaza, 1995-2000. J. Islamic Univer. of Gaza
the above results in Table(6) and Fig(6). These (Natural Sciences Series). 12(2):121-129.
results need more confirmation by examining the
nutritional status of individual examined in health Ferreira MR, Souza W, Perez EP, Lapa T,
center, and make more larger sample of Shekhan Carvalha AB ,Furtado A, Coutinho HB, Wakelin
district. D. 1998. Intestinal helminthiasis and anemia in
It is suggested that further study searching Youngsters from Matriz da Luz, district of Sao
for anemia in Shekhan especially as related to Lourenco da Mata state of pernambuco, Brazil.
nutritional status and searching other causes of Mem. Inst. Oswaldo Cruz Rio de Janeiro, 93(3):289
anemia such as chromic infection ,inflammatory -293.
disease and hemoglobin pathies like sickle-cell
anemia and thalassemia are needed of individual Greets S, Gryseels B. 2000. Drug resistance in
which could reveal more information about their human helminthes : current situation and lessons
occurrence, which later on facilitate its treatment. from livestock. Clin. Microbiol. Rev. 13:207-222.

Gillespie S, Kevany J, Mason J. 1991. Controlling
Al Abbady AIA. 2001. Epidemiology of intestinal Iron deficiency. Geneva: subcommittee of Nutrition
parasites among pupils of a number of primary (SCN).United Nations Adminstative Committee on
schools and kinerhation children in mosul city and Coordination(ACC).
Attempt to infection laboratory mice with
Enterobius vermicularis. A thesis submitted by Hama AA. 2007. Intestinal Parasites in relation to
college Sciences, mosul university . malnutrition among primary schoolchildren in Erbil

Journal of Research in Biology (2011) 5: 319-324 323
Al-Naemi et al.,2011

province, with evaluationm of some anti-parasitic Niazi AD, AlIssa TB, Khamis F. 1976. Studies
drugs. M.Sc. thesis, Salahaddin University-Erbil, on the Prevalence of Entamoeba histolytica in Iraq.
Iraq. Bulletin of Endemic Disease 17(1-4):127-141.

Koukounari A, Estamble BBA, Njag JK, Pothinpak N, Srivilairit S, Pengruksa Ch,
Chundill B, Aljanga A, Crudder C, Oyido J. Faithong S, Haohan O, Chalermrut K. 2005.
2008. Relationship between anemia and parasitic Health state: Malaria, Anemia and Intestinal
infections in Kenyan schoolchildren: a bayesian parasitic infections on the Thai- Myanmar border. J.
hierchical modeling approach, Int. J. Parasit., 38 Torp. Med. Parasitol. 28:26-30.
(14):1663-1671.
Roberts L, Janovy J. 2005. Gerald D. Schmidt and
Labiano Abel Ferreira MJ, Souza W, Perez El, Larry Roberts Foundation of Parasitology. McGraw
Lapa T, Larvalho AB, Furtado A, Coutinho HB, Hill, Higher Education.
Wakelin D. 1998. Intestinal helmianthiasis and
anemia in youngsters from Martiz da Luz district of Shubair ME, Yassin MM, Al Hindi AL, Al
Sao Loureco da Mata, State of Pernambuco, Brazil. Wahaidi AA, Jadallah SY, Anu-Shaaban NA.
Mem.Inst.Oswaldo Cruz, Rio de Janeiro. 93(3):289- 2005. Intestinal parasites in relation to haemoglobin
293. level and nutrionl status of school children in Gaza.
J. Egypt Soc. Parasitol., 30(2):365-373.
Labiano Abello N, Canese J, Velazquez ME,
Hawdon J ,Wilson M, Hotez PJ. 1999. Tsuynoka R , Bailey JW, Guimaraes AM,
Epidemiology of Hookworm infection in Itagua, Gurgel RQ, Cuevas LE. 1999. Anemia and
paroguoy: a cross sectional study. Mem. Inst. intestinal parasitic infections in primary school
Oswaldo Cruz, Rio de Janeiro, 94(5):583-586. students in Aracaju, sergipe, Brazil. Cad. Saude.
Publica. Rio. De. Janeiro. 15(2):413-421.
Le HT, Brower ID, Verhoef H, Nguyen KC, Kok
F. 2007. Anemia and intestinal infection in
schoolchildren in rural Vietnam. Asia Pac. J. Clin.
Nut., 16(4):716-723.

Lewis SM, Bain BJ, Bates I. 2000. Dacie and
Lewis Practical haematology. Churchill
Livingstone.

Lobo MR, Costa JM , Coutinhoo MA, Ferraz
N. 1998. Studies on prevalence of intestinal
parasites in Guine Bissau. ICOPA. 1.

Markell E, John D and Krotoski W. 1999.
Markell and Voges Medical Parasitology. 8th ed.
Saunders.

Mupfasoni D, Karibush B, Koukounari A,
Ruberanziza E, Kaberuka T. 2009. Polyparasite
helminth infections and their association to anemia
and undernutition in northern Rwanda. PLos
NEgl.Trop. Dis., 3(9):e517.

Neimiester R, Iohan AL, Egleton, JH, Kleger
B. 1990. Evaluation of direct wet mount
parasitological examination of preserved fecal
specimens. J. Clinical Microbiology 28(5):1082-
1084.
324 Journal of Research in Biology (2011) 5: 319-324
Journal of Research in Biology
An International Online Open Access
Original Research Paper Publication group

Vermiwash mixed diet effect on growth of Oreochromis mossambicus (Tilapia)
Journal of Research in Biology

Authors: ABSTRACT:
Rameshguru G1,
Senthilkumar P2 and
Govindarajan B3. Oreochromis mossambicusfries were collected from Kullursanthai reservoir
near Virudhunagar. They were acclimatized to the lab conditions at 29±1ºC for one
week. O. mossambicus receiving CFF (Control Fish Feed) diet exhibited a growth of
Institution:
1
Department of Zoology, 140±10.58 mg in 21 days. Maximum growth was observed in fish receiving FFV (Fish
VHNSN College, Feed with 100% Vermiwash) 100 diet and it was 460.67±06.03 mg. Thus vermiwash
Virudhunagar-626001, plays asignificant role in enhancing the growth.
Tamilnadu, India.
2
Entomo Pathology Lab,
Institute of forest genetics
and tree breeding,
Coimbatore-641002.
3
Manonmaniam Sundaranar
University, Thirunelveli- Keywords:
627012, Tamil Nadu, India. Oreochromis mossambicus, Vermiwash.

Corresponding author: Article Citation:
Rameshguru Rameshguru G, Senthilkumar P and Govindarajan B.
Vermiwash mixed diet effect on growth of Oreochromis mossambicus (Tilapia)
Journal of research in Biology (2011) 5: 335-340
Web Address:
http://jresearchbiology.com/ Dates:
Documents/RA0099.pdf. Received: 07 Sep 2011 /Accepted: 10 Sep 2011 /Published: 14 Sep 2011

© Ficus Publishers.
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Rameshguru et al.,2011

INTRODUCTION plate. The fluid can be pipetted out using a treated
Fish is the major source of protein for over pipette with fine nozzle. This is the pure vermiwash
one billion people around the world (Cathay et al., (Radha D.Kale., 2006).
2004). In aquaculture field, the edible fish has Preparation of feed
earned a significant position in the nutrition of man. Three different fish feeds, CFF (Control Fish
It is rich in protein and amino acids like lysine, Feed), FFV75 (Fish Feed with 75% Vermiwash),
methionine. It contains unique unsaturated fatty FFV100 (Fish Feed with 100% Vermiwash), were
acid like eicosapentaenoic acid which is known to prepared by using the same quantity of ingredients
control cholesterol in the blood. It is easily (Table 1).To the ingredients 1g of sodium alginate
digestible and cheaper than other animal flesh was added. It acts as a binder and does not allow
(Santhanam et al., 1987). Food intake is a vital the compounded feed to dissolve in water. The only
factor for fish growth and is dependent upon the difference in the feed was the quantity of
quality or chemical composition of food vermiwash mixed. In the control diet the feed
(Elankumaran et al., 1992). Fish requires diets ingredients were mixed with water. But in FFV75,
relatively higher in protein than those of other 75% of vermiwash and FFV100, 100% of
commercially cultured animals (NRC, 1981). vermiwash was used.
With the increasing research thrust on this Experimental Design
aspect during recent years there has been much The experiment was carried out to find out
interest on the possibility of using various non- the influence of different concentration of
conventional feed sources. Experiments with the vermiwash on growth, food consumption,
incorporation of materials of animal origin like assimilation, metabolism and feeding rate in fish.
silkworm pupae (Venkadesh et al., 1986; All the experiments were carried out in the
Nandeesha et al., 1988; Hossain et al., 1992), and laboratory at 29ºC±1ºC and 12:12 photoperiod. Fish
vermicompost (Mahalakshmi., 2004) hormones like was reared in round glass troughs containing 1 liter
t h yr ox i n e ( M a ha la ks h mi , 2 0 0 4 ) a nd of water. 15 fish, of similar size were chosen from
diethylstilbestrol (Nanjundappa & Varghese, 1989) the stock. They were weighed and divided into five
and Earthworm meal (Keshavappa et al., 1989; groups each containing three fish. Before the
Kostecka & Paczka, 2006) as substitutes for meal commencement of experiments, the fish were
has also been found encouraging. The objective of starved for 24 hours in order to facilitate to
the present study was to assess the growth evacuate their alimentary contents. They were
ofO.mossambicus by vermiwash adding diet. weighed to find out the initial live weight. The fish
were subjected to five types of experimental food
MATERIALS AND METHODS CFF, FFV75, FFV100 and all experiments were
Animal collection conducted simultaneously.
Healthy O.mossambicus fries were collected Fish in all groups were fed with 100mg of
from Kullursanthai reservoir near Virudhunagar. dry pelleted food. The fish were fed daily once at
They were acclimatized to the lab conditions at 10AM, the feed remains and faeces were collected
29±1ºC for one week. During acclimatization, the separately using pipettes and dried in a hot air oven
fish were fed ad libitum with fish meal contained and weighed in a monopan balance. Water was
control diet daily once at 10 AM and water also changed daily. The rearing experiment was carried
changed daily. out for a period of 21 days. The weight of the fish
Vermiwash preparation was found out on 0, 7th, 14th, and 21stday to assess
The vermiwash can be collected from the
body cavity of earthworms without causing any Table: 1 Feed preparation composition
harm to them. In this method of collecting the fluid, Ingredients Weight (g)
three to four earthworms were taken in an Dried fish powder 42g
approximately 10 cm diameter petri plate. Holding Ground nut oil cake powder 20g
the plate in a slanting position and keeping Blood meal powder 5g
earthworms pointing downwards, cold shock was Tapioca powder 15g
given to earthworms by gently moving a small Wheat flour 15g
beaker containing a few ice cubes over the body of Mineral mix 2g
worms. The vermiwash released due to cold shock Vitamins 1g
drips and gets collected at the lower side of the petri Sodium alginate 1g
336 Journal of Research in Biology (2011) 5: 335-340
Rameshguru et al.,2011

the growth. The fish were not subjected to any an assimilation rate of 81.67±01.53 mg dry wt/g
disturbances except during feeding and water live wt/day.The assimilation rates for fish receiving
changing operations. FFV75 and FFV100 diets were 106.33±02.08 and
113.67±00.47 mg dry wt/g live wt/day,
RESULTS respectively. Assimilation efficiency of fish in
Mass budget different groups did not differ much (Table 2, 3,
O. mossambicus were reared at 29±1ºC for and 4).
21 days. The fish were divided into five groups,
each group with three triplicates. The first group of DISCUSSION
fish received control diet (CFF). The second, third, Fish growth is influenced by the nutrients
fourth and fifth group of fish received FFV75 and levels of food. The optimum and maximum feeding
FFV100 diets. Mass budgets were prepared for all levels are influenced by the nature of the diet. It is
groups of fish (Table 2, 3 and 4). dependent on the type of the food offered. Increase
Growth in availability of food having higher protein that
O. mossambicus receiving CFF diet plays a major role in growth. From the culturists
exhibited a growth of 140±10.58 mg in 21 days. point of view, a good diet would be one which is
Maximum growth was observed in fish receiving readily available, cost effective, simple and
FFV100 diet and it was 460.67±06.03 mg.Fish versatile in application (Vigneeswaran 2005).
receiving CFF diet consumed 685.67±0.6.11 mg of Surendra et al 2005 reported vermiwash very much
dry food. The fish receiving FFV100 diet consumed useful in growth of legumes plants.
maximum food and it was 1088.33±05.86 mg and Several research peoples have studied the
showed an assimilation of 774.67±05.77 mg. growth of fish with conventional and
Feeding rate supplementary diet. Food supplement includes
Feeding rate of fish differed in different
groups of fish. Fish receiving CFF diet exhibited a Table: 3 Mass budgets of O. mossambicus fed with diet
containing 75% vermiwash (FFV75) at 29±1ºC for 21
feeding rate of 105.33±00.47 mg dry wt/g live wt/
days.
day. Fish receiving FFV75 diet exhibited a feeding
rate of 149.33±01.15 mg dry wt/g live wt/day and Parameters (mg dry weight/
FFV75
individual)
FFV100 diet exhibited a feeding rate of
159.33±01.53 mg dry wt/g live wt/ day. Growth (P)
Assimilation rate
Assimilation rate of fish differed in different Food consumption (C)
groups of fish. The fish receiving CFF diet showed
Faces (F)
Table: 2 Mass budget of O. mossambicus fed with
control diet (CFF) at 29±1ºC for 21 days. Food assimilation (A)
Parameters (mg dry weight/individual) Control
Metabolism (R)
Growth (P) 140.00±10.58
Food consumption (C) 685.67±06.11 Feeding rate (Cr)
(g live weight/day)
Faces (F) 155.67±10.26
Assimilation rate (Ar)
Food assimilation (A) 530.00±07.65 (g live weight/day)
Metabolism (R) 390.00±07.21 Conversion rate (Pr)
Feeding rate (Cr) (g live weight/day) 105.33±00.47 (g live weight/day)
Assimilation rate (Ar) (g live weight/day) 81.67±01.53
Metabolism rate (Mr)
(g live weight/day)
Conversion rate (Pr) (g live weight/day) 21.67±01.53
Metabolism rate (Mr) (g live weight/day) 60.00±01.00
Assimilation efficiency (Ae) (%)
Assimilation efficiency (Ae) (%) 77±01 Gross conversion efficiency
Gross conversion efficiency (K1) (%) 20±02
(K1) (%)
Net conversion efficiency
Net conversion efficiency (K2) (%) 17±01 (K2) (%)
Note: Each value ( ± S D) represents an average of 3 Note: Each value ( ± S D) represents an average of 3
individuals. individuals.

Journal of Research in Biology (2011) 5: 335-340 337
Rameshguru et al.,2011

earthworm (Stafford & Tacon 1984), earthworm receiving FFV100 diet showed 2.3 times more
powder and krill meal (Toshio Akiyama et al., growth than control.Radha D.Kale 2006 has
1984), earthworm meal (Nandeesha et al., 1988), observed that vermiwash is rich in amino acids,
earthworm and chara plant (Elankumaran et al., vitamins, and minerals. Similarly, in this research
1992), frozen earthworms (Oscar Pereira & Emidio fish receiving FFV75 and FFV100 over fish
F.Gomes 1995) and diets containing artemia, receiving control diet. This observation reveals that,
earthworms and liver mixed diet (James & coelomic fluid mixed with balanced diet to prepare
Kunchitham Sampath 2004). fish feed does not in any way act as a repellent in
In the present work O. mossambicus fries fish.
were fed with pelleted diet containing different However, Stafford& Tacon 1984 while
concentrations of vermiwash. However fish rearing trout on earthworm reported poor growth.
This is attributed to the coelomic fluid present in
Table: 4 Mass budgets of O. mossambicus fed with diet the earthworm which, according to him is
containing 100% vermiwash (FFV100) at 29±1ºC for unfavorable to fish. However, in the present study
21 days. instead of feeding the fish with earthworm, the
Parameters (mg dry weight/ coelomic fluid of earthworm has been used to
FFV100
individual) prepare balanced diet and this has proved to be a
Growth (P) good diet improving consumption, conversion and
assimilation in O. mossambicus. Satia 1974 and
Food consumption (C) Austrong & Reftsio 1979 have reported that
increase in body protein in rainbow trout with
Faces (F) increasing dietary protein levels.
Sakthivel 1994 reported 14% increase in
Food assimilation (A)
body protein with 16% increase in dietary
Metabolism (R) protein.In the present work also, increase in the
protein, lipid and carbohydrate in the diet, increased
Feeding rate (Cr) (g live the protein, lipid and carbohydrate of the carcass.
weight/day) Gross and net conservation efficiency also
Assimilation rate (Ar) (g live increased with increase in the amount of vermiwash
weight/day)
Conversion rate (Pr) (g live
(CFF) exhibited a gross and net conservation
weight/day) efficiency of 20% and 17%, respectively. From the
Metabolism rate (Mr) (g live above observation it is concluded that vermiwash
weight/day) can be used to prepare fish diets. It does not repel
Assimilation efficiency (Ae) the food consumption and decrease growth. Instead
(%) it enhances consumption, assimilation, growth and
Gross conversion efficiency conservation efficiency in fish. As it is rich in
(K1) (%) vitamins, minerals and amino acids, vermiwash can
Net conversion efficiency (K2) be used a safe and suitable medium for the
(%) preparation of fish diets.
Note: Each value ( ± S D) represents an average of 3
individuals.

Table: 5Growth of O. mossambicus reared in diet containing different concentrations of vermiwash.
Days CFF FFV75 FFV100
1st day 3018.67±19.86

7th day 3071.00±21.59

14th day 3111.67±11.24

21th day 3185.67±15.70

338 Journal of Research in Biology (2011) 5: 335-340
Rameshguru et al.,2011

BIBLIOGRAPHY AK and Radha D kale. 1988. Influence of
Austrong E and Refstio T. 1979. Effect of a earthworm meal on the growth and flesh quality of
varying dietary protein levels in different families common carp. Biological Wastes 26(3):189-198.
of rainbow trout. Aquaculture 18:145-156.
NRC. 1981. Nutrient requirements of cold water
Cathy A, Roheim, Robert J, Johnston, Jessica G fishes. Nutrient requirements of domestic animal
and Holger D. 2004. Consumer preferences for series. National academy of sciences, Washington.
ecolabeled sea food: Results of a connecticut DC. 83.
survey. Food marketing policy center Deaprtment
of Agriculture and Resource Economics College of Oscar Pereira J and Emidio F Gomes. 1995.
Agriculture and natural Resources, Uni. of Growth of rainbow trout fed a diet supplement with
Connecticut. 4-11. earthworms, after chemical treatment. Aquaculture
International 3(1):36-42.
Elankumaran S, Pandi B and Palanichami S.
1992. The dietary influence on food utilization in Partima Rao, Asblesha P and Anjali D. 2005.
the fresh water fish Oreochromis mossambicus and Consumption pattern of fish and sea food in a
Cyprinus carpio communis. J Ecobiol., 4(4):271- selected population in Hyderabad, Andra Pradesh. J
275. Aquatic biology 20(1):175-178.

Hossain M A, Islam MN and Alim MA. 1992. Radha D Kale. 2006. Vermicompost- Crown jewel
Evaluation of silkworm pupae meal as dietary of organic farming. Published by N D Kale,
source for cat fish (Heteropneustes fossilis) In: fish Bangalore-23.
nutrition in practice. Kaushik S J and Luquent P
(Eds). 785-791. Saktivel M. 1994. Growth, Carcass composition
and Haematological parameters in the fresh water
James R and Kunchitham Sampath. 2004. Effect fish Heteropneustes fossils fed at different dietary
of food type on growth and fertility in ornamental levels. J Ecobiol., 6(3):179-185.
fish, Xiphophorus helleri. Israeli J Aquaculture 56
(4):264-273. Santhanam R, Sukumaran N and Natarajan R.
1987. A manual of freshwater aquaculture. Oxford
Kesavappa GY, Devaraj KV and Seenappa D. and FBH publishing co.pvt.ltd, NewDelhi-2.
1989. Evaluation of three animal meal based feeds
for rearing catla fry. Proc. Nat. Freshwat. Aqua: Satia BP. 1974. Quantitative protein requirements
Bhubaneswar, India. 143-144. of rainbow trout. Proc. Fish. Cult., 36:80-85.

Kostecka J and Paczka G. 2006. Possible use of Stafford EA and Tacon GJ. 1984. Nutritive value
earthworm Eisenia fetida biomass for breeding of the earthworm Dendrodrilus subrubicundus
aquarium fish. European J Soil Biology 42(1):231- grown on domestic sewage in trout diets.
233. Agricultural wastes 9(4):249-266.

Mahalakshmi G. 2004. Effect of dietary thyroxine Surendra Suthar, Choyal RR, Sushma Singh
supplementation on the physiology of Oreochromis and Sudesh. 2005. Stimulatory effect of earthworm
mossambicus. M.Phil dissertation, Madurai coelomic fluid (vermiwash)on seed germination and
Kamaraj University, Madurai. seedling growth of two legumes. J Phytological
Research 18(2):291-222.
Nanjundappa T and Varghese TJ. 1989. Effect of
diethylstilbestrol on growth and food conversion of Toshio Akiyama, Takesh Murai, Yasuhiro
common carp Cyprinus carpio (Linn.).Indian Acad. Hirasawa and Takeshi Nose. 1984.
Sci., 98(2):85-88. Supplementation of varius meals to fish meal diet
for chem salmon fry. Aquaculture 37(3):217-222.
Nandeesha MC, Srikanth GK, Basavaraja N,
Keashavanth P, Varghese TJ, Kubra Bano, Ray

Journal of Research in Biology (2011) 5: 335-340 339
Rameshguru et al.,2011

Venkadesh B, Mukherji AP, Mukhopadhyay PK
and Dehadrai PV. 1986. Growth and metabolism
of the cat fish Clarias batrachus (Linn,) fed with
different experimental diets. Proc. Indian Acad.
Sci., 95(4):457-462.

Vigneeswaran M. 2005. Studies on food utilization
and fecundity of guppy (Lebistes reticulates) fed
with bioencapsulated Artemia franciscana, M.Phil
dissertation, V.H.N.S.N.College, Virudhunagar.

340 Journal of Research in Biology (2011) 5: 335-340
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An International Online Open Access
Original Research Paper Publication group

Temporal distribution of the transferrin alleles (Tf aand Tf b) in Amazon
Turtle (Podocnemis expansa Schweigger, 1812)
Journal of Research in Biology

Authors: ABSTRACT:
Moura AS1, Oliveira
PHG2, Klein GN3,
Tancredi NS4, Silva CA1 This study aims to evaluate the temporal distribution of allele frequencies at
and Teixeira AS1.
the codominant transferrin (Tf ) gene locus in three population samples of Amazon
turtle (Podocnemis expansa) collected from the Rio Uatumã, Rio Trombetas and Rio
Institution: Tapajós in the Amazon Basin, Brazil, over a period of 16 to 24 years ago. Confirming
1. Instituto Nacional de previous research, the Tf locus which was analyzed by starch gel electrophoresis
Pesquisas da Amazônia showed a diallelic polymorphism with the presence of theoretically expected
(INPA), Avenida André genotypes (Tfaa, Tfab and Tfbb), presumably encoded by the codominant alleles (Tfa and
Araújo 2936, 69060-001, Tfb). A good genetic fit according to Hardy-Weinberg expectations within and among
Manaus, AM, Brasil. all the population samples was also noticed. Contingency tests on the overall
2. Centro de Preservação e transferrin allele distributions by calculating the chi-square (x2) showed no statistically
Pesquisas de Quelônios significant temporal variation (x2 = 3.05, d.f. = 5, 0.70 > P > 0.50) in the population
Aquáticos (CPPQA), BR samples of P. expansa in the surveyed areas.
174, km101, AM 240, km
77, S/N, 69736-000,
Presidente Figueiredo, AM,
Brasil.
3. Instituto Chico Mendes de Keywords:
Conservação da Podocnemis expansa, Amazon Basin, transferrin allele frequencies,
Biodiversidade (ICMBio), eletrophoresis, temporal analysis.
Reserva Biológica (Rebio)
do Rio Trombetas, Praça da
Feirinha, S/N, 68.275-000,
Porto Trombetas, PA, Brasil.
4. Instituto Brasileiro do
Meio Ambiente e dos
Recursos Naturais
Renováveis (IBAMA),
Avenida Tapajós, 2267,
Bairro do Laguinho, Article Citation:
68040-000, Santarém, PA, Moura AS, Oliveira PHG, Klein GN, Tancredi NS, Silva CA and Teixeira AS.
Brasil. Temporal distribution of the transferrin alleles (Tfa and Tfb) in Amazon turtle
(Podocnemis expansa Schweigger, 1812)
Journal of research in Biology (2011) 5: 346-351
Corresponding author:
Teixeira AS
Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 15 Sep 2011
Email:
saturn@inpa.gov.br
© Ficus Publishers.

Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://
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commercial, distribution, and reproduction in all medium, provided the original work is properly
Documents/RA0082.pdf. cited.

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Moura et al.,2011

INTRODUCTION effects and processes associated with colonization
The community of Amazon aquatic turtles and population-genetic structure (Waples, 1989;
has 14 species, of which the representatives of the Kambhampati et al, 1990; Shikano et al, 2001;
genus Podocnemis are edible, commercially Williams et al, 2003; Jacquemyn et al, 2006;
exploited, and act as an important source of protein Chevolot et al, 2008; Lucentini et al, 2009).
for local people (Vogt, 2008). The Podocnemis However, this type of study has never been
species are listed in the Appendix II of the performed on P. expansa.
Convention on International Trade in Endangered The aim of this study was to evaluate the
Species of Wild Fauna and Flora (CITES), temporal distribution of transferrin allele
published as Annex II, Normative Instruction frequencies from three population samples of P.
Number 11/2005, available at the Brazilian Institute expansa collected from the Rio Uatumã, Rio
of the Environment and Renewable Natural Trombetas and Rio Tapajós in the Amazon Basin,
Resources (IBAMA). The Podocnemis expansa Brazil.
(Schweigger, 1812) known as the Amazon turtle
and the largest freshwater turtle of the genus MATERIAL AND METHODS
Podocnemis in South America (Alfinito, 1975; After obtaining license given by the
Pritchard & Trebbau, 1984) is no longer threatened; Brazilian Institute of Environment and Renewable
however, as it still shows low risk of extinction, it is Natural Resources (IBAMA) through the
considered a conservation dependent species Biodiversity Authorization and Information System
according to the criteria of the International Union (SISBIO License No. 13081-1 of 19/12/2007),
for Conservation of Nature and Natural Resources ninety five specimens of the Amazon turtle (P.
(IUCN). Of the three levels of biodiversity expansa) were collected from three geographical
conservation recognized by the IUCN (biological areas in the Amazon region, totalizing three
diversity, ecosystem diversity and genetic population samples: Maracarana, Rio Uatumã-AM
diversity), the genetic diversity is the result of (30); Trombetas, Rio Trombetas-PA (34) and
evolutionary history of populations, in which a loss Monte Cristo, Rio Tapajós-PA (31) (Table 1, Fig.
of genetic diversity can reduce their ability to adapt 1). Blood samples were taken from the femoral
in response to environmental changes (evolutionary artery and placed in 5 ml BD vacutainer tubes,
potential) (Frankham et al, 2002). containing 0.5 ml of 3.8% sodium citrate as
Research focusing on the population anticoagulant, and blood plasma specimens were
structure of Podocnemis expansa has already been separated by centrifugation at 3,000 rpm for 20
carried out by the plasma iron binding protein, minutes and stored at -25ºC, until electrophoresis
transferrin (Teixeira et al, 1996), allozymes (Bock run (Teixeira et al,1996). A rivanol precipitation
et al, 2001), mitochondrial DNA and microsatellites method was used for isolating plasma transferrin
(Sites et al, 1999; Pearse et al, 2006). molecules prior to starch gel electrophoresis
Analysis involving temporal variations in (Jamieson & Tuner, 1978), with modifications
allele frequencies of molecular genetic markers can performed by Teixeira et al. (1996), where the
be extremely useful for investigating genetic blood plasma specimens were treated with rivanol
variability, effective population size (Ne), founder solution (2%) at a ratio of 1:1. For detecting the
Table 1. The sampling localities from where population samples of the Amazon turtle (P. expansa) were caught
in the Amazon Basin.
Stage of Number of specimens
Locality and position of catch Date of catch
development tested
Maracarana, Rio Uatumã, AM
Hatchling 10.01.2008 30
(02º13’S; 58º15’W)
Trombetas, Rio Trombetas, PA
Hatchling 04.02.2008 34
(04°04’S; 55°38’W)
Monte Cristo, Rio Tapajós, PA
Hatchling 30.01.2008 31
(01°20’S; 56°45’W)

Total - - 95

347 Journal of Research in Biology (2011) 4: 346-351
Moura et al.,2011

Fig. 1. Map of the approximate locations from which the population samples of P. expansa, were captured from
the Amazon region: Maracarana, Rio Uatumã (1); Trombetas, Rio Trombetas (2) and Monte Cristo, Rio
Tapajós (3). Map modified from Teixeira et al. (1996).

transferrin molecules, the starch gels were stained population samples examined. Moreover, chi-
in 1% Amido Black for five minutes (Jamieson & square contingency tests were applied on the overall
Tuner, 1978). The Amido Black was diluted in a transferrin allele distributions to compare the
solution composed of water, methanol and acetic population samples tested in this study with those
acid (5:5:1), respectively. Then the gels were tested previously by Teixeira et al. (1996).
washed by successive immersions in plastic
containers containing the above-mentioned solution RESULTS AND DISCUSSION
(water, methanol and acetic acid) for approximately The transferrin gene locus (Tf) of P. expansa
15 hours, and the Tf alleles were identified and population samples (Uatumã, Trombetas and
classified according to Teixeira et al. (1996). At the Tapajós), which was analyzed by starch gel
end of the experiments all the animals were electrophoresis, showed a diallelic polymorphism
returned to their respective collection sites. with the presence of the genotypes (Tfaa, Tfab and
Chi-square (x2) and maximum log-likelihood Tfbb) theoretically expected, presumably encoded by
(G) tests for genetic balance assuming Hardy- codominant alleles (Tfa and Tfb) (Fig. 2). A good
Weinberg equilibrium were applied to compare the genetic balance within and among all the population
observed and expected numbers of transferrin samples was noticed (Table 2). The present
genotypes within and among the Amazon turtle findings are in accordance with the previous report

Journal of Research in Biology (2011) 4: 346-351 348
Moura et al.,2011

over a period of 16 to 24 years ago (Table 3).
Although the population samples of P. expansa
during this time have been suffering some degree of
predation for ma i nt ena nc e a n d/ or
commercialization (Alho, 1985; Alves & Santana,
2008), the locus Tf has preserved its integrity for
Tf a about two generations, since, according to Vogt
b
Tf (2008) this species takes eight to 12 years to reach
its reproductive maturity. This means that in theory,
during this interval of time (two generations),
Tf a neither the anthropogenic action mentioned above,
Tf b nor the evolutionary processes that normally
contribute to change the genetic composition of
populations: mutation, migration, genetic drift,
natural selection, among others (Ayala & Kiger,
1980; Hartl, 2008) were sufficient to cause
Fig. 2. Electrophoregram of Tf locus in P. expansa. significant changes at the locus Tf in P. expansa.
The genotypes are: lanes 1 and 5 (Tfaa); lanes 2, 4 and Apparently conservation programs of the Amazon
6 (Tfab) and lane 3 (Tfbb). turtle are contributing to maintaining the genetic
of Teixeira et al. (1996), whose data were variability of its natural populations over time, at
consistent with a free flow of genes among the least for the Tf locus.
population samples, supporting the hypothesis of a Routine analysis with historical series of
single stock of P. expansa in the geographic area molecular genetic markers examined so far in P.
examined. expansa, such as, transferrin (Tf) (Teixeira et al,
Contingency tests revealed no temporal 1996), allozymes (Bock et al, 2001), nuclear and
variation on the overall Tf allele distributions in the mitochondrial DNA (Sites et al, 1999; Pearse et al,
populations samples of P. expansa in the surveyed 2006) will serve as an effective tool in obtaining
areas, when the present data were compared with information about the genetic composition of its
those previously reported by Teixeira et al. (1996) natural stocks. This kind of approach is very
Table 2. The genotypes and transferrin allele frequency distributions in three population
samples of Amazon turtle (P. expansa). Chi-square (x2) and maximum log-likelihood (G)
statistical values assuming Hardy-Weinberg equilibrium are shown. The observed numbers in
each transferrin genotype showed good agreement with expected numbers shown in
brackets.
Population samples tested
Rio Uatumã Rio Trombetas Rio Tapajós Total
N = 30 N = 34 N = 31 N = 95
Tf genotypes
Tfaa 16 (16.0339) 22 (22.1642) 20 (19.2787) 58 (57.5556)
Tfab 12 (11.9322) 11 (10.6716) 9 (10.4426) 32 (32.8889)
Tfbb 2 (2.0339) 1 (1.1642) 2 (1.2787) 5 (4.5556)
Tf allele frequencies
Tfa 0.7333 0.8088 0.7903 0.7789
Tfb 0.2667 0.1912 0.2097 0.2211
Hardy-Weinberg test
d.f. 1 1 1 1
x2 0.001022 0.0345 0.6332 0.0708
P 0.9745 0.8527 0.4262 0.7902
G 0.001024 0.0355 0.5824 0.0697
P 0.9745 0.8504 0.4454 0.7918
349 Journal of Research in Biology (2011) 4: 346-351
Moura et al.,2011

Table 3. Chi-square contingency tests used to examine the temporal distribution of the transferrin alleles (Tfa
and Tfb) among the population samples of P. expansa from three geographical areas in the Amazon region.
Expected numbers of alleles are shown in parentheses.

Rio Uatumã Rio Trombetas Rio Tapajós
Teixeira Teixeira Teixeira
This This This
et al. (1996)1 et al. (1996)2 et al. (1996)3 Total
report report report
46 44 85 55 98 49
Tf a 377
(47.92) (47.92) (81.47) (54.31) (95.85) (49.52)
14 16 17 13 22 13
Tf b 95
(12.08) (12.08) (20.53) (13.69) (24.15) (12.48)
Total 60 60 102 68 120 62 472
(x2 = 3.05, d.f. = 5, 0.70 > P > 0.50)
1
Sample collected in the Rio Uatumã (Balbina Hydroelectric Power Plant Dam) from 13.04.1988 to 22.05.1988
(adults).
2
Samples collected in the Rio Trombetas in 1984 (juvenile), in 24.11.1988 (adults), in 10.01.1990 and 04.01.1992
(hatchlings).
3
Sample collected in the Rio Tapajós in 04.01.1992 (hatchlings).

important for monitoring, preservation and Alho CJR. 1985. Conservation and management
management programs of this species which are strategies for commonly exploited Amazonian
now being carried out by the Chico Mendes turtles. Biol Conserv., 32:291-298.
Institute for Biodiversity (ICMBIO) and the Center
for Conservation and Management of Reptiles and Alves RRN and Santana GG. 2008. Use and
Amphibians (RAN) in the Amazon region. commercialization of Podocnemis expansa
(Schweiger 1812) (Testudines: Podocnemididae)
ACKNOWLEDGMENTS for medicinal purposes in two communities in
This research was partially financed by the North of Brazil. J Ethnobiol Ethnomed., 4:1-6.
Instituto Nacional de Pesquisas da Amazônia
(INPA), Manaus, AM, Brasil through the Ayala FJ and Kiger Jr. JA. 1980. Modern
Institutional Project Programme (PRJ12.01). The Genetics. The Benjamins/Cummings Publishing
authors wish to thank the Coordenação de Company, Inc., Menlo Park, California.
Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Brasília, DF, Brasil and Fundação de Bock BC, Páez VP and White MM. 2001. Genetic
Amparo à Pesquisa do Estado do Amazonas population structure of two threatened South
(FAPEAM), Manaus, AM, Brasil for the Master of American river turtle species, Podocnemis expansa
Science (MSc) scholarship given to Arlisson Silva and Podocnemis unifilis. Chelonian Conserv Bi.,
de Moura, the Centro de Preservação e Pesquisas de 4:47-52.
Quelônios Aquáticos (CPPQA), Presidente
Figueiredo, AM, Brasil, the Instituto Brasileiro do Chevolot M, Ellis JR, Rijnsdorp AD, Stam WT
Meio Ambiente e dos Recursos Naturais and Olsen JL. 2008. Temporal changes in allele
Renováveis (IBAMA), Santarém, PA, Brasil and frequencies but stable genetic diversity over the
Instituto Chico Mendes de Conservação da past 40 years in the Irish Sea population of
Biodiversidade (ICMBio), Porto Trombetas, PA, thornback ray, Raja clavata. Heredity 101:120-126.
Brasil for the logistical support during the catch of
the turtle specimens in the field, and Mr. George Frankham R, Ballou JD and Briscoe DA. 2002.
Nakamura who kindly revised the preliminary Introduction to conservation genetics. Cambridge
English version of the manuscript. Univerity Press, Cambridge, UK.

REFERENCES Hartl DL. 2008. Princípios de genética de
Alfinito J. 1975. A preservação da tartaruga população. Third edition. Editora FUNPEC,
amazônica. Brasil Florestal 7:49-53. Ribeirão Preto, SP.

Journal of Research in Biology (2011) 4: 346-351 350
Moura et al.,2011

Jacquemyn H, Honnay O, Van Looy K and Pritchard PCH and Trebbau P. 1984. The Turtles
Brine P. 2006. Spatiotemporal structure of genetic of Venezuela. Society for the study of Amphibians
variation of a spreading plant metapopulation on and Reptiles, Contributions in Herpetology 2.
dynamic riverbanks along the Meuse River. Fundación de Internados Rurales, Caracas,
Heredity 96:471-478. Venezuela.

Jamieson A and Tuner RJ. 1978. The extended Shikano T, Chiyokubo T and Taniguchi N. 2001.
series of Tf alleles in Atlantic cod, Gadus morhua. Temporal changes in allele frequency, genetic
In: Battaglia B., Beardmore J.A. (Eds). Marine variation and inbreeding depression in small
Organisms: genetics ecology and evolution, NATO populations of the guppy, Poecilia reticulate.
Conference Series IV: Marine Sciences, No. 2. Heredity 86:153-160.
Plenum Press, New York & London. 699-729.
Sites Jr. JW, Fritzsimmons NN, Da Silva Jr. NJ
Kambhampati S, Black IV WC, Rai KS and and Cantarelli VH. 1999. Conservation genetics of
Sprenger D. 1990. Temporal variation in genetic the giant Amazon river turtle (Podocnemis expansa:
structure of a colonising species: Aedes albopictus Pelomedusidae) Inferences from two classes of
in the United States. Heredity 64:281-287. molecular markers. Chelonian Conserv Bi., 3:454-
463.
Lucentini L, Palomba A, Gigliarelli L, Teixeira AS, Jamieson A, Raposo JCP and
Sgaravizzi G, Lancioni H, Lanfaloni L, Natali M Vieira AA. 1996. Transferrin polymorphism in
and Panara F. 2009. Temporal changes and Amazon Turtle (Podocnemis expansa) stocks. Braz
effective population size of an Italian isolated and J Genet., 19:559-564.
supportive-breeding managed northern pike (Esox
lucius) population. Fish Res., 96:139-147. Vogt RC. 2008. Tartarugas da Amazônia. Gráfica
Biblos, Lima, Peru.
Pearse DE, Arndt AD, Valenzuela N, Miller BA,
Cantarelli V and Sites Jr. JW. 2006. Estimating Waples RS. 1989. A generalized approach for
population structure under nonequilibrium estimating effective population size from temporal
conditions in a conservation context: continent- changes in allele frequency. Genetics 121:379-391.
wide population genetics of the giant Amazon river
turtle, Podocnemis expansa (Chelonia: Williams CL, Blejwas K, Johnston JJ and
Podocnemididae). Mol Ecol., 15:985-1006. Jaegert MM. 2003. Temporal genetic variation in a
coyote (Canis latrans) population experiencing
high turnover. J Mammal., 84:177-184.

351 Journal of Research in Biology (2011) 4: 346-351
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Study on display sequences in Cotton Pygmy-Goose Nettapus
Coromandelianus Coromandelianus Gmelin
Journal of Research in Biology

Authors: ABSTRACT:
Upadhyaya S1 and
Saikia PK2.

The pairing and display sequence of Cotton Pygmy-goose (Nettapus
Institution:
coromandelianus coromandelianus Gmelin) in Eastern Assam was observed from April
1. Asstt. Prof., Dept. of
Zoology, Tyagbir Hem 2007 to July 2008. The frequency of head movement (up-down and left-right) are
Baruah College, found to be 0.288 and 0.28 per second. Courtship bouts occurred in groups averaging
Karchantola, Dist.-Sonitpur 5.7 birds, with a male: female ratio of 1.1:1. Several new displays and vocalizations are
(Assam), India. PIN-784189. described. Early pair bonds appeared tenuous and were continually tested until nest-
2. Department of Zoology, searching activities began in May. Birds in groups displayed and vocalized more than
Gauhati University, Assam paired birds, and birds displayed and vocalized more or less equally throughout the
(India). period with mild rainy climate. Early pairing may be related to a bird’s condition,
climatic condition, success in obtaining a nesting site, and increased productivity.

Corresponding author:
Sanjib Upadhyaya

Keywords:
Email: Burping, coquette call, chin-lifting, display shake, inciting, preening,
sanjib1970@sify.com vocalizations, whistling jerks.

Web Address: Article Citation:
http://jresearchbiology.com/
Documents/RA0089.pdf.
Upadhyaya S and Saikia PK.
Study on Display Sequences in Cotton Pygmy-Goose Nettapus Coromandelianus
Coromandelianus Gmelin.
Journal of research in Biology (2011) 5: 341-345

Dates:
Received: 21 Aug 2011 /Accepted: 27 Aug 2011 /Published: 15 Sep 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

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with the river Brahmaputra.
INTRODUCTION Scan and ad libitum sampling methods were
Most anatids confine their displays to the used to study the nesting behavior of the Cotton
water (or land) surface, since their heavy weight Pygmy-goose, as per Altmann (1974) and
relative to their wing area dictates continuous Brockelman (1975). Field observations were
flapping and makes complex maneuvers, such as conducted continuously for three days from April
hovering and soaring, difficult or impossible. Aerial 2007 to July 2008 on every week in each month of
communication is thus largely restricted to short, the year at Kadamani wetland and Dagaon.
ritualized flights (ordinarily close to the water Courtship was recorded from a portable tree blind
surface) and vocalizations, including contact calls or while concealed in vegetation or in the open. The
that help to maintain flock coherence in these rapid day samplings were taken total of 2 – 5 hours and
fliers that often go long distances between landings. totaling 50 hours devoted in a week. The sampling
Most of the studies on display behavior of anatids patterns followed for the study time viz., first day:
are confined to Mallards and a few other species. 05:00 to 08:00 hours and 09:00 to 11:00 hours,
The ethogram of the Cotton Pygmy-goose second day: 08:00 to 09:00 hours, and 11:00 to
(CPG, Nettapus coromandelianus coromandelianus 14:00 hours, third day: 14:00 to 16:00 hours.
Gmelin) is incomplete, so to say not studied at all. Cotton Pygmy-goose behavior before and after
A description of Wood duck vocalizations and courtship and were observed using a pairs of
courtship displays is given by Heinroth (1910) and binoculars (10 X 50 and 20 x 50) and data were
Lorenz (1951 - 1953). Display behavior in Blue- recorded and sketches were drawn. No birds were
billed Dick has been studied by Johnsgard and marked. The frequency of head movement during
Nordeen (1981). Males court one female with social feeding and searching of nesting trees were also
display relative to Anas species (Lorenz 1951 – recorded. The display behaviors were categorized
1953, Wishart 1983). Pair formation, courtship and as per Lorenz (1951-1953).
display in musk duck and mallards were also
studied (Bossema and Roemers 1985, Fullagen and RESULTS & DISCUSSION
Carbonell 1986). The Cotton Pygmy-goose generally found
Information is lacking on the chronology of to select big hole or cavity bearing trees for nesting
pair formation and strength of pair bonds on CPG, purposes during the breeding season. The initiation
because most published studies have concerned of searching the nesting tree was made by the pair
only spring courtship behavior in Wood ducks before 10 – 12 days of egg laying. Both the male
(other than CPG), Mallards (Lebret 1961; and female birds circling in an around the selected
Weidmann & Darley 1971) and a few other species. nesting tree, might be to confirm the security status
CPGs breeds during the monsoon season (Ali & of the trees and as well as the anthropogenic
Ripley 1983). This study was designed to determine activities. They perform several rounds of flights in
the chronology and mechanism of pair formation various hours of the day near nesting trees and then
and display in CPG. they select the nest sites. The monitoring flight rate
STUDY AREA & METHODS was found to be high on a rainy day between 07:00
The study was carried out in the Sonitpur – 10:00 hours. They move around the nesting tree
district of Assam (240 09/ N to 27058/ N and 89042/ for nesting territory with a mean duration of 3.28
E to 96001/ E) during 2007 - 2008. The Sonitpur hour/day (n = 7, Table 1). The selection of nesting
district of Assam, with an area of 5,324 km2, is tree was an unique phenomenon of CPG which
located in between 9302/80// E to 930 57/1// E prefers to move out during rainy or mild raining
longitude and 260 22/1// N to 260 42/ 2// N latitude. condition and the frequency was found to be 0.056
The district is bounded by Hawajan tributary in the rounds per seconds.
east, Pachnoi tributary in the west, the mighty river Display behavior and pair formation
Brahmaputra in the south and the state Arunachal During field observations for display
Pradesh (previously North Eastern Frontier Area or behavior and pair formation, altogether 110 Cotton
NEFA) in the north. Physiographically, major parts Pygmy-goose were encountered on 10 occasions.
of the district are plain area with a number of Courtship was observed twice (once at Kadamani
tributaries like Pachnoi, Mora-Bhoroli, Jia-Bhoroli, on 21st May 2007 for 1.4 min and once in an
Ghiladhari, Burigang, Borgang, Buroi, and Satrang agricultural field on 12th June 2008 for 1.1 min near
arose from the hills of Arunachal Pradesh and joins Dagaon, Biswanath Chariali). Display bouts
342 Journal of Research in Biology (2011) 5: 341-345
Upadhyaya et al.,2011

Table 1 Movement pattern of Cotton Pygmy-goose during nesting period (prior to nest selection) 2007;
(location- Kadamani; Tree species- Sowalo Litsea polyantha)
Freq. of
Weather Active period Total time Birds Mov. Social
Obs. Dates movement
condition (in hours) (in hours) (no. of tips) Structure
/sec.
1 06/7/ 2007 Cloudy 06.30 -09.30 3.00 hrs 8 P 0.044
2 14/7/ 2007 Raining 07.10 -10.00 2.40 hrs. 11 P 0.069
3 15/7/ 2007 mild raining 09.45 -11.45 2.00 hrs 8 P 0.067
4 15/7/ 2007 mild raining 07.30 -10.30 2.00 hrs 5 P 0.042
5 16/7/ 2007 --do-- 11.00 -14.00 3.00 hrs 0 0 0
6 07/8/ 2007 mild raining 07.00 -09.00 2.00 hrs 7 S (male) 0.058
7 07/8/.2007 --do-- 09.30 -11.30 2.00 hrs 0 0 0
Average --- --- 3.28 hrs/day 7.8 tips/day --- 0.056 / second

consisted of a series of sequences in which the (Fig. 1g–i; Fig. 2). Similar types of behavioral
intensity of sexual behavior progressively observations were also made by Lorenz (1951 - 53),
increased. Various types of display bouts were Johnsgard (1960), McKinney (1970), Armbruster
observed, which were categorized as: (i) (1982) and others in various species of anatids.
vocalizations, (ii) head-up-down movement, (iii) Increased intensity of vocalizations and aggressive
head-turn, (iv) burping, (v) Whistling jerks or bill behavioral postures increases the sexual excitement
shaking, (vi) Coquette call, (vii) chin-lifting, (viii) again. The length of display sequences ranged
display shake, (xi) wing-and-tail-flash, (x) inciting between 2 - 6 minutes interspersed with 10 - 65
and (xi) preening and feeding etc. minutes of preening or feeding. These types of
A typical display sequences were usually behavioral displays were frequently seen during
starts with vocalizations or agonistic postures from breeding season. The display behaviors were
male (Fig. 1a). The male sat quietly on the water; observed during early hours of the morning and late
with the head turn back (Fig. 1b) and slowly afternoon. The displayed vocalization was almost
changed positions around the female. The male similar with that of domestic pigeon before mating,
starts whistling or possess bill jerks or shakes their viz. gor...r...gor...r...gor...r.....r........The up-down
bills (Fig. 1c). The female produce the Coquette postures, left-and-right side movement of head are
call (Fig. 1d). The female move the bill down and the common display behavior of CPG. The
up as it drinking water and lifts the chin up (Fig. frequency of head movement was found to be 0.288
1e), and make a display shake and whistled and 0.28 per second (total duration =204.29
frequently along with the wing-and-tail-flash minutes; n =7) respectively during the study period
(Fig.1f) until a male began to inciting or males (Table 2). Fox and Madsen (1981) published more
starts fighting among them. Following an agonistic or less similar results in Greenland White-fronted
possession between them, both usually bathed. This goose (Anser albifrons) while studying its pre-
behavior leads to a session of preening on breast or nesting behavior.
back or feeding, and then they completed display

Table 2. Frequency of head movement of Cotton Pygmy-goose during breeding season
(Location: Kadamani wetland, date: 02/07/2007 and 03/07/2007).
Sl. Nos. Distance of Duration of Up and down movement Lt. and Rt. movement of
obs. (m) obs. (sec.) of the head the head
No. of times Freq/sec No. of times Freq/sec
1 18.00 60 33 0.55 31 0.517
2 20.00 120 40 0.333 36 0.300
3 22.00 120 31 0.258 36 0.300
4 26.00 150 36 0.24 33 0.220
5 22.00 150 49 0.327 47 0.313
6 23.00 230 37 0.161 37 0.161
7 32.00 600 87 0.145 88 0.147
Average 23.29 204.29 44.71 0.288 44 0.280

Journal of Research in Biology (2011) 5: 341-345 343
Upadhyaya et al.,2011

Fig. 1 Diagrammatic representation of courtship behavior of Cotton Pygmy-goose observed during the
study period.

The results of the present study shows with the breeding season and availability of habitats
that, in Cotton Pygmy-goose the pair formation for the growing ducklings. This might be due to
starts from the month of May and retains up to the their requirement of extra as well as stored energy
month of July, though they pass most of the time of for nesting period. Pair formation takes place
the year in flocks, they form pair with a male and a throughout the year for the adult Cotton Pygmy-
female during the breeding season. The Cotton goose. However, the most active period of display
Pygmy-goose moves out of the foraging site during and copulation found during the present observation
breeding period in search of nesting tree. The was May to July. It is important to mention here
frequency of group formation was found to be 5.4 that, the growing of breeding plumage will
birds with maximum 8 pairs during the third week encourage to formation of pairs. The Cotton Pygmy
of May and second week of June 2007 (Table 3). -goose acquires alternate plumage in November
The study shows that, the Cotton Pygmy- retains up to February, pair by May to July, lay first
goose prefers to nest during June – September. eggs in second half of the May. The breeding
Higher frequency of nesting was found in the plumage might help in courtship. Though early
month of August, though the pairing and courtship pairs are frequently unstable, but firm bonds are
display starts in the months of May to July during distinguishable only after many weeks of pairing
the study period (n =52). The nest site selection and activity.
display behavior in Cotton Pygmy-goose are related
344 Journal of Research in Biology (2011) 5: 341-345
Upadhyaya et al.,2011

Table 3. Pair formation in Cotton Pygmy-goose around the Kadamani wetland, 2007.
Months Duration No. of pairs No. of solitary male/female
May 2007 1/5/2007 to 7/5/2007 3 -
8/5/2007 to 14/5/2007 4 -
15/5/2007 to 21/5/2007 8 -
22/5/2007 to 28/5/2007 6 1M
29/5/2007 to 31/5/2007 6 1M
June 2007 1/6/2007 to 7/6/2007 6 1M
8/6/2007 to 14/6/2007 8 -
15/6/2007 to 21/6/2007 4 1M
22/6/2007 to 28/6/2007 6 1M
29/6/2007 to 30/6/2007 6 1M
ACKNOWLEDGEMENT 101-156.
Authors are very much thankful to Mr. C. K.
Bora, the then DFO, Sonitpur Forest Division Johnsgard PA. 1960. Comparative behaviour of
(East), and Mr. Biren Sahani, Kadamani for their the Anatidae and its evolutionary implications.
active support during the study period. Authors are Wildfowl 11:31-45.
also thankful to UGC (NERO) for providing
financial assistance in the form of Minor Research Johnsgard PA and Nordeen C. 1981. Display
Project. behavior and relationships of the Argentine Blue-
billed Duck. Wildfowl 32:5-9.
REFERENCES
Ali S and Ripley SD. 1983. Handbook of the Birds Lebret T. 1961. The pair formation in the annual
of India and Pakistan. Compact Edn. Oxford Univ. cycle of the Mallard (Anas platyrhynchos L.).
Press, New Delhi (revised edition). xlii+737. Ardea 49:97 -158.

Altmann J. 1974. Observational study of Lorenz K. 1951-1953. Comparative studies on the
behaviour: sampling methods. Animal Behav., behavior of Anatidae. Aviculture Manage., 57:157-
49:227-267. 182; 58:8-17, 61-72, 86-94, 172-184; 59:24-34.

Armbruster JS. 1982. Wood Duck displays and McKinney F. 1970. Displays of four species of
pairing chronology. The Auk., 99:116-122. blue-winged ducks. Living Bird, 9:29-64.

Bossema I and Roemers E. 1985. Mating strategy Weidmann U and Darley J. 1971. The
including mate choice including mate choice in synchronization of signals in the “social display” of
Mallards. Ardea 73:147-157. Mallards. Rev. Comp. Anim., 5:131-135.

Brockelman WY. 1975. Competition, the fitness of Wishart RA. 1983. Pairing chronology and mate
o f f s p r i n g a n d o p t i m a l c l u t c h - s i z e. selection in the American Wigeon (Anas
American Naturalist 109:677-699. americana). Can. J. Zool., 61:1733-1743.

Fox AD and Madsen J. 1981. The pre-nesting
behavior of the Greenland White-fronted
Goose. Wildfowl 32:48-54.

Fullagen PA and Carbonell M. 1986. The display
postures of the male Musk Duck, Wildfowl. 37:142
-150.

Heinroth O. 1910. Beobachtungen bei
Einbiirgerungsversuch mit der Brautente
(Lampronessa sponsa). Journal of Ornithology 1:

Journal of Research in Biology (2011) 5: 341-345 345
Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Some observations on demography and edible plants of Lion-tailed Macaques
(Macaca silenus) in the rain forest fragmented habitats of Anamalai Hills, Western Ghats.
Journal of Research in Biology

Authors: ABSTRACT:
Narasimmarajan K, Some observations on demography and edible plants species of endangered
Nagarajan R and Lion-tailed Macaques (LTM) (Macaca silenus) were studied between January 2008 and
Kumaraguru A. July 2008 in the rain forest fragments of Annamalai Hills, Southern India. Totally, 14
different fragments were surveyed, in which LTM existence was observed in 10
fragments. LTM were noticed in Iyyerpady, Uralikkal, Chinnakalar, Manompoly,
Sekalmudy, Korangumudi, Pannimedu, Varattuparai, Puthuthottam and Andhiparai.
18 different troops of LTM numbering 279 individuals were observed in 10 different
fragments. The group size ranged from smallest of five individuals at Pannimedu to
highest number of 62 individuals at Puthuthottam. In total, 48 adult males, 86 adult
Institution: females, 24 sub-adult males, 41 sub-adult females, 49 juveniles and 30 infants were
Deportment of Zoology and observed for the entire study period. It was observed that the group size and number
Division of Wildlife of troops varied in these fragments which could be attributed to the food availability
Biology, AVC College, in each fragments and other factors such as anthropogenic pressures, competition
Mayiladuthurai– 609001, between sympatric primates. Other variations such as diversity of vegetation, mean of
South India. tree density, Girth at Breast Height (GBH), canopy spread and tree height noticed in
these fragments were also analyzed for their implication on the distribution pattern.
Based on this study we give some essential suggestion (1) Ecotourism is one of the
serious threats for the LTM populations, (2) People knowingly feeding the plastics and
food items to LTMs, which are yet to have existing on the road side. (3) We suggest
that tourists traveling between Azhiyar to Valparai should be checked properly with
mobile patrol and spot penalty is to be needed (Fig; 2) for the control of these
Corresponding author:
activities.
Narasimmarajan K
Keywords:
Demography; Lion-tailed Macaque, Fragmentation, Edible plants, Ecotourism
and Anamalai
Abbreviations:
LTM –Lion-tailed Macaque.
Email:
narasimma@wii.gov.in Article Citation:
Narasimmarajan K, Nagarajan R and Kumaraguru A.
Some observations on demography and edible plants of Lion-tailed Macaques
(Macaca silenus) in the rain forest fragmented habitats of Anamalai Hills, Western
Ghats.
Journal of research in Biology (2011) 5: 352-362

Web Address: Dates:
http://jresearchbiology.com/ Received: 29 Aug 2011 /Accepted: 16 Sep 2011 /Published: 20 Sep 2011
Documents/RA0093.pdf.

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INTRODUCTION (Singh, et.al, 2002). Due to extensive plantation of
Tropical rain forests are the centers of bio- beverages; these rain forests are highly fragmented.
diversity. These forests cover about 6% of the land Hence, the LTM‟s habitat is highly fragmented and
surface of the world (McNeely et al., 1990). surrounded by tea estates (Singh, et.al. 2006). Some
Extensive removal of these forests is therefore a of the basic Demographic parameters and edible
matter of global concern, and especially isolation plants species were studied based on the following
process continuous the progressive lose an objectives.
endangered species from these fragments 1. To identify some of the major demographic
(Kumaraguru, 2005). Furthermore the effects of parameters and distribution pattern of Lion-
fragmentations on many forests could ultimately tailed macaque.
have the same consequences as extinction (Cutler, 2. To assess the vegetation characteristic features
1991). Wilcox and Murphy (1985) stated that, for selected fragmented habitats.
“Habitat fragmentation is the most serious threat to Study Area
biological diversity and forms the primary cause of The study was conducted in rain forest
the present extinction crisis”. At the current rate of fragments in and around Anamalai Tiger Reserve
habitat loss and fragmentation of tropical rain (formaly Indira Gandhi Wildlife Sanctuary) in
forests, the survival of significant components of Anamalai hills, Southern India. It was extensively
bio-diversity would depend on the retention and clear felled between 1860s and 1930s for
management of a network of fragmented forest cultivation of tea, coffee and cardamom; later teak
(Saunders et. al., 1991). and eucalyptus plantations leaving behind
Ecological and behavioural changes form fragments varying in area from less than 10 hectares
the first phase of the interactions between a species to more than 200 hectares (Singh et.al, 2002). The
and fragmentation of its habitat (Umapathy, 1998). Anamalai Tiger Reserve (10°11‟08”N to 10°
These include alterations in the activity pattern, 33‟27”N and 76°49‟02”E to 77°21‟09”E) isolated
feeding ecology, changes in predation pressure, in the state of Tamil Nadu is one of the largest
inter-specific competitions and host-parasite sanctuaries in south India (Fig.1) with an area of
relationships (Strunsaker, 1976). The demography 987sq km. Rainfall varies from 800 mm to 1000
consequences are the final response of the species mm. The day temperature varies from 23°C to 40°C
to habitat fragmentation leading earlier to its at the foothills, and from 20°C to 30°C at higher
decline and gradually to the extinction. The elevations (above 1800m).
demographic changes in primates might be due to Extensive clear-felling has reduced the
habitat degradation and severe resource depression tropical rain forest in the sanctuary and adjoining
(Umapathy and Kumar, 2000b; Struhasker, 1976; areas to 30 fragments ranging in the area from less
Sugiama and Oshawa, 1982; Decker, 1994; Johns than 10 hectare to about 2500 hectares (Singh et.al,
and Skorupa, 1987). Parasites and pathogens 2002). Only five fragments are more than 200
(Holmes, 1996), stochastic and inbreeding (Quinn hectares in area. Most of the smaller fragments
and Hastings, 1987) also cause demographic occur as islands surrounded by tea estates and are
changes in fragmented population. Dispersal can private owned viz., the larger fragments (more than
increase the persistence and stability of fragmented 100 hectares) of the sanctuary are bordered by teak
population through various ways (Hanski and and eucalyptus plantations and in some cases viz.,
Gilpin, 1991, Arnold et. al., 1993). However, there by tea plantations on one side. This area has a town
is a considerable variation among species in their (Valparai) with a human population of nearly 2,
ability to disperse among habitat islands (Singh, et. 00,000 people (Umapathy and Kumar, 2000a).
al. 2001).
Hence we studied the demographic and food METHODS
plant species of endangered, close to extinct (IUCN, The Valparai town was taken as the centre
2007), Lion-tailed Macaque (LTM) (Macaca and 14 fragments within the radius of 30Km were
silenus) due to habitat fragmentation. We examined taken for the intensive study. The fragments were
major demographic parameters in selected Iyyerpady, Uralikkal, Chinnakalar, Manompoly,
fragments. Fourteen fragments of the Indira Gandhi Sekal mudy, K or angumudi , Panni medu,
Wildlife Sanctuary in the Anamalai hills were Varattuparai, Puthuthottam, Andhiparai, Solayar
selected as the main study area. Anamalai hills have Dam, Kavarakkal, Sangarankudi and Akkamalai
the largest extent of rain forests in Western Ghats respectively. The survey was conducted following
353 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

(Merenlender et al. 1998), the method, which that approximate age in groups that have been studied
relies on repeatedly identifying social groups, and for many years. Individuals in a group were
obtaining demographic data on all the identified classified as infants (up to 1year old), juveniles (1
groups. At most sites, data were collected on pre- to 4 years), sub-adults (4 years to first birth at about
existing forest trails, abundant forest roads and 6.5 years for females; 5 years to about 8 years old
nallah (marked at 5 km intervals) that had either for males), adult females (females that have given
been created by forest Dept. or by researchers birth at least once) and adult males (>8 years old)
working in the area. Each trail roads and nallah (Singh, et.al, 2002; Umapathy and Kumar, 2000b).
were surveyed from early morning to early Vegetation in these fragmented habitats was
afternoon, at a slow pace. During the study period, assessed using parameters such as tree density,
all detectable groups were identified to count total Girth at Breast Height (GBH) of the trees, canopy
numbers and determine the age, sex classes (adults, spread, and tree height. These were estimated from
sub adults, juveniles, and infants), whenever forty to fifty 10×10 meters quadrates in selected
encounters LTM, the details of feeding activities eight fragmented habitats. The vegetation plots
were also recorded. Data collections were repeated were done in Iyyerpady, Uralikkal, Chinnakalar,
4-5 times at each site, over 3-day period intervals Varattuparai, Korangumudi, Puthuthottam,
(R. J. Rao & Abhishek Bhatnagar 2001). Andhiparai and Akkamalai fragments respectively.
Size of these 14 fragments and altitude were In each quadrates all trees were enumerated the
recorded from the survey maps and earlier literature measurement of GBH in each quadrate the number
(Umapathy & Kumar, A 2000b). The group counts of tree species, number of food species, number of
were taken when the entire group had to move one individuals of each species and basal area were
or very few canopy, across the road, steams or assessed. The various edible plant portions such as
canopy openings. The age/sex classification was leaf, flower, fruit, mesocarp, seed coat and seed
based on a comparison with individuals of known were recorded from all the food trees by observing
Fig 1. Map showing the rain forest fragments of Anamalai Hills, Western Ghats.

Journal of Research in Biology (2011) 5: 352-362 354
Narasimmarajan et al.,2011

LTM. The trees used by LTM for roosting were males, 86 adult females, 24 sub-adult males, 41 sub
also recorded. LTM were observed through Nicon -adult females, 49 juveniles and 30 infants were
Action series binoculars 10X. observed for the entire study period. Pannimedu
had the smallest troop (5 individuals) followed by a
RESULTS troop in Puthuthottam which had six individuals in
Among the 14 fragments LTM was observed one troop. No sub-adult female was observed in one
in only 10 fragments. Troop size of LTM observed troop of Iyyerpady, one troop of Puthuthottam, and
in these fragments is given in Table 1. The altitude Varatupparai and Pannimedu. In Andhipparai, both
of the fragment ranged from 750 – 1300m, troops didn‟t have any sub-adult females. No
fragments ranged from 24 ha – 2000 ha (Table 1). juvenile was observed in one troop of Uralikkal,
On the other hand, the LTM was not observed in one troop of Sekalmudy and troops of Manompoly,
Solayar Dam, Kavarakkal, Sangarankudi and Varatupparai, and Pannimedu. The highest number
Akkamalai. These fragments had different of juveniles was observed in one troop of
vegetations and forest types such as evergreen Puthuthottam (15 juveniles). Two troops of
forest, rain forest, moist deciduous forest, wet Iyyerpady, Andhipparai and Sekalmudy, one troop
evergreen, dry evergreen forest, and also the of Uralikkal, Varatupparai and Pannimedu didn‟t
Cardamom (Elettaria cardamomum) and Coffee have any juveniles. Two troops of Iyyerpady and
(Coffea arabica) plantations. Totally 18 different Puthuthottam, one troop of Sekalmudy,
troops were observed in 10 different fragments. The Andhipparai and Manompoly didn‟t have any
fragments Chinnakalar, Manompoly, Korangumudi infants (Table 2).
and Varattuparai had one troop each. A maximum A total of 51 edible plant species and one
of four troops were observed in Iyyerpady whereas unknown species were recorded from 8 different
in Puthuthottam three troops were observed. The fragments of Anamalai Hills during this study
fragments viz., Uralikkal, Sekalmudy, and period. Chinnakalar had maximum species of plants
Andhipparai had two troops each (Table 1). (23 species) followed by Iyyerpady (19 species),
The demography of LTM in different Akkamalai (18 species) and Korangumudi (15
fragments is given in Table 2. A total of 279 species). Uralikkal had the lowest number of
individuals were observed in 10 different species of (12 species). Cullenia excellrata was the
fragments. The group size ranged from smallest of only species found in all the fragments except
five individuals at Pannimedu to highest number of Akkamalai. Agalia roxburghina, Cardia mixa,
62 individuals at Puthuthottam. In total, 48 adult Mimusops elengii, was observed only in

Table. 1: List showing the Fragments, with Forest type, Area, Altitude, Presence / Absence of Lion -
tailed Macaque and the Number of Troops observed
S. Fragment Area No. of Total no. of
Forest type Altitude (m)
No Name (ha) Troops Individuals
1 Iyyerpady Evergreen 750 - 850 1800 4 47
Cordomomum
2 Uralikkal 850 - 950 400 2 44
Plantation
3 Chinnkalar Riveraine 1000 - 1100 1300 1 15
4 Monamboly Evergreen 1000 - 1100 500 1 13
5 Sekalmudy Evergreen 1000 - 1100 600 2 25
6 Korangumudi Coffee plantation 1100 - 1150 35 1 27
7 Pannimedu Moist deceduous 1100 - 1150 50 1 5
8 Varattupparai Coffee plantation 1100 - 1150 24 1 8
9 Puthuthottam Coffee plantation 1100 - 1200 65 3 77
10 Andhiparai Wet evergreen 1200 - 1300 185 2 20
11 Soliyar Dam Dry evergreen 850 - 900 700 0 0
12 Kavarakkal Evergreen 900 - 1100 350 0 0
13 Sangarankudi Evergreen 1100 - 1200 180 0 0
14 Akkamalai Evergreen 1200 - 1300 2000 0 0
355 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Table. 2: Demography of Lion -tailed Macaque in different forest fragments in Annamali Hills
Sub Sub
Adult Adult Group
Fragment Name Adult Adult Juvenils Infants
Male Female Size
Males Females
1 2 1 2 1 1 8
4 2 1 1 2 0 9
Iyyerpady
2 2 0 2 4 0 12
4 8 2 0 3 1 18
2 4 2 5 0 4 17
Uralikkal
4 9 2 5 3 4 27
Chinnkalar 2 5 1 2 2 1 13
2 5 1 3 2 2 15
Sekalmudy
3 5 0 2 0 0 10
Monamboly 3 5 0 3 2 0 13
Varattupparai 1 4 1 0 0 2 8
Pannimedu 1 2 0 0 0 2 5
Korangumudi 3 6 2 5 6 5 27
1 1 0 1 3 0 6
Puthuthottam 1 2 0 0 6 0 9
10 15 7 10 15 5 62
2 6 2 0 0 3 13
Andhiparai
2 3 2 0 0 0 7

Akkamalai, Varatupparai and Puthuthottam (Umapathy and Kumar, 2000b) and competition
respectively (Table 3). between two males which are equal in size and
The mean tree density, Girth at Breast strength which leads to the splitting up of groups
Height (GBH), Diameter at Breast Height (DBH), (Kumar, 1995). Hence the variations in the group
Canopy spread and Tree height of different size and number of troops in these fragments were
fragments are given in Table 4. Among the assessed with reference to food availability and
fragments Akkamalai had the highest tree density dominance of individual males in the group.
(1200 ± 424.26/ha) followed by Iyyerpady (1140 ± In Chinnakalar, with an area of 1300 ha,
24.64/ha) and the lowest plant density was observed only one troop was observed. Such low population
in Andhiparai (560 ± 80.07/ha). The GBH (239 ± maybe attributed due to less availability of edible
20.22cm), Tree height (68 ± 19.94ft) were highest plants. Another reason could be of competition
in Iyyerpady. On the other hand the lowest GBH between LTM and Nilgiri Langur (Trachypithecus)
(51 ± 10.58cm), and Tree height (12 ± 1.73ft) were recorded in this area (Kumaraguru 2005).
observed in Varatupparai. The canopy spread was Furthermore this area faces huge disturbance due to
highest in Iyyerpady, Uralikkal and Puthuthottam. ecotourism, human settlements, and monoculture
The canopy spread was lowest in Varatupparai (5 ± practices tea plantation in vast areas by private
2.64cm) (Table 4). estates for commercial uses (Sharma, 2002).
The portions of different plant species which Earlier, Umapathy and Kumar (2000a) found
were fed by LTM was tabulated in Table 5. LTM that the size of the area would be important when
consumed leaves of seven different plant species, the area was small. But in Chinnakalar, in spite of
flower and fruits of eight different plant species, vast area, group size and number of troops was
mesocarp of 17 different plants, seed coat of four found to be less, which could explain size of the
different plant species and seed of 15 different plant area was not a determining factor for the group size
species were given in (Table 5). and number of troops were found to be less, which
could explain size of the area was not a determining
DISCUSSION factor for the group size and number of groups.
The number of troops and group size are Furthermore Umapathy and Kumar (2000b)
mainly determined by the food resource availability confirmed that the density of trees and canopy

Journal of Research in Biology (2011) 5: 352-362 356
Narasimmarajan et al.,2011

Table. 3: Presence/ Absence of Tree species in different Fragments of Annamali Hills.
'√'- indicates presence the respective species in the fragament where as 'x'- indicates
the absence.
Plants species 1 2 3 4 5 6 7 8
Aglaia roxburghina X X x x x X √ √
Artocarpus heterophyllus X X √ x x √ x √
Beasa indicum √ X √ √ √ X x X
Bischofia javanica X √ x √ x √ x √
Cardia mixa X X x x √ X x
Calophillium sp. √ √ √ x x X x X
Cambendra sp. X X √ x x X x X
Carcinia cambogia X X √ x x X x √
Cassia sp. X X x x x X x X
Colikkarna sp. √ X √ x x X x X
Cinnamomum malabaricum X X x √ √ X x X
Cinnamomum zeylanicum √ X √ x x X x √
Coffea Arabica X X x x √ √ √ √
Cullenia excellrata √ √ √ √ √ √ √ X
Dalbergia sissu √ X x x x X x X
Disocyleum sp. X X √ x x X √ X
Divesperous sp. √ X √ x x X x X
Eucalyptus sp. X √ x x x √ x X
Eleaocarpus tuberculatus X X x x x √ √ X
Ficus bengalensis X X x x x √ x X
Ficus glomerata X X x x √ X x X
Ficus microcarpa X X √ √ x X x X
Harppulia imbricate X X x x x X x X
Kitnocarpus sp. X X √ √ x X √ X
Lechea globerata √ X x x √ X x X
Litsea deccanenis √ X √ √ x X √ X
Litsea oleoides X X √ x x X √ X
Maccaranga peltata X X x √ x √ √ X
Maccaranga roxburghii X X x x √ √ x √
Mealeosma sp. X √ x √ √ X x X
Measa indigum √ X √ x x X √ X
Measua ferrea √ X √ √ x √ √ √
Mimusops elengii X X x x x √ x √
Mimusops eleatum X X x x x X x X
Myristica beddomi X √ √ x x X x √
Myristica indicum √ X √ √ x X √ X
Myristica malabaricum X √ x x x X √ X
Palaquium ellipticum √ √ √ x x √ x √
Randia dumetorum X X x x √ X x √
Sandalam album X X x x x X x √
Sembcarprus sp. X √ x x √ X x √
Semicarpus anacardium √ X x √ x X x X
Sheleichera oleosa √ √ √ √ √ √ √ X
Shorea talura X √ x x x X √ X
Sterculia guttata X X x x x X x X
Syzygium cumini √ X x x x √ x X
Syzygium lanceolatum X X x x x X x √
Toona ciliate √ X x x x X √ √
Tripatacean sp. X X √ √ x X x √
Vateria indica X X x x x X x X
Vernonia sp. √ X √ x x X x X
Unknown √ √ √ √ √ X √ √
1=Iyyerpady; 2=Uralikkal; 3=Chinnakalar; 4=Korangumudi; 5=Varatupparai;
6=Puthuthottam; 7=Andhiparai; 8=Akkamalai.

357 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Table. 4: List showing the Mean (± S.D) Density of Tree, and Girth at Breast Height
(GBH), Canopy spread and Tree Height in 8 fragments of Anamalai Hills.
Tree GBH of the Canopy Tree height
Fragment Name
Density / ha tree (cm) spread (m) (m)
Iyyerpady 1140 ± 24.66 239 ± 201.29 3.05 ± 1.49 20.72± 6.07
Uralikkal 620 ± 116.61 101 ± 44.64 3.05± 1.79 14.93± 3.40
Chinnakalar 1080 ± 213.54 167 ± 114.77 2.74± 1.02 17.37± 7.20
Varatupparai 620 ± 231.51 51 ± 10.58 1.52± 0.80 3.65± 0.52
Korangumudi 820 ± 240.01 117 ± 46.68 2.13± 0.52 15.84± 5.48
Puthuthottam 960 ± 412.79 179 ± 139.30 3.04± 1.87 18.28 ± 6.58
Andhipparai 560 ± 80.07 123 ± 112.86 2.74± 1.38 11.58± 7.65
Akkamalai (Shola) 1200 ± 424.26 104 ± 76.66 2.13± 1.10 13.10 ± 6.18

height were the best predictors of the presence of private owned lands such as tea and coffee gardens,
LTM in a forest area. Although the Chinnakalar had have a much lower resource base than the relatively
high density of trees, the canopy height was shorter undisturbed rainforest complex (Ramachandran and
than that of the other LTM preferred fragments such Joseph, 2000). The major demography changes due
as Iyyerpady, and Puthuthottam. to fragmentation were in birth rate and proportion
The mesocarp of Cullenia excellrata, a plant of juveniles in the group, and an increase in the
recorded in all of these fragments, was one of the number of adult males and variability in group size
most preferred food items of LTM. The dispersal of adult sex- ratios with decreasing area and increasing
Cullenia excellrata in different fragments might be disturbance levels (Malcom, 1991). Vegetation
due to LTM. Cullenia excellrata, Eleaocarpus status and fragment area are highly correlated
tuberculatus, Cinnamomum malaricum, (Umapathy and Kumar, 2000b) which explains
Cinnamomum zeylanicum are used as roosting trees similar variation in the demographic parameters
by LTM viz, the height of these plants and the with different habitat parameters. On comparison
presence of primary and secondary branches with the demographic pattern of LTM in these
favored roosting. And the canopy provides an fragments, it was concluded that the group size and
opportunity to hide to themselves to the predators. number of troops or the individual population of
The variations in the canopy spread mean of tree LTM in this study area are mainly determined by
Density, Girth at Breast Height, and Tree height in the availability of food and other secondary factors
different fragments could be associated with includes disturbance, competition, predation.
altitudinal variations and also the variations in the Moreover the ecotourism is one of the serious
climate conditions. threats for LTM populations, people knowingly
Perhaps LTM was not observed in Solayar feeding the plastics and food items to LTMs, which
Dam, Kavarakkal, Sangarankudi. All these areas are existing on the road side. We suggest that
have extensive Cardamom (Elettaria cardamomum) tourists traveling between Azhiyar to Valparai,
plantations, human settlements, no water source and should be checked properly with mobile patrol and
low density of food species, which could be the spot penalty is to be needed (Fig; 2) for the control
reason for the lack of LTM populations in these of these activities.
areas. Akkamalai fragment was geographically
varied from other fragments since it consists of vast ACKNOWLEDGEMENT
grasslands and many small island pocket shola We acknowledged Tamil Nadu Forest
forest which was not habitat for arboreal mammals Department and all the Forest staffs of Anamalai
such as LTM (Menon and Poirier, 1996). Tiger Reserve. I personally thanks to my Guru Dr.
Furthermore the dynamite explosions in quarry K. Thiyagesan, Reader in A.V.C College, We
mined in this area could also be a reason. express our sincere thanks to Principle, HOD and
We attribute this unusual growth rate in our All Staff members of department of Wildlife
study group to its feeding habitats and the nature of Biology, A.V.C. College. Author thanking the field
available resources. It is reported that most of the Asst. Mr. Ravi and Mr. Prakash without whom this
forest fragments, especially those that are located in was not possible.

Journal of Research in Biology (2011) 5: 352-362 358
Narasimmarajan et al.,2011

Table 5: List showing the Plant species and Edible portions of Lion-tailed Macaque
‘√ '- indicates that the respective part of that plant was eaten where as ' x ' - indicates that
the parts was not eaten
Plants species Leaf Flower Fruit Mesocarp Seed coat Seed
Aglaia roxburghina x X x √ X x
Artocarpus heterophyllus x X x √ X x
Beasa indicum x X x √ X x
Bischofia javanica x X x X X √
Cardia mixa x X x X √ x
Calophillium sp. x X x X x √
Cambendra sp. x X x X x √
Carcinia cambogia x √ x X x √
Cassia sp. x √ x X x √
Colikkarna sp. x x x X x √
Coffea arabica x x √ X x x
Cullenia excellrata x x x √ x x
Dalbergia sissu x x x X x x
Disocyleum sp. x x x X √ √
Divesperous sp. x x x X x √
Ficus bengalensis x x √ X x x
Ficus glomerata x x √ X x x
Ficus microcarpa x x √ X x x
Harppulia imbricata x √ x √ x x
Kitnocarpus sp. √ x x X x x
Lechea globerata x x x √ x √
Litsea deccanenis x x x √ x x
Litsea oleoides x x x √ x x
Maccaranga peltata x x x √ x x
Maccaranga roxburghii √ x x X x x
Mealeosma sp. √ √ x X x x
Measa indigum x x x √ x x
Measua ferrea x x √ X x x
Mimusops elengii x x x √ x x
Mimusops eleatum x x x √ x x
Myristica beddomi x x x X √ x
Myristica indicum x √ x √ x x
Myristica malabaricum x x x √ x x
Palaquium ellipticum x x x √ x x
Randia dumetorum x x x X x √
Sembcarprus sp. √ √ x X x x
Semicarpus anacardium x x x X √ x
Sheleichera oleosa x √ x X x √
Shorea talura x x √ X x x
Sterculia guttata x x x X x √
Syzygium cumini x x x X x √
Syzygium lanceolatum x x x √ x x
Toona ciliata x x √ X x x
Tripatacean sp. √ √ x X x x
Vateria indica x x x X x √
Vernonia sp. √ x x X x x
Unknown √ x √ √ x √
359 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Fig 2. (A). LTM in its natural habitat; (B).LTM consuming fruits from the tree, Cullenia excellrata ; (C&D).
LTM consuming plastics due to effects of ecotourism and loss of their natural habitat & behaviour; (E&F). Due
to ecotourism and habitat loss, LTM is losing its arboreal nature and original feeding behavior.

Journal of Research in Biology (2011) 5: 352-362 360
Narasimmarajan et al.,2011

REFERENCE Merenlender A, Kremen C, Rakotondratsima M
Arnold GW, Steven DE, Weeldenberg JR, Smith and Weiss A. 1998. „Monitoring impacts of natural
EA. 1993. Influences of remnant size, spacing resource extraction on lemurs of the Masoala
pattern and connectivity on population boundaries Peninsula, Madagascar.‟ Conservation Ecology
and demography in Euros macropus robustus (online). 2:2- 5.
living in a fragmented landscape. Biol Conserv.,
64:219-230. Menon S, Poirier FE. 1996. Lion-tailed Macaque
(Macaca silenus) in a disturbed forest fragment:
Cutler A. 1991. Nested faunas and extinction in activity patterns and time budgets. Int J Primatol.,
fragmented habitats. Conserv Biol., 5:496-505. 17:969-985.

Decker BS. 1994. Effects of habitat disturbance on Quinn JF, Hastings A. 1987. Extinction in sub-
the behavioral ecology and demographics of the divided habitats, Conservation Biology 1:198-208.
Tana River Red Colobus (Colobus badius
rufomitratus) Int J Primatol., 15:703-737 Ramachandran KK, Joseph GK. 2000. Habitat
utilization of Lion-tailed Macaque (Macaca silenus)
Hanski I, Gilpin ME. 1991. Metapopulation in Silent Valley National Park, Kerala, India.
dynamics: brief history and conceptual domain. Primate Report 58:17-25.
Biol J Linn Soc., 40:3-16
Rao RJ, Bhatnagar A. 2001. Primates of the
Holmes JE. 1996. Parasites as threats to Amarkantak Forests, Madhya Pradesh Envis
biodiversity in shrinking ecosystems. Biodiversity Bulletin: Wildlife and Protected Areas 1(1):120-
Conserv., 5:975-983 123.

IUCN. 2007. 2007 IUCN Red List of Threatened Saunders DA, Hobbs R, Margules CR. 1991.
Species. Accessed 27 Feb. www.iucnredlist.org Biological consequences of ecosystem
Johns, A.D. Skorupa, J.P. (1987) Responses of rain- fragmentation: a review. Conserv Biol., 5:18-32.
forest primates to habitat disturbance: a review. Int
J Primatol 8:157-191. Sharma AK. 2002. A Study of Reproductive
Behaviour of Lion-tailed Macaque (Macaca
Kumar A, Umapathy G, Prabhakar A. 1995. A silenus) in the Rain forest of Western Ghats. Ph.D
study on the management and conservation of the dissertation ,University of Mysore, Mysore.
small mammals in fragmented rain forests of
Western Ghats, South India. A Preliminary report Sharma AK, Singh M, Kaumanns W, Krebs E,
Primate Conservation 16:53-58. Singh M, Kumar MA, Kumara HN. 2006. Birth
patterns in wild and captive Macaca silenus, Int J
Kumaraguru A. 2005. The influence of prey Primatol., 27:1429-1439.
species diversity and densities in different
vegetation types on the foraging ecology and Singh M, Kumara HN, Kumar MA, Sharma AK,
community structure of large carnivores in Indira Kaumanns W. 2002. Distribution, population
Gandhi Wildlife Sanctuary and National Park, structure and conservation of Lion-tailed Macaques
South India. Ph.D dissertation, Bharathidasan (Macaca silenus) in the Anamalai Hills, Western
University, Tiruchirappalli, Tamilnadu, India. Ghats. India. Am J Primatol., 57:91-102.

McNeely JA, Miller KR Reid WV, Mittermeier Singh M, Kumara, HN, Kumar MA, Sharma
RA, Werner TB. 1990. Conserving the World‟s AK. 2001. Behavioural responses of Lion-tailed
Biological Diversity. IUCN, World Resources Macaque (Macaca silenus) to a changing habitat in
Institute, Conservation International, WWFUS and a tropical rainforest fragment in the Western Ghats,
the World Bank: Washington, DC. India. Folia Primatol., 72:278-279.

Malcom JR. 1991. The small mammals of Struhsaker TT. 1976. A further decline in numbers
Amazonian forest fragments: pattern and process. of Amboseli vervet monkeys. Biotropica 8:211-214.
Ph.D dissertation, University of Florida, Florida.
361 Journal of Research in Biology (2011) 5: 352-362
Narasimmarajan et al.,2011

Sugiyama Y Ohsawa H. 1982. Population
dynamics of Japanese macques with special
reference to the effect of artificial feeding. Folia
Primatol., 39:238-263.

Umapathy G. 1998. Impacts of habitat
fragmentation on the arboreal mammals in the wet
evergreen forests of Anamalai hills in the Western
Ghats, South India. Ph.D. dissertation, Bharathiar
University, Coimbatore, India.

Umapathy G, Kumar A. 2000a. The occurrence of
arboreal mammals in the rain forest fragments in
Anamalai Hills, South India. Biol Conserv., 92:311-
319.

Umapathy G, Kumar A. 2000b. The demography
of Lion-tailed Macaque (Macaca silenus) in
rainforest fragments in the Anamalai Hills, South
India. Primates 41:119-126.

Wilcox BA. Murphy DD. 1985. Conservation
strategy: the effects of fragmentation on extinction.
Am Nat., 125:879-887.

Journal of Research in Biology (2011) 5: 352-362 362
Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Species diversity of water birds in Deepor Beel, Assam
Journal of Research in Biology

Authors: ABSTRACT:
Jyotismita Das1 and
Saikia PK2.
Study has been carried out to assess the water bird diversity in Deepor Beel
Ramsar site of Assam from March 2007 to March 2010. Study revealed the presence
Institution:
1 of 39 species of water birds belonging to 16 families. The family Anatidae represented
Research Scholar,
Department of Zoology, highest of eight species followed by seven species in the family Ardeidae whereas
Gauhati University. other 14 species represented by rest of the family. However, there were marked
2
Associate Professor, variation of species abundance in different years of study, of which, the family
Department of Zoology, Anatidae represented the highest abundant family throughout the study period. The
Gauhati University. analysis of species diversity index using Shannon diversity index showed that the
diversity was highest during 3rd year study period. Study also highlighted the threat
factors prevailing in the Deepor Beel Ramsar Site.
Corresponding author:
Jyotismita

Keywords:
Email: Diversity, water birds, Shannon Diversity index, Threats factor,
deeporbeel@gmail.com anthropogenic, Deepor Beel.

Web Address: Article Citation:
http://jresearchbiology.com/ Jyotismita Das and Saikia PK.
Documents/RA0103.pdf. Species diversity of water birds in Deepor Beel, Assam
Journal of research in Biology (2011) 5: 363-369

Dates:
Received: 12 Sep 2011 /Accepted: 16 Sep 2011 /Published: 20 Sep 2011

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INTRODUCTION Birdlife International has also declared Deepor Beel
Wetlands are widely recognized as fragile as an Important Bird Area (IBA). At maximum
ecosystems with diverse attributes including distinct flooding the Beel becomes above 4 meters in deep
avifauna (Burger, 1985). Wetlands have many and during the dry season the depth drops to about
ecological roles and are of importance to birds 1-1.5 meter. Deepor Beel (Coordination: 26°03′26″
(Gill, 1994). Wetlands are highly important because –26°09′26″N and 90°36′39″–90°41′25″E) is
they serve as critical breeding, staging and situated on the Southern bank of the river
wintering grounds for wide array of globally Brahmaputra and Village Maj Jalukbari, Pachim
important bird species (Kristen and Brander, 2004). Jalukbari, Dharapur and National Highway No.37
The It has been estimated that freshwater wetlands lie on the North; Dakhin Jalukbari, Tetelia and
hold more than 40% of bird species of the entire Pachim Baragoan to the East; Gorbhanga Reserve
world and 12% of all animal species (Zakaria et al. Forest, Chakardew Hill and Chilla Hill to the South
2009).Wetlands are important because they serve as West and the Village Azara and Kahikuchi to the
critical breeding, staging and wintering grounds for west. Owing to its high biological diversity, this
wide array of globally important bird species was included in the Directory of Asian wetlands.
(Kristen and Brander, 2004). The species richness Deepor Beel has a meso-thermal climate,
and relative abundance of birds depend upon characterized by high humidity and moderate
wetland characteristics such as size, water level, temperature. The temperature ranges between 10.6°
quality of water, availability and distribution of C to 30°C. The pre-monsoon season (March-May)
food resources, presence of suitable roosting and has a maximum temperature of 27° C and minimum
nursery sites (Wiens,1989).Variation in habitat of 24° C, and relative humidity between 50.5-76.8
condition may also cause changes in relative %. (Saikia and Kakati, 2010). The monsoon season
abundance of bird species composition (Garcia et (May -September) has a maximum temperature of
al.2007;Caziani and Derlindati,2000). Birds are bio 32°C and minimum of 27.3°C. The relative
-indicators as they notify us of certain changes humidity is 82.5%. The retreating monsoon
occurring in our environment. They are among the (September to October) has maximum and
numerous fauna that may be at risk by the use of minimum temperatures of 27° and 25° C
Agricultural pesticides (Mineau et al. respectively. (Saikia and Kakati, 2010). The
1990).Besides, the beauty of birds but also, relative humidity is 82% and the rainfall gradually
particularly, water birds has made bird watching a decreases to average as the season advances. The
very useful way of spending leisure and gathering winter season begins in November and continues
revenue from both local and international tourists. until January. The average field temperature during
Avian distribution and abundance are expected to this period remains at 20 ± 2°C and the relative
respond seasonal changes in wetlands (Colwell and humidity measures about 77.5%. January is the
Dodd, 1997; Farmer and Parent, 1997). coldest month, with a lowest temperature of 7 oC
but sometimes the temperature falls to 6 oC. At least
MATERIAL AND METHODS 232 species of avian fauna from 72 different
Study area families were recorded in previous studies from this
Deepor Beel is a large natural wetland wetland (Saikia and Kakati, 2010). Apart from
having great biological and environmental these; the Beel is associated with a rich variety of
importance (Deka and Goswami, 1992). This large other flora and fauna. Deepor Beel appears to be
water body is a great food source and breeding relatively high with respect to the biodiversity of
ground for a variety of migratory birds, amphibians, free floating, emergent and submerged aquatic
reptiles, insects, micro and macrophytes, terrestrial macrophyte (Saikia and Bhattacharjee, 1987).The
weeds and important taxa of ecological and free floating plant species are identified as Eicchornia
economic importance (Bera et al., 2008). This is crassipes, Azolla pinnate, Pistia stratiotes, Lemna
endowed with both floral and faunal biodiversity. minor, Lemna major. Again the emergent water plants
The Deepor Beel Ramsar site has a total area of 40 so far recorded are Trapa bispinosa, Utricularia
Km2 of which 4.14 Km2 had declared as a Bird flexuosa, Eleocharis pantaginea, Nelumbo nucifera,
Sanctuary. In November 2002, it was listed as a Nymphaea alba, Euryale ferox. Ipomea reptans. The
Ramsar site owing to its rich wetland biodiversity submerged plants which are so far identified are
and sociocultural importance. Again, considering Potamogeton crispum, Valisnaria spiralis, Hydrilla
the varieties of bird species found in the Beel, verticillata, Najas foveolata.

364 Journal of Research in Biology (2011) 5: 363-369
Jyotismita et al.,2011

Data Collection
Avian data were collected from March 2007
to March 2010.Data were collected in the early
morning (5a.m-9 p.m) and in the afternoon time
(4p.m-5p.m).For data collection four to five days
were allotted in one month. For watching, counting
and identifying birds Binocular (10X50), telescope
(25-40X), camera (Cannon 110 PS), note book,
guide book, pen, pencil etc were used.
Meteorological data (Temparature, Rainfall, and
Humidity) were collected from the Central
Meteorological Department, Borjhar, Assam.
Photographs and videos were taken to justify the
species type for those species that are difficult to
identify. Birds were identified by seeing their Figure 1. Water bird composition of the study area
characteristics feature in accordance with the During the study period declining trend was
identification keys involved in Ali and Ripley noticed regarding the presence of total numbers of
(1983), Grimmett et al. (1999). Counts were not individuals of the species in the three successive
made on days with fog, rain or strong wind to years. In 2007-08 the total numbers of individuals
lessen the bias caused by the effect of extreme of water birds recorded were 14,104 numbers. In
weather (Verner, 1985). 2008-09 total numbers of individuals of species
Data analysis were recorded as 11,508 numbers and in 2009-10
The diversity of water birds were analyzed the total number of birds were 10,380 (Fig. 2).
using species diversity and richness software (3.02
version).The Shannon diversity index (H) was
calculated to analyze the water bird diversity in the
study area. Bootstrap method was used to calculate
95% confidence intervals. In order to test the
difference in diversity between the three successive
years of study (First year, Second year and third
year) pair-wise randomization test was carried out
based on 10,000 re-sample of species abundance
based on Solow (1993).

RESULTS Figure 2. Total numbers of individuals recorded in
Water bird composition of the study area three successive years
Altogether 35,555 individuals from 39
species of 16 different families were found during Percent occurrence of water birds in three
the study period (Fig.1). The highest number of successive years
species (8) was found to be in the bird family While calculating the percentage of water
Anatidae which is followed by the family Ardeidae birds in the study area in three successive years it
containing seven numbers of species. The families was seen that in the first year the percent
Ciconiidae, Alcidinidae and Rallidae were occurrence of the Anatidae family was found to be
represented by three numbers of species highest (67.6%) and the same value was found to be
respectively. The families Accipitridae, Jacanidae lowest (0.13%) in Passeridae family (Fig.3). Again
and Passeridae are represented by two numbers of in the second year the percent occurrence of the
species respectively. Again one species of each of Anatidae family was highest (69.2%) as compared
the families Phalacrocoracidae, Corvidae, to the other families and the same was calculated to
Charadridae, Apodidae, Hirundinidae, Upopidae, be lowest (0.15%) in Passeridae family (Fig.4). In
Meropidae and Laridae were reported during the the third year also the percent occurrence of
study period. Anatidae family was highest (69.2%) than other
families (Fig.5). The percent occurrence of the

Journal of Research in Biology (2011) 5: 363-369 365
Jyotismita et al.,2011

Table 1. The values of Shannon diversity indices (H) of
three successive years
Variance Lower Upper
Sample H
H 95% 95%
First year 1.269 0.0001381 1.247 1.293

second year 1.228 0.0001703 1.2 1.253

Third year 1.335 0.0001902 1.308 1.36

Comparing Shannon index (H) between first
Figure 3. Percent occurrence of water birds in first year year and second year showed that the first year was
more diverse than the second year, at 5% level (pair
wise randomized test based on 10,000 random
samples; SI: First year: H=1.3; second year: H=1.2;
^=0.04). Again the comparison between second and
third year had showed that in the third year of study
diversity was found to be higher as compared to the
second year, at 5% level (pair wise randomized test
based on 10,000 random samples; SI: second year:
H=1.2,third year: 1.3; ^=0.10). Again while
comparing the diversity between first year and third
year it was seen that the third year was more diverse
than the first year, at 5% level (pair wise
randomized test based on 10,000 random samples;
Figure 4. Percent occurrence of water birds in second year SI: first year: H=1.2, third year: H=1.3;
^=0.06).Overall, the study showed that Shannon
Diversity Index was reported to be highest (H=1.3)
in third year of study period (Fig.6).

Figure 5. Percent occurrence of water birds in third year Figure 6. Shannon diversity indices (H) of avian fauna
of three successive years
Passeridae family was reported to be lowest
(0.15%) during the study period. Overall it was DISCUSSION
seen that the percent occurrence of Anatidae family The results clearly showed the presence of
was reported to be highest in all the three 39 species of water birds from 16 different families
successive years. in the study area. The present findings contradict
Comparison of diversity indices between first, the views of Saikia (2005) who had reported the
second and third year presence of 232 species of aquatic avifauna from
In these section Shannon diversity index (H) different families. Likewise, A. U. Choudhury
of the three successive years had been worked out (2000) had also recorded 150 species of birds in and
to analyze the species diversity of water birds and it around the Sanctuary. Barman (1995) had reported
was presented in Table 1. 62 species of water birds of which 16 species were
of Anatidae family. Interestingly, they had also
365 Journal of Research in Biology (2011) 5: 363-369
Jyotismita et al.,2011

mentioned the presence of 1,018 individuals of Markitan (2001), Houdkova (2003), Horn et al,
Aythya baeri in their survey in 1988-89, but (2008), Phillips (2008). Likewise year wise
subsequently the population declined to 250 in 1989 declining trend in population also might be
- 90, 3 in 1990-91, 135 in 1991-92, and none in attributed to different threat factors within Beel
1992-93. The decline in species content as reveled ecosystem. The percent occurrence of Anatidae
from the present study might be due to degradation family was reported to be highest in three
and habitat fragmentation due to anthropogenic successive years which was also supported by
activities in and around the Beel ecosystem. Four Saikia (2005). Among the members of Anatidae
factors impact on migratory birds population at family the population of both lesser whistling teal
their stop-over sites and winter quarters: restriction (19,661) and large whistling teal (3,540) were
of habitats, hunting and trapping, disturbance, and reported to be highest in three successive years.
effects of biocides (Berthold, 1994). Similarly the Saikia (2005) also had reported the highest
major threats to the water birds of Deepor Beel occurrence of large whistling teal in Deepor Beel.
were encroachment, habitat fragmentation, hunting The high density of aquatic vegetation might be
and trapping of water birds, Soil digging, over contributed in the high abundance of whistling teal
fishing, Application of pesticides in the agricultural in the study area as makhana act as a special food
field. Likewise in Yunnan province habitat component of both the species of whistling teal.
destruction and over-hunting were the major threats Saikia and Bhattacharjee (1987) had also reported
to the wetland species (Wen et al. 1995). Bharatha high density of free floating, emergent and
Lakhmi (2006) had also revealed in his study that submerged aquatic macrophyte in Deepor Beel.
anthropogenic pressure affects the habitat of water Shannon diversity index was reported to be
birds. Apart from this hunting and trapping of water highest in third year of study (H=1.3).This may be
birds can also play a major role in declining of the due to the fact that the diversity of wetland
water birds number in an alarming level. Poaching component and the adaption of different aquatic
of water birds also greatly contributed in declining avian fauna to exploit the resources of wetland
water bird species. This fact was also supported by ecosystem might be the cause of supporting high
Barman (1995) in his work by mentioning the diversity of water birds in the third year. Again
highest threat factors in Deepor Beel as compared different vegetation types as well as abundant food
to other Beel. Saikia and Kakati (2010) had also resources also might be playing a greater role in
mentioned about the heavy destruction process difference in habitat preference by bird species. The
which was prevailing within the Beel periphery rich and high vegetation might be providing
since last few years. Saikia (2005) had mentioned heterogeneous and suitable site for foraging,
about the recent soil digging of the Beel bed in roosting and nesting of birds. Similar findings of
number of locations in northern boundaries and high species diversity were reported by Macdonald
heavy encroachments for settlements caused (1977), Weller (1978) and Puttick (1984) in their
tremendous loss of wetland area in their works. work. Similar findings regarding the affect of
Apart from these he also mentioned different vegetation diversity and richness on the bird
anthropogenic factors which promotes in population richness were studied by Karr and Roth
degradation of the Beel in a quite high rate. Apart (1971), Cody (1981), Canterbury et al. (1999),
from this the Expert Team constituted by the Soderstrom and Part (1999), Paracuellos (2006) had
Planning Commission, Government of India, to mentioned in his work that the bird assemblage is
Review the status of implementation of the National affected by various factors like food availability, the
Wetland Conservation and Management size of the wetland etc. Zakaria et al. (2009) had
Programme (NWCMP) of the Ministry of reported that diversity of flora subsequently
Environment & Forests in 2008 had also reported affected the abundance and diversity of birds,
that the past two decades had witnessed a lots of insects, amphibians, fishes, reptiles and small
transformation in the ecological and social character mammals. Again, he mentioned that the abundance
of Deepor Beel. of insects, amphibians, reptiles and small mammals
Similar findings for the declining trend in had also attracted waders and raptors. Similar
various waterfowls in many regions of the world findings were documented by the work of Lee &
were observed by Korschgen and Dahlgren (1992), Rotenberry (2005) where they had reported that the
Vaisanen and Solonen (1996), Lebedeva and distribution and abundance of many bird species are

Journal of Research in Biology (2011) 5: 363-369 366
Jyotismita et al.,2011

determined by the composition of the vegetation Bharatha Lakshmi B. 2006. Avifauna of Gosthani
that forms a major element of their habitats. As estuary near Visakhapatnam, Andhra Pradesh.
vegetation changes along complex geographical and J.Natcon., 18(2):291-304.
environmental gradients, a particular bird species
may appear, increase or decrease in number, and Burger J. 1985. Habitat Selection in Temperate
disappear as the habitat changes. Smith (1992) had Marsh-Nesting Birds. In: (Eds) Cody M.L. Habitat
also worked out that differences in feeding habits Selection in Birds. Academic Press, Orlando 253-
and habitats could also increase diversity, evenness 281.
and species richness.
Canterbury GE, Martin TE, Petit DR, Petot LJ
CONCLUSION and Branford DF. 1999. Bird communities and
Deepor Beel which is the lone Ramsar site habitat as ecological indicators of forest condition
in Assam is very rich in biodiversity. Apart from in regional monitoring. Conserv. Bio., 14:544-558.
the extensive destruction processes which are
continuously prevailing within the Beel, the water Caziani SM and Derlindati EJ. 2000. Abundance
birds are still visiting the wetland body for its rich and habitat of High Andean flamingos in
biodiversity. So, proper management practices Northwestern Argentina. Waterbirds 23 (Special
should be taken for the proper conservation of the Publication 1):121-133.
wetland ecosystem as well as the water birds.
Choudhury AU. 2000. The Birds of Assam.
ACKNOWLEDGEMENT Gauhati: Gibbon Books and World Wide Fund for
Authors are very much helpful to Mr. Nature 240.
S.Upadhaya, Assistant Professor, T.H.B College,
Sonitpur for providing necessary information. Cody ML. 1981. Habitat selection in birds: The
Again the authors are grateful to Dr. Hemen Deka, role of vegetation structure, competitors and
Assistant Professor, B.B.K College and some local productivity. Bioscience 31:107-113.
peoples for providing the immediate helps in proper
completion of the work. The Authors express proud Colwell MA and Dodd SL. 1997. Environmental
thanks to UGC for providing financial help as well and habitat correlates of pasture use by nonbreeding
as Head of the Department Zoology, Gauhati shorebirds. Condor 99:337-344.
University for providing help for proper completion
of the work. Farmer AH and Parent AH. 1997. Effects of the
landscape on shorebird movements at spring
REFERENCES migration stopovers. Condor 99:698-707.
Ali S and Ripley SD. 1983. Handbook of the Birds
of India and Pakistan. Oxford Univ. Press 48-49. Deka SK and Goswami DC.1992. Hydrology,
Sediment Characteristics and Depositional
Barman R and Saikia SJ. 1995. Length-Weight Environment of Wetlands: A case study of Deepor
Relationship and Some Ecological Observation of Beel, Assam. J. of Assam Scien. Socie., 34(2):62-
Cirrhina reba (Ham.) from the Wetlands of Assam, 84.
India. Environ. and Ecol., 13(3):721-724.
García Borboroglu P and Yorio P. 2007.
Berthold P and Francisco P. 1994. Heritability of Breeding habitat requirements and selection by
migratory activity in a natural bird population. The Olrog’s Gull (Larus atlanticus) a threatened
Royal society, Proceedings: Biological sciences species. Auk 124:1201-1212.
257(1350):311-315.
Gill FB. 1994. Ornithology. 2nd edition, New
Bera SK, Dixit S, Basumatary SK and Gogoi R. York.
2008. Evidence of biological degradation in
sediments of Deepor Beel Ramsar Site, Assam as Grimmett R, C Inskipp and Inskipp T.1999.
inferred by degraded palynomorphs and fungal Pocket guide to the Birds of Indian Sub- Continent.
remains. Current science 178(2):178-180. Oxford Univ. Press 384.

367 Journal of Research in Biology (2011) 5: 363-369
Jyotismita et al.,2011

Horn KV, Benton K and Gatti. 2008. Waterfowl Puttick GM. 1984. Foraging and Activity Patterns
Breeding Population Survey for Wisconsin, 1973- in Wintering Shorebirds. Behavior of Marine
2007. Wisconsin Department of Natural Resources Animals, Shorebirds: Migration and Foraging
(WDNR), Great Lakes Indian Fish and Wildlife Behaviour (Eds) Burger J and BL Olla. Plenum
Commission (GLIFWC) and U.S. Fish and Wildlife Press, New York, London.1-7.
Service (USFWS), Madison Wisconsin.
Soderstrom, B and Part T.1999. Influence of
Houdkova B. 2003. Trends in numbers of Coot landscape on farmland bird breeding in semi-
(Fulica atra) in Czech Republic in 1998-2000. natural pastures. Conserv. Bio., 14:522-533.
Ornis Hungarica 12-13:283-288
Smith RL. 1992. Elements of Ecology. 3rd edition.
Karr JR and Roth RR. 1971. Vegetation structure Harper Collins Publishers Ltd. London.
and avian diversity in several new world areas. Am.
Naturalist 105: 423-435. Saikia PK and Bhattacharjee PC. 1987. A study
of the Avifauna of Deepor Beel a Potential Bird
Korschgen CE and Dahlgren RB. 1992. Human Sanctuary in Assam. Wetland and Waterfowl
Disturbance of Waterfowl: Causes, Effects and Conservation in Asia (Eds) Parisah, D. and
Managements: In Management Hand Book, Fish Prentice, R. C. Asian Wetland Bureau/ International
and Wildlife Leaflet 1-7. Waterfowl & Wetlands Research Bureau, Kuala
Lumpur. 52:188-195.
Kirsten S and Brander L. 2004. The Economic
Values of the World’s Wetlands. Living Waters, Saikia PK. 2005. Qualitative and Quantitative
Amsterdam. Study of Lower and Higher Organisms and Their
Functional role in the Deepor Beel Ecosystem.
Lebedeva NV and Markitan LV. 2001. Problems Funded by North Eastern Space Applications
of Population dynamics of White-Eyed Pochard Centre, Department of Space, Government of India,
(Aythya nyroca Guld., 1770) in the Eastern sea of Umium, Meghalaya, Shillong 1-97.
Azov region. Russian J. of Ecology 32:425-431.
Saikia PK and Kakati M. 2010. Biodiversity in
Lee P and Rotenberry JT. 2005. Relationships Deepor Beel Ramsar Site of Assam India: Faunal
between bird species and tree species assemblages Diversity. Lap Lambert Academic Publishing 132.
in forested habitats of eastern North America. J. of
Biogeography 32:1139-1150. Smith RL. 1992. Elements of Ecology. 3rd edition.
Harper Collins Publishers Ltd, London.
Macdonald KB. 1977. Plant and Animal
Communities of Pacific North American Salt Solow AR. 1993. A simple test for change in
Marshes. Wet Coastal Ecosystems (Eds) Chapman, community structure. J.of Animal Eco., 62:191-
V.J. Elsevier Scientific Publication Co., 193.
Amsterdam.
Vaisanen RA and Solonen T. 1996. Summary:
Mineau P, Sheenan PJ and Baril A. Population Trends of 100 Winter Bird Species in
1990.Pesticide and waterfowl on the Canadian Finland in 1957-1996. Finnish Museum of Natural
Prairies, need for research and History, University of Helsinki, Finland. 70-97.
monitoring.IWRB.Tech. Pub. U.K. 133-134.
Verner J. 1985.Assessment of counting
Paracuellos M. 2006. How can habitat selection techniques. Current ornithol., 2:247-302
affect the use of a wetland complex by water birds.
Bio. and Conserv., 15:4569-4582. Weller NW. 1978. Management of Freshwater
Marshes for Wildlife. Fresh Water Wetlands.
Phillips JH. 2008. Decline of the Pintail-Part II, Ecological Processes and Management Potential
MadDuk- The Conscience of Waterfowl (Eds) R.E .Good, D.F. Whigham and R.L. Simpson.
Conservation. <www.madduk.org>. Academic Press, New York. 267-284.

Journal of Research in Biology (2011) 5: 363-369 368
Jyotismita et al.,2011

Wen X, Yang L and Yang X. 1995. Status of
waterbirds in wetlands of Yunnan plateau, China
(Eds) Y. Chen Study of Wetlands in China. Jilin
Science Technology Press, Chang Chun. 248-255

Wiens JA. 1989. The Ecology of the Bird’s
Communities. Cambridge University Press,
Cambridge (2):8-9.

Zakaria M, Rajpar MN and Sajap AS. 2009.
Species diversity and feeding guilds of birds in
Paya Indah wetland reserve, peninsular Malaysia.
International J. of Zoological Research 5(3):83-
100.

369 Journal of Research in Biology (2011) 5: 363-369
Journal of Research in Biology

Original Research Paper An International Online Open Access
Publication group

Feasible use of rock oyster ( Crassostrea commercialis) and seaweeds (Gracilaria salicornia
and Caulerpa lentillife) as biofilter in a laboratory - scale closed recirculating system for
juveniles spotted babylon (Babylonia areolata)
Journal of Research in Biology

Authors: ABSTRACT:
Nilnaj Chaitanawisuti1,
Wannanee Santhaweesuk1, This study was conducted to assess the feasible use of rock oyster
Sirusa Kritsanapuntu 2 (Crassostrea commercialis) in biofiltration and two seaweeds (Gracilaria salicornia and
Caulerpa lentillife) as nutrient absorbant in a laboratory - scale recirculating system for
growing of juveniles spotted babylon (Babylonia areolata). The experiment was
Institution:
1. Aquatic Resources carried out in triplicates over a period of 90 days. The experiment was a complete
Research Institute, randomized design with three growth trials: Treatments 1: without oyster and
Chulalongkorn University, seaweed biofilter used as a control; Treatment 2: Oyster biofiltration (1,500 g per
Phya Thai Road, Bangkok, tank) and seaweed (G. salicornia) absorption (250 g per tank); and Treatment 3:
Thailand 10330. Oyster biofiltration (1,500 g per tank) and seaweed (C. lentillife) absorption (250 g per
2. Faculty of Science and tank). No significant differences (P>0.05) in final shell length, final body weight, body
Industrial Technology, weight gains, shell length increment and growth rate among all treatments. Growth
Prince of Songkla rate in shell length and body weight of spotted babylon ranged from 0.33 – 0.34 cm
University, Surattani mo-1 and 0.62 – 0.67 g mo-1, respectively. Significant differences (P<0.05) in final
province, Thailand 84100. survival rate of spotted babylon were found among treatments, ranging from 86.72 to
86.98 % compared with those of the control (84.27%). There were no significant
differences (P>0.05) in water temperature, salinity, pH, dissolved oxygen, ammonia-
Corresponding author: nitrogen, nitrite-nitrogen, nitrate-nitrogen and phosphate-phosphorus among the growth
Nilnaj Chaitanawisuti trials but not for alkalinity. This study can conclude that G. salicorniand C. lentillifera can
be used as nutrient biofilter for regulating of water quality in a closed recirculating
system for growing juveniles spotted babylon but not suitable on using oyster.
Email:
nilnajc1@hotmail.com; Keywords:
saenisa479@hotmail.com; Seaweed, biofilter, recirculating culture, water quality, Gracilaria salicornia,
wannanee295@hotmail.com Caulerpa lentillifera, Babylonia areolata.

Web Address: Article Citation:
http://jresearchbiology.com/
Documents/RA0078.pdf.
Nilnaj Chaitanawisuti, Wannanee Santhaweesuk, Sirusa Kritsanapuntu.
Feasible use of rock oyster (Crassostrea commercialis) and seaweeds (Gracilaria
salicornia and Caulerpa lentillife) as biofilter in a laboratory - scale closed recirculating
system for juveniles spotted babylon (Babylonia areolata).
Journal of research in Biology (2011) 5: 370-375

Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 23 Sep 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

370-375 | JRB | 2011 | Vol 1 | No 5
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Chaitanawisuti et al.,2011

INTRODUCTION: nutrients, in particularly nitrogen in the term of
Commercial growing of juveniles spotted ammonia (Buschmann 1996, Neori et al. 1996,
babylon, Babylonia areolata, in Thailand are Chow et al. 2001, Hernandez et al. 2002, Matinez-
cultivated under the flow-through seawater system. Aragon et al. 2002, Msuya et al. 2006). Thus, we
This type of culture needs about 100% water have developed a simpler recirculating system for
exchange per day to maintain water quality and also the production of juvenile spotted babylon available
had some criticism regarding its environmental use for commercial applications. This study was
impact due to its tendency to release waste effluents conducted to assess the capable use of rock oyster
containing elevated levels of nitrogen or (Crassostrea commercialis) as biofiltration and two
phosphorus – rich compounds which may be seaweeds (Gracilaria salicornia and Caulerpa
considered as water pollutants. One of the main lentillife) as nutrient absorption in a laboratory -
drawbacks of spotted babylon culture lies in the scale closed recirculating system for growing of
wastes derived from feed and their metabolic juveniles spotted babylon (Babylonia areolata)
products. The main products are uneaten food,
faeces and excreted dissolved inorganic nutrients MATERIALS AND METHODS
which are transported in the water at various This study was conducted at the Spotted
concentrations (Chaitanawisuti, Krisanapuntu and Babylon Aquaculture Research Unit, Aquatic
Natsukari 2005). Many valuable marine species can Resources Research Institute, Chulalongkorn
be successfully grown and have high growth and University, Petchaburi province, Thailand. The
survival rates in recirculating aquaculture system laboratory experiment was designed to test the use
(RAS) due to the high-quality culture water. Water of rock oyster (C. commercialis) and seaweeds (G.
reuse methods require maintaining good water salicornia and C. lentillife) as biofilter in a
quality with a system that easily operated at a low laboratory - scale closed recirculating system for
cost. RAS have attractive much attention because growing juveniles spotted babylon (B. areolata).
they consume less water than flow-through systems The experiment was a complete randomized design
and also reduce water exchange rates. Therefore, with three replications as following: Treatment 1:
RAS is biologically and technically feasible and Without oyster and seaweed biofilter used as a
secondly that it will produce worthwhile income to control; Treatment 2: Oyster biofiltration (1,500 g
the producers. It has been used extensively for per tank) and seaweed (G. salicornia) absorption
rearing and maintaining adult and juvenile marine (250 g per tank); and Treatment 3: Oyster
species. Pagand et al. (2000) reported that the use of biofiltration (1,500 g per tank) and seaweed (C.
recirculating water systems is one approach used to lentillife) absorption (250 g per tank). Each
limit the impact of aquaculture on the aquatic experimental unit was designed as an independent
environment. Although the total quantity of closed - recirculating system consisting of a rearing
nutrients released is similar in open and tank, an oyster filtration tank and a seaweed
recirculating systems, the small volumes of absorption tank. Cylindrical plastic tanks of 300 l
concentrated effluents that are produced should be capacity were used for each tank of the recirculating
easier to deal with, although proper technical and unit. The bottom area of the rearing tank was 0.78
economical solutions have to be implemented. The m2 and was covered with a 5 cm layer of coarse
use of seaweeds for the treatment of marine sand (0.5 – 1.0 mean grain size) to serve as a
aquaculture wastes has been proposed by a number substrate. The seaweed absorption tank contained
of authors since marine seaweeds have high one of the marine seaweeds as a biofilter. All
capacities for absorption and metabolism of N and experimental units were located in an indoor
P – rich compound excreted by marine animals. In hatchery, but the roof over all seaweed absorption
addition to improving the water quality, seaweeds tanks was transparent and permitted about 80% of
can provide an additional source of income from a sunlight to pass thus enabling seaweed
valuable by-product in the process. Jones, Dennison photosynthesis. Seawater was pumped from an
& Preston (2001) suggested that nutrient removal earthen pond, filtered mechanically, and circulated
efficiency of macroalgae may be improved with through the experimental unit. The water depth in
increased light, particularly for nitrate uptake, all tanks was 30 cm. Water flowed from the
increased water flow to reduce the boundary layer, biofilter tank through the rearing tank and was
and higher stocking densities. The special interest is maintained in a small submersible pump at a
the use of seaweeds for recycling some dissolved
371 Journal of Research in Biology (2011) 5: 370-375
Chaitanawisuti et al.,2011

constant flow rate of 530 l h-1 before it was returned intervals, and shell length and whole body weight
to the rearing tank. No seawater was exchanged were determined. Shell length was measured with
throughout the experimental period of 90 days. The calipers to the nearest millimetre from the
tanks were moderately aerated by air diffusers maximum anterior to posterior distance of a shell,
placed at the bottom of each tank. Water and the whole weight was measured after air drying
temperature was maintained at room temperature for a period of 10 min before weighing and then
±1.5oC. Salinity was monitored daily, as necessary, returned to the tank. The number of dead
to keep the variation within ±1.0 ppt by addition of individuals was recorded at 15-day intervals.
fresh water to correct for any increased salinity due Seaweeds from each tank were also weighed to
to water evaporation. Photoperiod was a natural 12 determine the increase in biomass, and specific
l:12 day:night. growth rates were calculated at 15-day intervals.
Juvenile spotted babylons, B. areolata, used Seaweeds were placed in a plastic basket to drain
in growth and survival experiments were produced off excess water. Visible epiphytes were carefully
at a private hatchery in Rayong province, located on removed. The experiment was terminated after a 90
the eastern coast of the Gulf of Thailand. days during May to June, 2009. Average shell
Individuals from the same cohort were sorted by length increments, body weight gains and growth
size to prevent possible growth retardation of small rates were calculated after the method of
spotted babylons when cultured with larger ones. Chaitanawisuti and Kritsanapuntu 1999). Body
Their initial shell length and whole body weight weight gains (BWf - BWi), and monthly growth
averaged 1.23 ± 0.005 cm and 0.37 ± 0.01 g, n = 30, rates for body weight (BWf - BWi)/T) were
respectively. Mean shell lengths and body weights calculated, in which BWf = mean final body
used in each treatment were not statistically weight, BWi = initial body weight, and T = time in
different, and hence, treatments could be compared months. The specific growth rate (SGR, % day-1) =
statistically. Juveniles were held in the experimental 100 9 [ln (final weight, g) - ln (initial weight, g)/
rearing tanks at a stocking density of 300 individual (culture period, days). Mortality, expressed as the
m-2 or 192 snails per tank. The rock oyster percentage of the initial stocking density, was
Crassostrea commercialis with average body calculated from the difference between the number
weight of 4.0 - 10.0 g were collected from the of snails stocked and harvested. The following
oyster farm at Ang-sila, Cholburi province. Two seawater quality parameters in each rearing tank
seaweeds Gracilaria salicornia and Caulerpa were analysed weekly: water temperature (Hg
lentillifera were used as biofilters. The first was thermometer), salinity (portable multiparameter
collected from intertidal pools along the coastal metre model YSI#63), conductivity (portable
water of Samui Island, Surattani province, and the multiparameter metre model YSI#63), pH (pH
latter was collected from broodstock-rearing ponds metre), total alkalinity (phenolphthalein methyl
of an intensive shrimp farm. Oysters and seaweeds orange indicator), dissolved oxygen (DO metre
were placed in a plastic basket of 25.0 x 35.0 x 25.0 model YSI#52), total ammonia–nitrogen (phenate
cm which contained numerous pores of 1.5 cm2 (4 method), nitrite–nitrogen (colorimetry method),
holes cm-2) at each side and were suspended 30 cm nitrate–nitrogen (cadmium reduction) and
above the bottom of biofilter tanks. Oyster and orthophosphate (ascorbic acid method) (APHA et
seaweeds in all tanks were not harvested throughout al. 1998).
the experiments. Data on growth performance and water
The snails were fed ad libitum with fresh quality were analyzed using the SPSS statistical
trash fish once daily at 10:00 h. The amount of food software package (version 10). Analysis of variance
consumed was recorded daily, and uneaten food (ANOVA) was used to test the interaction of
was removed immediately after the animals stopped seaweeds and stocking density at α = 0.05, and
feeding and air dried for a period of 10 min before differences between means were compared using
weighing. Size grading of snails in all treatments Tukey’s test at α = 0.05.
was not done throughout the grow-out period. No
chemical and antibiotic agents were used RESULTS
throughout the entire experimental period. To Growth in shell length and body weight of
determine growth performance, 50% of snails were juvenile spotted babylon (Babylonia areolata) in a
sampled randomly from each treatment at 15 days laboratory - scale closed recirculating system using

Journal of Research in Biology (2011) 5: 370-375 372
Chaitanawisuti et al.,2011

oyster and two seaweeds as biofilters over 90 days commercialis) and seaweeds (G. salicornia and C.
is shown in Figure 1. One-way ANOVA showed no lentillifera) as biofilters over 90 days is shown in
significant differences (P>0.05). in final shell Table 2. There were no significant differences
length, final body weight, body weight gains, shell (P>0.05) in water temperature (24.2 – 24.60C),
length increment and growth rate among all salinity (30.5 – 30.7 psu), pH (8.35 – 8.42), dissolved
treatments (Table 1). Shell length increments of oxygen (6.5 – 6.7 mg/l), ammonia - nitrogen (0.0766
spotted Babylon ranged from 0.98 to 1.00 cm and – 0.0922 mg/l), nitrite -nitrogen (0.1555 – 0.1728 mg/
1.88 to 2.03 g for those of body weight gains. l), nitrate - nitrogen (0.8584 – 0.9073 mg/l) and
Growth rate in shell length and body weight of phosphate - phosphorus (0.4863 – 0.5185 mg/l)
spotted babylon ranged from 0.33 – 0.34 cm mo-1 among the growth trials but not for total alkalinity
and 0.62 – 0.67 g mo-1, respectively. Significant (77.0 – 81.0 mg/l) (P<0.05).
differences (P<0.05). in final survival rate of
spotted babylon were found among rearing DISCUSSION
treatments (P<0.05), ranging from 86.72 to 86.98 This study showed that no significant
% compared with those of the control (84.27%). differences in final shell length, final body weight,
Growth of seaweeds (G. salicornia and C. body weight gains, shell length increment and
lentillifera) were used as biofilters in a laboratory growth rate among all treatments. Growth rate in
scale, closed recirculating system for juveniles shell length and body weight of spotted babylon.
spotted babylon (B. areolata) for 90 days is shown Significant differences in final survival rate of
in Figure 2. There were significant differences spotted babylon were found among treatments,
(P<0.05) in absolute growth rates between the two ranging from 86.72 to 86.98 % compared with
seaweed biofilter treatments. Weight gain of C. those of the control (84.27%). There were no
lentillifera (716.0 g) was significantly higher than significant differences in water temperature, salinity,
those of G. salicornia (353.0 g mo -1). Growth rate pH, dissolved oxygen, ammonia-nitrogen, nitrite-
of C. lentillifera (238.6 g mo -1) was significantly nitrogen, nitrate-nitrogen and phosphate-phosphorus
higher than those of G. salicornia (117.6 g mo -1). among the growth trials but not for alkalinity. This
However, growth of rock oyster (C. commercialis) study can conclude that G. salicorni and C. lentillifera
is very low with less than 0.05 g mo-1 for all can be used as nutrient biofilter for regulating of
treatments but final survival was high (100%) for water quality in a closed recirculating system for
Treatment 1 and 2. growing juveniles spotted babylon but not suitable
Water quality parameters in a closed use of oyster. As compared to the study of
recirculating system using rock oyster (C. Chaitanawisuti, Kritsanapuntu and Natsukari

-
-

373 Journal of Research in Biology (2011) 5: 370-375
Chaitanawisuti et al.,2011

Table 1. Growth of juveniles spotted babylon (B. areolata) in a laboratory - scale closed recirculating system
using oyster and two seaweeds as biofilters for 90 days
Parameters Treatment 1 Treatment Treatment
1.23±0.02 1.23±0.01 1.23±0.001
0.30±0.02 0.29±0.01 0.30±0.004
2.23±0.04 2.23±0.16 2.21±0.10
2.32±0.11 2.30±0.57 2.17±0.27
1.00+0.03 1.00+0.15 0.98+0.10
2.03+0.11 2.01+0.58 1.88+0.27
0.33+0.01 0.34+0.05 0.33+0.03
0.67+0.04 0.67+0.19 0.62+0.09
84.27b 86.72a 86.98a
Values are means +standard deviation. Means with different superscript in the same row are significantly
different (P<0.05).

(2005), growth rate of B. areolata in this study taxifolia almost doubled internal nitrogen in
(0.62 to 0.67 g mo-1) is the same as those cultured nutrient – rich water), Caulerpa species have
in the recirculating system used oyster shells as application in bioremediation of intensive tank-
biofilter (0.62 to 0.68 g mo-1) but lower than those based aquaculture and perhaps treated pond
cultured in flow-through system (1.05 to 1.25 g mo- aquaculture effluent. As compared to other
1
) and This study agreed with the study of Chow et seaweeds commonly used for water treatment in
al. (2001) who indicated that Gracilaria chilensis mariculture purposes, Msuya et al. (2006) reported
culture was highly efficient at biofiltration of the that seaweed, Ulva reticulate, grew at an average of
soluble nutrients but had little effect on particulate 4.0% day-1 at the fishpond outflow with a biomass
emission. The best growth of G. chilensis occurred yield averaging 46 g m-2 day-1, compared with
in the ammonium – rich effluent from the fish averaging 2.5% and 2.7 g m-2 day-1at the fishpond
culture. Jones, Dennison & Preston (2001) also inflow. Martinez-Aragon et al. (2002) indicated that
recommended that nutrient removal efficiency of seaweeds (Ulva rotundata and Enteromorpa
macroalgae may be improved with increased light, intestinalis and Gracilaria gracilis removed
particularly for nitrate uptake, increased water flow efficiently the phosphate dissolved in the waste
to reduce the boundary layer, and higher stocking water from the fish culture tanks. Removal
densities. Paul & Nys (2008) reported that seaweed efficiency was highest in U. rotundata (99.6%) and
from the genus Caulerpa culture will not be easily lowest in G. gracilis (62.2%). In addition, the
integrated into settlement ponds in tropical maximum uptake rate of phosphate occurred in U.
aquaculture. However, because some species of rotundata, slightly greater than that for E.
Caulerpa grew well in tank-based system (C. intestinalis, while G. gracilis showed the lowest
racemosa grew at > 7% day-1) and others are uptake rate. Hernandez et al. (2002) also
capable of luxury uptake (C. serrulata and C. indicated that U. rotundata and E. intestinalis and
Table 2. Seawater quality in a laboratory - scale closed recirculating system for juveniles spotted babylon (B.
areolata) using oyster and two seaweeds as biofilters for 90 days
Parameters Treatment 1 Treatment Treatment
Water temperature ( C) 24.2±2.4 24.3±2.4 24.6±2.3
Salinity psu) 30.6±1.3 30.7±1.7 30.5±1.2
pH 8.42±0.11 8.37±0.12 8.35±0.15
Alkalinity (mg/L) 81±9 a 74±8c 77±9b
Dissolved oxygen (mg/L) 6.5±0.7 6.7±0.6 6.5±0.5
Ammonia - nitrogen (mg-N/L) 0.0766±0.0590 0.0900±0.0538 0.0922±0.0604
– - 0.1728±0.0404 0.1638±0.0809 0.1555±0.0802
– - 0.9073±0.3112 0.8584±0.3036 0.8827±0.3230
– - 0.4863±0.2109 0.5185±0.2516 0.4922±0.2643
Values are means +standard deviation. Means with different superscript in the same row are significantly
different (P<0.05).

Journal of Research in Biology (2011) 5: 370-375 374
Chaitanawisuti et al.,2011

G. gracilis removed efficiently the ammonium through and recirculating seawater system.
dissolved in the waste water from the fish culture Aquaculture International 13:233-239.
tanks. U. rotundata and E. intestinalis showed the
highest ammonium disappearance (97.7%), whereas Chow FY, Macchivello J, Santa Cruz S, Fonck E.
G. gracilis removed 93.2% of the nutrient during 2001. Utilization of Gracilaria chilensis (Rhodophyta:
the incubation. In addition, the minimum uptake Gracilariaceae) as a biofilter in the depuration of
rate of ammonium occurred in G. gracilis. Wang et effluents from tank cultures of fish, oysters and sea
al. (2007) showed that growth rate of Ulva pertusa urchin. Journal of World Aquaculture Society 32:215-
was 3.3% day-1 in a indoor recirculating system for 219.
production of juvenile sea cucumber (Apostichopus
Hernandez I, Martinez-Aragon JF, Tovar A, Perez
japonicas). U. pertusa was efficient in removing
-llorens JL and Vergara JJ. 2002. Biofiltering
toxic ammonia and in maintaining the water quality efficiency in removal dissolved nutrients by three
within acceptable levels for sea cucumber culture. species of estuarine macroalgae cultivated with sea
U. pertusa removed 68% of the total ammonia bass (Dicentrarchus labrax) waste waters 2 Ammonia.
nitrogen and 26% of the orthophosphate from the Journal Applied Phycology 14:375-384.
sea cucumber culture effluent; the macroalgae
biofilter removed ammonia at an average rate of Jones AB, Dennison WC, and Preston NP. 2001.
0.459 g N m-2 day-1. This study can conclude that G. Integrated treatment of shrimp effluent by
salicorniand C. lentillifera can be used as nutrient sedimentation, oyster filtration and macroalgal
biofilter for regulating of water quality in a closed absorption: a laboratory scale study. Aquaculture
recirculating system for growing juveniles spotted 193:155-178.
babylon but not suitable use of oyster. However, the
stocking density and production of G. salicorniand Martinez-Aragon JF, Hernandez I, Perez-llorens
C. lentillif should be considered for optimal use in JL, Vazquez R, Vergara JJ. 2002. Biofiltering
the recirculating system for the spotted Babylon. efficiency in removal dissolved nutrients by three
species of estuarine macroalgae cultivated with sea
ACKNOWLEDGES bass (Dicentrarchus labrax) waste waters 1
This research was funded by Chulalongkorn Phosphate. Journal Applied Phycology 14:365-374.
University in the fiscal year of 2009. The authors
thank to Aquatic Resources Research Institute, Msuya FE, Kyewalyanga MS, and Salum D. 2006.
The performance of the seaweed Ulva reticulate as a
Chulalongkorn University, in particular Mr.
biofilter in a low-tech, low cost, gravity generated
Yongyudth Chankaew for his assistances in the
water flow regime in Zanzibar, Tanzania. Aquaculture
hatchery and collecting of oysters and seaweeds. 254:284-292.
The authors also thank to Associated Professor Dr.
Somkiat Piyatiratitiworakul for valuable comments Neori A, Krom MD, Ellner SP, Boyd CE, Popper
during the experiments and preparation of this D, Rabinovitch R, Davison PJ, Dvir O, Zuber D,
manuscript. Ucko M, Angel D, and Gordin H 1996. Seaweed
biofilters as regulators of water quality in integrated
REFERENCES fish-seaweed culture unit. Aquaculture 141:183-199.
APHA, AWWA, WPCF. 1998. Standard methods
for the examination of water and wastewater, Pagand P, Blancheton JP, Lemoalle J, and Casellas
American Public Health Association, American C. 2000. The use of high rate algal ponds for the
Water Works Association, 20th ed. Water Pollution treatment of marine effluent from a recirculating fish
Control Federation: Washington, DC. 2-24-4-159. rearing system. Aquaculture Research 31:729-736.

Buschmann AH. 1996. An introduction to Paul NA, and de Nys R. 2008. Promise and pitfalls of
integrated farming and the use of seaweeds as locally abundance seaweeds as biofilters for integrated
biofilters. Hydrobiologia 326(327):59-60. aquaculture. Aquaculture 281:49-55.

Chaitanawisuti N, Krisanapuntu S, Natsukari Y. Wang H, Liu CF, Qin CX, Cao SQ, and Ding J.
2005. Growth of hatchery-reared juvenile spotted 2007. Using a macroalgae Ulva pertura biofilter in a
recirculating system for production of juvenile sea
babylon (Babylonia areolata Link 1807) to
cucumber Apostichopus japonica. Aquaculture
marketable size at four stocking densities in flow-
Engineering 36:217-224.
375 Journal of Research in Biology (2011) 5: 370-375
Journal of Research in Biology

Original Research Paper An International Online Open Access
Publication group

The combined effects of temperature and salinity on hatching success and survival of
early life stages in the economically candidate marine mollusks : Spotted babylon
Babylonia areolata
Journal of Research in Biology

Authors: ABSTRACT:
Nilnaj Chaitanawisuti,
Sirinun Nunim and The combined effects of temperature and salinity on the hatching success
Wannanee Santhaweesuk. and larval and juvenile survival of the spotted babylon Babylonia areolata were
investigated in a 3x3 complete factorial experimental design employing three
temperatures (25, 30 and 350C) and three salinities (27, 30 and 33‰). The results
showed that hatching of egg capsules B. areolata was only significantly affected by
Institution: salinity but not for temperature. No significant interaction between both factors
Aquatic Resources Research occurred. However, survival of larvae and early juvenile were significantly affected by
Institute, Chulalongkorn temperature and salinity. A significant interaction between both factors occurred in
University, Phya Thai Road, this experiment. The highest survival of early juveniles was founded at the lowest
Bangkok, Thailand 10330. temperature (250C) with the highest salinity (33‰) and the highest temperature
(250C) with the highest salinity (33‰). From an aquaculture point of view, high
hatching of egg capsules B. areolata was obtained at temperature of 29 -350C and
salinity above 32‰ to 33‰. As well as the high survival of larvae was obtained at
temperature of 29 -30.50C and salinity above 32‰ to 33‰ and 29 -350C and 32 - 33‰
Corresponding author: for early juveniles.
Nilnaj Chaitanawisuti

Email:
nilnajc1@hotmail.com,
Keywords:
saenisa479@hotmail.com, Spotted babylon, Babylonia areolata, temperature, salinity, hatching,
wannanee295@hotmail.com. survival.

Web Address: Article Citation:
http://jresearchbiology.com/ Nilnaj Chaitanawisuti, Sirinun Nunim and Wannanee Santhaweesuk.
Documents/RA0079.pdf. The combined effects of temperature and salinity on hatching success and survival of
early life stages in the economically candidate marine mollusks : Spotted babylon
Babylonia areolata
Journal of research in Biology (2011) 5: 376-384

Dates:
Received: 11 Aug 2011 /Accepted: 27 Aug 2011 /Published: 23 Sep 2011

© Ficus Publishers.
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creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
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cited.

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Chaitanawisuti et al.,2011

INTRODUCTION: April 2011 in indoor air condition room at Sichang
Temperature and salinity are considered to Marine Science Research and Training Station,
be the most important physical factors influencing Chulalongkorn University, located on Sichang
marine organisms, and the biological effects of Island Cholburi province, Thailand. To verify the
these factors are complex and wide ranging. combined effects of temperature and salinity on
Temperature is one of the most critical external various life stages of the spotted babylon resistance,
factors of ontogeny in the early life stages of fish. a 3x3 complete factorial design was used for the
There are two ways in which temperature affects experiments including three temperatures (25, 30
ontogeny. Firstly, temperature, if within a viable and 350C) and three salinities (27, 30 and 33‰).
range, strongly affects the rate of ontogeny. A Each of the nine different temperature–salinity
temperature beyond this range is lethal for the combinations was conducted in three replicates. A
species. Secondly, temperature affects the hatch total of 27 experimental units were set up initially.
rate, incubation period, the size of the newly The experiment followed a completely randomized
hatched larvae, larval yolk absorption and design, using the egg capsule, larvae and juveniles
utilization, larval feeding behavior, larval survival from a single spawn to minimize variability
and larval growth (Shi et al. 2010). Tolerance and between experimental units. This experiment was
resistance tests can be used to determine the conducted in the air condition room with normal
survival capacity of various life stages of a species temperature control at 25 0C. Seawater used in all
in relation to certain factors. The combined effects treatments was filtered (10 µm) and dechlorinated
of temperature and salinity on the survival of for two days before use to ensure no chlorine
marine animals have been demonstrated in many residue. Seawater with low salinities was made up
marine organisms, mainly fish (Shi et al. 2010, daily by diluting a source of natural seawater with
Berlinsky et al. 2004, Cook et al. 2005), crustaceans de-chlorinated freshwater and high salinities water
(Dawirs 1979, Ponce-Palafox et al. 1997, Mene et was made by evaporating seawater. Temperature
al. 1991, Nagaraj 1988, Albuquerque et al. 2009), were maintained within +0.2 0C using 3 identical
echinoderms (Asha & Muthiah 2005, Dong & Dong 22.0 L thermostatically controlled water baths (L x
2006, Ji et al. 2008, Lavitra et al. 2010), bivalves W x H = 50 x 30 x 15 cm). Each water bath
(Taylor et al. 2004, Robert et al. 1988, Tettelbach et contained nine experimental units (1000 mL glass
al. 1981), and gastropods (Lu et al. 2004, Davis beaker). Gently aeration was provided during larval
2000, Chen & Chen 2004, Zheng et al. 2000). culture, a normal photoperiod of 12L:12D was
However, there is scant information on the effects adopted throughout the experiment. Water
of environmental fluctuation, such as temperature, exchange was not done during the experiment for
salinity on growth and survival of Babylonia all aquaria. Salinity and temperature in all
areolata particularly the combined effects of experimental units were measured every six hours
temperature and salinity. This is the case for early using a portable refractosalinometer and a mercury
life stage of Babylonia areolata that may be more thermometer, respectively. All experimental units
sensitive to environmental change. Albuquerque et were covered with aluminum foil to prevent
al. (2009) stated that salinity could modify the evaporation. The experiments lasted after 96 h.
effects of temperature and alter the temperature Experiment 1: Effect of temperature and salinity
range of many biological processes. In turn, on the hatching success
temperature can also modify the effects of salinity. After spawning, the egg capsules were
The objective of this study is to investigate the transferred to experimental units maintained at
combined effect of temperature and salinity on the different temperature – salinity combinations. The
eggs, larvae and early juvenile of spotted Babylon mean size (+SD) of egg capsules at the start of the
Babylonia areolata to determine the optimal experiment was 1.2+0.03 cm in length. For each
temperature and salinity for incubation and larval temperature and salinity combination, each
culture of this species. The results of this study will experimental unit consisted of 100 egg capsules
be useful in increasing the production of this acclimated in a 1000 mL glass beaker filled with
species through incubation and larval culture. 700 mL seawater. Each unit was stocked with low
stocking density of 10 egg capsules per unit so that
MATERIALS AND METHODS stocking density was not a limiting factor for
Experimental design survival. Hatching –out of veliger larvae from egg
The study was conducted from February to capsule usually was observed by naked eyes and
377 Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

considered as hatched or unhatch egg capsule. each experimental unit (1000 mL glass beaker filled
Hatched egg capsules expressed by no fertilized with 700 mL seawater) was stocked with low
eggs inside or partial retain of fertilized eggs. The stocking density of 50 larvae per unit so that
number of hatched capsules and duration of stocking density was not a limiting factor for
incubating period in each experimental unit were survival. Juveniles were fed with fresh meat of trash
recorded. The mean percentage of hatching rate was fish at once daily during the experiment. Uneaten
calculated by combining the data from three food was removed immediately after they stopped
replicates at the end of the experiment. eating to prevent water degradation. Then tested
Experiment 2: Effect of temperature and salinity juveniles were fed so that food availability was not
on the survival rate of veliger larvae a limiting factor for survival. Each experimental
Within 6 h after hatching, aeration was unit was initially examined for the dead juveniles
stopped, and the veliger larvae were allowed to after 6 h, and every 12 h thereafter. The number of
concentrate at the water surface; they were then juveniles which did not response to the touch of a
collected from the hatching tanks for use in the needle were observed by naked eyes and considered
experiments. The mean size (+SD) of larvae at the as dead. The mean percentage of survival rate was
start of the experiment was 428+0.01 µm in shell calculated by combining the data from three
length. For each temperature and salinity replicates at the end of the experiment.
combination, each experimental unit consisted of Data analysis
100 larvae reared in a 1000 mL glass beaker filled To investigate the effect of temperature and
with 700 mL seawater. Each unit was stocked with salinity on hatching of egg capsule, and survival of
low stocking density of 100 larvae per unit so that larvae and juveniles, a two-way analysis of variance
stocking density was not a limiting factor for (ANOVA) (fixed factors: temperature and salinity)
survival. Larvae were fed with a single algal diet with a 95% confidence interval was used. All data
(Chaetoceros calcitrans) once daily during the were tested for normality and homoscedasticity. On
experiment. Optimal concentrations of algal cells significant difference indication, Tukey test was
(6x104 cell ml-1) were fed so that food availability used to verify the difference among the treatments.
was not a limiting factor for survival and was The correlation between the survival and the
adjusted for each experiment according to the size temperature and salinity was estimated by multiple
of larvae (Chaitanawisuti and Kritsanapuntu 1997). regression analysis.
Each experimental unit was initially examined the
dead larvae after 6 h, and every 12 h thereafter. The RESULTS
number of larvae which sink down to bottom of the Experiment 1: Effect of temperature and salinity
aquaria / empty shell or no movement of velum on the hatching success
were observed microscopically and considered as Hatching of egg capsules B. areolata over
dead. The mean percentage of survival rate was the three temperatures and three salinity
calculated by combining the data from three combinations for 96 h was presented in Table1.
replicates at the end of the experiment. The highest hatching of 86.67% and 100% were
Experiment 3: Effect of temperature and salinity founded at the lowest temperature (25 0C) with the
on the survival rate of early juveniles highest salinity (33‰) and the highest temperature
Within 48 h after settlement, the mean size (350C) with the highest salinity (33‰),
(+SD) of juveniles was 1393.33+8.18 µm in shell respectively, while the lowest hatching 63.33% was
length and are used at the start of the experiment. founded at the lowest temperature (25 0C) with the
For each temperature and salinity combination, lowest salinity (27‰).
Table 1. Percentage hatching of egg capsules B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature Salinity (‰)
(0C) 27 30 33
25 63.33+25.20 83.33+15.30 86.67+5.70
30 76.67+11.50 86.67+15.30 96.67+20.80
35 76.67+20.80 83.33+15.30 100
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)

Journal of Research in Biology (2011) 5: 376-384 378
Chaitanawisuti et al.,2011

Two-way ANOVA showed that hatching of Experiment 2: Effect of temperature and salinity
egg capsules B. areolata was only significantly on the survival rate of veliger larvae
affected by salinity (F = 5.102; P = 0.018) but not Survival of veliger larvae B. areolata over
for temperature (F = 1.085; P = 0.359). No the three temperatures and three salinity
significant interaction between both factors were combinations for 96 h was presented in Table4.
occurred in this experiment (F = 0.220; P = 0.924). The highest survival of veliger larvae of 78.67%
Tukey test showed that hatching of egg capsules and 47.00% were founded at the lowest temperature
were significantly different between the salinity of (250C) with the highest salinity (33‰), and the
27‰ and 33‰ (Table 2). highest temperature (350C) with the highest salinity
Multiple regression analysis showed that (33‰), respectively, while the lowest survival of
only salinity (P = 0.002) were statistically veliger larvae of 23.33% and 17.67% were founded
significant, showing a negative correlation with the at the lowest temperature (25 0C) with the lowest
percentage hatching of egg capsules B. areolata salinity (27‰), and the highest temperature (35 0C)
(Table 3). In additional, standard coefficient (Beta) with the lowest salinity (27‰), respectively.
of salinity (0.570) was higher than that of Two-way ANOVA showed that survival of
temperature (0.228), indicating a higher correlation veliger larvae B. areolata was significantly affected
between salinity and hatching. The higher salinity by temperature (F = 1662.01; P = 0.000) and
provided the higher hatching. The multiple salinity (F = 488.96; P = 0.000). A significant
regression equation on hatching of egg capsules B. interaction between both factors were occurred in
areolata over the combined effects of temperatures this experiment are observed (F = 152.69; P =
and salinity were estimated as follows: 0.000). Tukey test showed that survival of veliger
Hatching = -54.074 + 0.889 Temperature + 3.704 larvae were significantly different one another
Salinity among temperature and salinity treatments (Table
The response surface plots summarizing 5).
percentage hatching under nine different Multiple regression analysis showed that
combinations of temperature and salinity over 96 h both temperature (P = 0.020) and salinity (P =
showed that high hatching (95%) was obtained at 0.012) were statistically significant, showing a
temperature of 29 -350C and salinity above 32‰ to negative correlation with the percentage survival of
33‰. Lower temperatures and salinities reduced veliger larvae B. areolata (Table 6). In additional,
hatching markedly and higher salinities had a standard coefficient (Beta) of salinity (0.445) was
marked positive effect at lower temperatures (Fig higher than that of temperature (0.406), indicating a
1). higher correlation between salinity and survival. It
also indicated that the higher salinity provided the
higher survival. The multiple regression equation
on survival of veliger larvae B. areolata over the
combined effects of temperatures and salinity was
estimated as follows:

Survival = -13.037 - 2.789 Temperature + 5.093 Salinity

The response surface plots summarizing
percentage hatching under nine different
combinations of temperature and salinity over 96 h
showed that high hatching (90%) was obtained at
temperature of 29 -30.50C and salinity above 32‰
to 33‰. Lower temperatures and salinity reduced
hatching markedly but higher temperature and
salinities had a marked positive effect (Fig 2).
Experiment 3: Effect of temperature and salinity
Fig 1. Response surface plots with estimating the mean on the survival rate of early juveniles
percentage hatching of egg capsules B. babylonia after Survival of early juvenile B. areolata over
96 h under 9 different combinations of temperature the three temperatures and three salinity
and salinity. combinations for 96 h was presented in Table 7.
379 Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

salinity (F = 49.060; P = 0.000). A significant
interaction between both factors occurred in this
experiment are observed (F = 36.376; P = 0.000).
Tukey test showed that survival of early juveniles
were significantly different one another among
temperature and salinity treatments (Table 8).
Multiple regression analysis showed that
both temperature (P = 0.002) and salinity (P =
0.026) were statistically significant, showing a
negative correlation with the percentage survival of
early juvenile B. areolata (Table 9). Standard
coefficient (Beta) of temperature (0.818) was higher
than that of salinity (0.251), indicating a higher
correlation between temperature and survival. The
multiple regression equation on survival of early
juvenile B. areolata over the combined effects of
temperatures and salinity was estimated as follows:
Survival = 165.176 – 6.148 Temperature + 3.148
Fig 2. Response surface plots with estimating the mean Salinity
percentage survival of veliger larvae B. babylonia after The response surface plots summarizing
96 h under 9 different combinations of temperature
percentage survival under nine different
and salinity.
combinations of temperature and salinity over 96 h
The highest survival of early juveniles of showed that high survival (100%) was obtained at
100% and 65.56% was founded at the lowest 29 -350C and 32 - 33‰. Higher temperatures at all
temperature (250C) with the highest salinity (33‰), salinities reduced hatching markedly (Fig 3).
and the highest temperature (25 0C) with the highest
salinity (33 ‰), respectively, while the lowest DISCUSSION
survival of early juveniles (15.56%) was founded at Our results showed that hatching of egg
the highest temperature (35 0C) with the lowest capsules B. areolata was only significantly affected
salinity (27‰). by salinity but not for temperature. No significant
Two-way ANOVA showed that survival of interaction between both factors occurred in this
early juvenile B. areolata was significantly affected experiment. The highest hatching was founded at
by temperature (F = 605.192; P = 0.000) and the lowest temperature (25 0C) with the highest

Table 2. ANOVA comparing the effects of temperature and salinity on percentage hatching of egg capsules B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 189170.37 1 189170.37 865.69 0.000
Temperature 474.07 2 237.03 1.08 0.359
Salinity 2229.63 2 1114.81 5.10 0.018
Temperature x salinity 192.59 4 48.14 0.22 0.924
Error 3933.33 18 218.51

Table 3. Multiple regression analysis of data on hatching of egg capsules B. areolata through 96 h under 9
different combinations of temperature and salinity on (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept -54.074 36.676 -1.474 0.153
Temperature 0.889 0.627 0.228 1.417 0.169
Salinity 3.704 1.046 0.570 3.542 0.002
Standard error of estimation = 13.310
R = 0.614 R2 = 0.377 Adjusted R2 = 0.326
F = 7.275 P = 0.003

Journal of Research in Biology (2011) 5: 376-384 380
Chaitanawisuti et al.,2011

Table 4. Percentage survival rate of veliger larvae B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature Salinity (‰)
(0C) 27 30 33
25 23.33+1.53 65.67+2.08 78.67+1.53
30 81.33+3.51 84.00+3.00 87.67+1.53
35 17.67+1.15 18.67+1.53 47.00+2.65
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)

salinity (33‰) and the highest temperature (25 0C)
with the highest salinity (33 ‰). Multiple
regression analysis showed a negative correlation
with the percentage survival of early juvenile B.
areolata as well as indicating a higher correlation
between temperature and larval survival, as well as
salinity and early juvenile survival. The response
surface plots showed that high survival of larvae
was obtained at temperature of 29 -30.50C and
salinity above 32‰ to 33‰ and 29 -350C and 32 -
33‰ for early juveniles. The results of this study
agreed with various studies that salinity had
strongly effect on larvae of various mollusks
(Doroudi et al. 1999, Tettelbach & Rhode 1981,
Davis 2000 and Dove & O’Oconner 2007. Doroudi
et al. (1999) reported that optimal conditions for
maximum larval survival of the black-lip pearl
oyster Pinctada margaritifera were 26-290C and 28
-32‰. Temperature of 350C or greater were lethal
Fig 3. Response surface plots with estimating the mean
percentage survival of new settled juvenile B.
for larvae and at all temperature tested, larval
babylonia after 96 h under 9 different combinations of survival were lowest at a salinity of 40‰.
temperature and salinity. Tettelbach & Rhode (1981) reported that optimum
combination of temperature and salinity for survival
salinity (33‰) and the highest temperature (35 0C) of the Northern Bay scallop Argopecten irradians
with the highest salinity (33‰). Multiple larvae from 2 to 5 day after fertilization, as
regressions showed a higher correlation between estimated from the response surface plot, was
salinity and hatching. High hatching was obtained 18.70C and 28.1‰. Temperatures of 35 0C or
at temperature of 29 -350C and salinity above 32‰ greater and / or salinities of 10‰ or less were lethal
to 33‰. Survival of larvae and early juvenile B. for all life stages of this species. Davis (2000)
areolata were significantly affected by temperature showed that at the end of 0 to 7 day interval,
and salinity. A significant interaction between both percent mortality of tropical gastropod veligers
factors occurred in this experiment was observed. Strombus gigas was highest for veligers grown at
The highest survival of early juveniles was founded 20 and 240C and at salinity of 45‰, while percent
at the lowest temperature (25 0C) with the highest mortality was low and not different for veligers

Table 5. ANOVA comparing the effects of temperature and salinity on percentage survival of veliger larvae B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 84896.14 1 84896.14 19760.31 0.000
Temperature 14280.96 2 7140.48 1662.00 0.000
Salinity 4201.40 2 2100.70 488.95 0.000
Temperature x salinity 2624.14 4 656.03 152.69 0.000
Error 77.33 18 4.29
381 Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

Table 6. Multiple regression analysis of data on survival of veliger larvae B. areolata through 96 h under 9
different combinations of temperature and salinity on (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept -13.037 65.309 -.0200 0.843
Temperature -2.789 1.117 -0.406 -2.496 0.020
Salinity 5.093 4.862 0.445 2.735 0.012
Standard error of estimation = 23.701
R = 0.603 R2 = 0.364 Adjusted R2 = 0.311
F = 6.855 P = 0.004

Table 7. Percentage survival rate of early juvenile B. areolata through 96 h under 9 different combinations of
temperature and salinity.
Temperature Salinity (‰)
(0C) 27 30 33
25 93.33+1 98.89+1.92 100
30 92.23+1.93 92.23+3.05 92.23+1.93
35 15.56+5.09 26.67+5.77 65.56+7.70
Mean values are calculated from a total of 3 replicate experiments
Values with different superscript letters are significantly different (P<0.05)

grown at 240C and at salinity of 30, 35 and 40‰. optimum temperature and salinity conditions for
Dove & O’Oconner (2007) showed that salinity had survival of P. vannamei postlarvae coincides the
a significant effect on D-veliger larval survival of best at 28 to 300C and 33 to 40‰. Jackson &
Sydney rock oysters Saccostrea glomerata whereas Burford (2003) showed that salinity did not have a
temperature significantly affected survival of both significant effect on survival of larval shrimp
D-veliger and pediveliger larvae. There was an Penaeus semisulcatus above 28‰. At 28‰,
interaction between salinity and temperature for D- survival rate decreased, while temperature had a
veliger larval survival. While spat survival was substantial and regular influence on growth rate.
significantly affected by salinity only and no Zacharia & Kakati (2004) showed that salinity
interaction was detected between salinity and exerted a greater influence than temperature on the
temperature for spat survival. survival and development of larvae Penaeus
In addition, results of this study also agreed merguiensis. The best temperature – salinity
with the combined effects of temperature and combination for hatching and larval survival was
salinity on larvae and juveniles of various obtained at 330C and 35‰, and a salinity range of
organisms; crab (Paula et al. 2003, Nurdiani & 30-35‰ is ideal for larval development. Paula et al.
Zeng 2007) and shrimp (Palafox et al. 1997, (2003) indicated that for all zoeal stages of
Jackson & Burford 2003 and Zacharia & Kakati mangrove crab Parasesarma catenaata, the highest
2004). Palafox et al. (1997) indicated that good survival was obtained at temperature of 25 0C and
survival (80 to 90%) of Penaeus vannamei salinity of 35‰, but survival decreased towards the
postlarvae was obtained below 30 0C and below lower and higher salinity. Nurdiani & Zeng (2007)
40‰. Higher temperatures reduced survival showed that temperature and salinity as well as the
markedly. Higher salinities only had a marked interaction of the two parameters significantly
negative effect at higher temperatures. The affected the survival of zoeal larvae of mud crab

Table 8. ANOVA comparing the effects of temperature and salinity on percentage survival of early juvenile B.
areolata (95% confidence interval).
Parameters Sum of square df Mean square F-value P-value
Intercept 152616.90 1 152616.90 8826.82 0.000
Temperature 20927.67 2 10463.84 605.19 0.000
Salinity 1696.51 2 848.25 49.06 0.000
Temperature x salinity 2515.77 4 628.94 36.37 0.000
Error 311.22 18 17.29
Journal of Research in Biology (2011) 5: 376-384 382
Chaitanawisuti et al.,2011

Table 9. Multiple regression analysis of data on survival of early juvenile B. areolata through 96 h under 9
different combinations of temperature and salinity (95% confidence interval).
Parameters B Standard error Beta t-value p-value
Intercept 165.176 46.503 3.552 0.002
Temperature -6.148 0.796 -0.818 -7.728 0.000
Salinity 3.148 1.326 0.251 2.347 0.026
Standard error of estimation = 16.876
R = 0.855 R2 = 0.731 Adjusted R2 = 0.709
F = 32.679 P = 0.000

Scylla serrate. At low salinity, both high and low Berlinsky DL, Taylor JC, Howell RA, Bradley
temperature led to mass mortality of newly hatched TM and Smith TIJ. 2004. The effects of
larvae, while the low temperature and high salinity temperature and salinity on early life stages of black
combination of 250C and 35 gL-1 resulted in the sea bass Centropristis striata. Journal of the World
highest survival to the megalopal stage. Aquaculture Society 35:335-344.
This study is the first study to investigate
salinity and temperature effects on hatching Chaitanawisuti N and Kritsanapuntu A. 1997.
success, and survival of larvae and early juvenile B. Laboratory spawning and juvenile rearing of the
areolata. Although the results suggest the marine gastropod: Spotted babylon, Babylonia
possibility that the culture and natural population of areolata Link, 1807 (Neogastropoda: Buccinidae)
this species in Gulf of Thailand may suffer in Thailand. Journal of Shellfish Research 16:31-
mortality from higher water temperature due to 37.
climate change and lower salinity from heavy
rainfall. Cook MA, Guthriel KM, Rust MB and Plesha
PD. 2005. Effects of temperature and salinity
ACKNOWLEDGEMENT during incubation on hatching and development of
This research was a part of the Research lingcod Ophiodon elongates Girard, embryos.
University Program funded by Chulalongkorn Aquaculture Research 36:1298-1303.
University (CC103A). The authors thank Sichang
Marine Science Research and Training Station, Chen JC and Chen WC. 2000. Salinity tolerance
Aquatic Resource s Re searc h Institute, of Haliotis diversicolor supertexta at different
Chulalongkorn University, in particular Mr. salinity and temperature levels. Aquaculture
Soontorn Thepmoon for help in the hatchery and 181:191-203.
providing of algae. The authors also thank to
Associated Professor Dr. Gullaya Wattayakorn for Doroudi MS, Southgate PC and Mayer RJ. 1999.
valuable comments and coordinations during the The combined effects of temperature and salinity on
preparation of this research project. embryos and larvae of the black-lip pearl oyster
Pinctada margaritifera. Aquaculture Research
REFERENCES 30:271-277.
Albuquerque EF, Meurer B and Netto GCG.
2009. Effects of temperature and salinity on the Dawirs RR. 1979. Effects of temperature and
survival rates of coxicerberus ramosae salinity on larval development of Pagurus
(Albuquerque, 1978), an interstitial isopod of a bernhardus (Decapoda, Paguridae. Marine Ecology
sandy beach on the coast of Brazil. Brazilian Progress Series 1:323-329.
Archives of Biology and Technology 52:1179-
1187. Davis M. 2000. The combined effects of
temperature and salinity on growth, development,
Asha PS & Muthiah P. 2005. Effects of and survival for tropical gastropod veligers of
temperature, salinity and pH on larval growth, Strombus gigas. Journal of Shellfish research 19:
survival and development of the sea cucumber 883-889.
Holothuria spinifera Theel. Aquaculture 250:823-
829. Dong Y. and Dong S. 2006. Growth and oxygen
consumption of the juveniles sea cucumber
383 Journal of Research in Biology (2011) 5: 376-384
Chaitanawisuti et al.,2011

Apostichopus japonicus at constant and fluctuating 1897 (Brachyura: Sesarmidae). Western Indian
water temperature. Aquaculture research 37:1327- Ocean Journal of Marine Science 2:57-63.
1333.
Ponce-Palafox J, Martinez-Palacios CA and
Dove MC. and O’Oconner WA. 2007. Salinity Ross LG. 1997. The effects of salinity and
and temperatures tolerance of Sydney rock oysters temperature on the growth and survival rates of
Saccostrea glomerata during early ontogeny. juvenile white shrimp, Penaeus vannamei, Boone,
Journal of Shellfish Research 26:939-947. 1931. Aquaculture 157:107-115.

Ji T, Dong Y and Dong S. 2008. Growth and Taylor JJ, Southgate PC and Rose RA. 2004.
physiological responses in the sea cucumber, Effects of salinity on growth and survival of silver-
Apostichopus japonicus : Aestivation and lip pearl oyster, Pinctada maxima spat. Journal of
temperature. Aquaculture 283:180-187. Shellfish research 23:375-377.

Jackson CJ. and Burford MA. 2003. The effects Tettelbach ST. and Rhodes EW. 1981. Combined
of temperature and salinity on growth and survival effects of temperature and salinity on embryos and
of larval shrimp Penaeus semisulcatus (Decapoda: larvae of the northern bay scallop Argopecten
Penaeoidea). Journal of Crustacean Biology 23:819 irradiant irradians. Marine Biology 63: 249-256.
-826.
Robert R, His E and Dinet A. 1988. Combined
Lavitra T, FohyN, Gestin PG, Rasolofonirina R effects of temperature and salinity on fed and
and Eeckhaut L. 2010. Effect of water temperature starved larvae of the European flat oyster Ostrea
on the survival and growth of endobenthic edulis. Marine Biology 97:95-100.
Holothuria scabra (Echinodermata : Holothuridae)
juveniles reared in outdoor ponds. SPC Beche-de- Shi YH, Zhang GY, Zhu YZ, Liu JZ and Zang
mer Information Bulletein 30:25-28. WI. 2010. Effects of temperature on fertilized eggs
and larvae of tawny puffer Takifugu flavidus.
Lu J, Lin Q, Sun Y, Sheng J and Chen Q. 2004. Aquaculture research 41:1741-1747.
Effect of temperature on the early development of
Haliotis diversicolor Reev. Journal of Shellfish Zheng H, Zhu J, Ke C, Zhou S and Li F. 2000.
research 23:963-966. Effects of temperature and salinity on embryonic
development of Babylonia formosae habei
Mene L, Alvarez-Ossorio MT, Gonzalesz- (Gastropoda: Buccinidae). Journal of Oceanography
Gurriaran E and Valdes L. 1991. Effects of in Taiwan Strait 19:1-5.
temperature and salinity on larval development of
Necora puber (Brachyura : Portunidae). Marine Zacharia S and Kakati VS. 2004. Optimal salinity
Biology 108:73-81. and temperature for early developmental stages of
Penaeus merguiensis De man. Aquaculture 232:373
Nagaraj M. 1988. Combined effects of temperature -382.
and salinity on the complete development of
Eurytemora velox (Crustacea : Calanoidea). Marine
Biology 99:353-358.

Nurdiani R and Zeng C. 2007. Effects of
temperature and salinity on the survival and
development of mud crab Scylla serrata larvae.
Aquaculture Research 38:1529-1538.

Paula J, Mendes RN, Mwaluma J, Raedig C and
Emmerson W. 2003. Combined effects of
temperature and salinity on larval development of
the mangrove crab Parasesarma catenaata Ortman,

Journal of Research in Biology (2011) 5: 376-384 384
Journal of Research in Biology

Original Research Paper An International Online Open Access
Publication group

Biorestraining potentials of marine macroalgae collected from
Rameshwaram, Tamil nadu
Journal of Research in Biology

Authors: ABSTRACT:
Anandhan S and
Sorna kumari H.
The marine ecosystem is the treasure place for many natural resources. In
this study, about five different marine macroalgae were chosen to study the
antibacterial activity and larvicidal activity of Aedes sp. About five different stranded
strains of bacteria have been selected to detect the antibacterial activity of collected
Institution:
Sri Sankara Arts & algae. Among the five algae collected, Gracilaria crassa and Hypnea valentia have
Science College -Enathur, shown maximum antibacterial activity in methanolic extraction by antibiosis of Kirby-
Tamilnadu. Bauers method. The active biocompound from methanol was determined by using
different solvent systems in TLC. The partially purified antibacterial compound was
identified as saponins by phytochemical tests. Larvicidal bioassay was carried out with
the two algae Gracilaria crassa and Hypnea valentia. The LC50 determined by the
Gracilaria crassa and Hypnea valentia was noted as 52.2 and 53.4 respectively.
Corresponding author:
Sorna kumari H

Email: Keywords:
sornask@gmail.com Larvicidal bioassay, LC50, antibiosis.

Web Address: Article Citation:
http://jresearchbiology.com/
Anandhan S and Sorna kumari H.
Documents/RA0100.pdf.
Biorestraining potentials of marine macroalgae ollected from Rameshwaram,
Tamil nadu.
Journal of research in Biology (2011) 5: 385-392

Dates:
Received: 09 Sep 2011 /Accepted: 16 Sep 2011 /Published: 23 Sep 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly
cited.

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Anandhan et al.,2011

INTRODUCTION structurally diverse secondary metabolites. The
Man relied on natural products in general secondary metabolites offer a defense against
and plants in particular to promote and maintain herbivores, fouling organism and pathogens. They
good health and fight sickness, pain and disease also play a role in reproduction, protection from UV
since in time immemorial. India is an important radiation and as allelopathic agent. There is an
country in world where ambient system of medicine urgent need to search for alternatives to synthetic
such as ayurveda, siddha and unani has been in antibiotics. The revalution of the discovery of new
practice for many years. In common all the above groups antimicrobial peptides make natural
mentioned system of medicine are directly antibiotics the basic elements of novel generation
dependent upon resources such as plants with the drugs for the treatment of bacterial and fungal
advances in experimental methods in infection. Marine algae have become recognized as
phytochemistry and pharmacology, several potential source of antibiotic substances (Zheng Yi
medicinal plants were screened for active principles et al., 2001). The antibacterial activity of six marine
and biological activites (Prashant Kumar et al., algae belonging Rhodophyceae and phaeophyceae
2006). Marine environment is a rich source of were studied against pathogenic microbes
biological and chemical diversity. The diversity has (Staphylococcus aureus, E.coli, Micrococcus,
been a unique source of chemical compounds of Enterobacter aerogens, Enterococcus faecalis were
potential for pharmaceuticals, cosmetics, dietary studied (Taskin et al., 2007). Mosquito transmits
supplements and agrochemicals (Chau Van Minh et serious human diseases like Malaria, Filariasis,
al., 2005). In recent years, a significant number of Japanese encephalitis, dengue, hemorrhagic fever
novel metabolites with potent pharmacological and yellow fever causing millions of death every
properties have been discovered from the marine year. Extensive use of chemical insecticides for
organisms. control of vector borne diseases has created
(Narisnh et al., 2005).Marine algae were problems related to physiological resistance to
also reported to have some antioxidant properties vectors, adverse environmental effects, high
(Faten et al., 2009) Commercially available operational cost and community acceptance,
varieties of marine macro algae are commonly numerous plant products have been reported either
referred to as seaweeds. Seaweeds have some of as insecticides for killing larva or adult mosquit oes
valuable medicinal value compounds such as or as repellent for mosquito biting and are one the
antibiotics, laxatives, anticoagulants, antiulcer best alternatives for mosquito control (Rajkumar et
products and suspending agents in radiological al., 2009). The revolution therapy of infection
preparation (Rajasulochana et al., 2009).Fresh and disease by the use of antibacterial drugs has certain
dry seaweeds are extensively consumed by people limitation due to changing pattern of resistance in
especially living in coastal areas. Seaweeds are pathogens and side effects they produced. These
classified as rhodophyta (red algae) or phaeophyta limitation demands for search of new antimicrobial
(brown algae) or chlorophyta (green algae) compounds for development of drugs. Seaweeds are
depending on their nutrient and chemical used alternative and traditional remedies in many
composition (Cox et al ., 2010).As a consequence parts of the world.
of an increasing demand in screening for new The production on inhibitor substance by
therapeutic drugs from natural products, there is a seaweeds has antibacterial actions and some of their
greater interest towards marine organisms. Several substances have potential use in mosquito control
marine organisms produce bioactive metabolites in (Nagi et al., 2010).Use of synthetic insecticides to
response to ecological pressure such as competition control vector mosquitoes has caused physiological
for space maintenance of unfolded surface resistance and adverse environmental effects in
deterrence of predation and the ability to addition to high operational cost (Virendra et al.,
successfully reproduce (Fangming Kong et al., 2009). The treatment of this disease becoming more
1994). The antibacterial agent found in the algae difficult since Plasmodium falciparum parasite has
include terpenoid, phlorotannins, acryl acid, developed resistance to wide range of anti malarial
phenolic compounds, steroid, halogenated ketone drugs (Anne Platt Mc Ginn et al., 2002). Secondary
and alkaline, cylic polysulphides and fatty acid. In a metabolites of many marine algae species isolated
number of marine algae antimicrobial activites are from Canaria Island were shown to have a wide
attributed to the presence of acryl acid (James et al., antimicrobial activity (Antonio Gonzalez et al.,
1975). Seaweeds provide a rich source of 2001). Thus the screening of marine organism for a
386 Journal of Research in Biology (2011) 5: 385-392
Anandhan et al.,2011

variety of biological activities with in aim approximately 5mm in diameter and allowed at
identifying novel with interesting and potentially room temperature for few minutes. After that the
therapeutic activities continues till this date. disc were placed on the Muller Hinton agar plates
(Sreenivasan Sasidharan et al., 2010). by using sterile forceps. All the plates were
incubated at 37ºC for 24 hours. After incubation of
MATERIALS AND METHODS 24 hours at 37°C a clean zone around the disc was
Collection of algae samples evidence of antimicrobial activity (Rajasulochana et
Marine algae samples were collected from al., 2009)
the coastal region of Rameshwaram. Algae were Partial purification of selected algae extract
washed with sea water to remove extraneous Based on the antibacterial activity of potential algae
materials and brought to the laboratory in plastic extracts further investigations like partial
bag containing sea water to prevent evaporation. purification by thin layer chromatography were
Sample preparation done.
After collection of sample, it was brought to Partial purification of crude extract by thin
the laboratory. Algal samples were washed in layer chromatography (TLC)
running tap water to remove any associated debris Analytical TLC
and then with the distilled water. After washing, the The crude extract was purified by using thin layer
samples were dried in a blotting paper for two chromatography. For determining the best solvent
weeks. After drying, the samples was grinded in to system for good separation of crude compound,
powder form, which was then stored in 4ºC for solvents such as methanol, chloroform, n-hexane,
further studies. (Rajasulochana et al., 2009). diethyl ether, n-butanol, ethyl acetate and acetic
Preparation of different solvent extracts acid were used in the following ratio.
One gram of each algal sample was B u t a n o l : A c e t i c a c i d : w a t e r
extracted with different solvents systems 10ml of [30:45:25,45:30:25,50:35:15],hexane:ethylacetate
methanol, Hexane, and ethyl acetate in a beaker for [90:10,80:20,70:30,60:40,50:5
24 hours at room temperature. Then the solvent 0],hexane:diethylether:aceticacid
portion was centrifuge at 5000rpm for 10minutes. [80:10:10,70:20:10,60:30:10], hexane:methanol
The supernatant was collected from the centrifuge [70:30,60:40,50:50], meth anol:ethylacetate:hexane
tube and the solvent were evaporated. Finally crude [70:20:10,60:30:10,75:20:5].The crude extract was
extract was obtained. The extracts were collected in dissolved in 200μl of methanol. With the help of
plastic vials and stored in the refrigerator for further capillary tube the sample was spotted on the silica
studies. (Aseer et al., 2009) gel coated slide and placed in the developing
Disc preparation chamber which contain solvent mobile phase,
The solvent extracts were used for disc covered with the watch glass in order to prevent the
diffusion assay to test for antibacterial activity. The evaporation of solvents. The solvent was allowed to
discs were prepared by using Whatman filter paper run till it reaches about half a cm below the top of
approximately 5mm in diameter and then it was the plate. After running the slide was kept in room
soaked in extracts and then dried. Then the discs temperature for the complete drying of the plate.
were stored and used for further works. Then the slide was kept closed in iodine chamber to
Antibacterial assay visualize the separated compound as clear spots
The stranded strains of Bacillus subtilis, (Jebakumar Solomon et al., 2008). The RF value
Staphylococcus aureus, Klebsiella pneumoniae, were determine by RF=Movement of the solute
Pseudomonas aeruginosa and E.coli cultures were from the origin /movement of solvent from the
collected from the Department of Microbiology, Sri origin.
Sankara Arts and Science College, Enathur, Preparative TLC
Kanchipuram. The antibacterial activity of algae Preparative TLC was performed to get
extracts were studied by disc diffusion methods by partially purified compound.TLC plate was
using Muller Hinton Agar (MHA). 18hours old prepared by spreading the slurry of silica gel evenly
broth culture was prepared and inoculated on on the plate. The plate was activated at 100ºC for
Muller Hinton agar plates by using sterile cotton 15 minutes. Crude extract was applied on the plate
swab. After the swabbing, 20μl of crude extract disc as a single line and the chromatogram was
were added in to sterile filter paper disc performed with the solvent system Hexane:

Journal of Research in Biology (2011) 5: 385-392 387
Anandhan et al.,2011

ethylacetate solvent system (50:50).After the present study, among the five different algae
separation, the active spot band was scrapped, with varying extracts tested by disc diffusion
mixed with methanol and centrifuged at 3000rpm method, the methanolic extract of all algae have
for 15 minutes. Supernatant was collected in a pre satisfactory inhibition properties. Stranded strain of
weighed vial and kept for evaporation. The partially Bacillus subtilis had shown maximum sensitivity to
purified compound obtained from preparative TLC the algae Gracilaria crassa and Hypnea valentia
was tested for antibacterial activity against stranded with a maximum zone of inhibition of about
organisms by disc diffusion method. 14mm.Similarly, in a study conducted on
Antimicrobial Activity of TLC fractions a nti mi crobial acti vitie s of mic roal gae
After the swabbing the stranded organism, 20μl of Trichodesmium erythraeum, the hexane extracts
TLC extracts disc were added on to the sterile filter have shown good inhibitory activities (Kasinathan
paper disc approximately 5mm in diameter and thillairajasekar et al., 2009). The crude methanolic
allowed at room temperature for few minutes. After extract of different algae like Asparagopsis,
that the disc were placed on the Muller Hinton agar Laurencia, and Hypnea showed significant
plates by using sterile forceps. All the plates were antimicrobial activity(Nagi et al., 2010). It was
incubated at 37ºC for 24 hours. After incubation of observed that kappaphycus, a red sea weed algae
24 hours at 37°C a clean zone around the disc was showed maximum activity against Pseudomonas
evidence of antimicrobial activity. flouresences, Staphylococcus aureus and less
Phytochemical test inhibition on Vibrio cholera and Proteus mirabilis
The partially purified compound obtained (Rajasulochana et al., 2009). In a study it was
from preparative TLC was subjected to qualitative observed that the crude butanol extract of Isochrysis
phytochemical analysis for the identification of galbana, marine algae had shown a satisfactory
compound present in the purified extract. All the inhibitory effect against the selected bacterial
tests were carried out using standard methods pathogens (Srinivasakumar et al., 2009).Among the
(Aliyu et al., 2008). sea weeds highest inhibitory activities was
Larvicidal bioassay documented among the members of red, green and
Mosquito larva was collected from ditch brown algae against both the gram positive and
area near Enathur, in Kanchipuram district and it gram negative (Vallinayagam et al., 2009).In this
was examined by experts for the confirmation of study, the second highest sensitivity is shown in
mosquito larva of Aedes sp. The algal extracts of Enteromorpha intestinalis against Pseudomonas of
Gracilaria crassa and Hypnea valentia were an inhibition zone about 13mm as shown is table 1.
volumetrically diluted to obtain the test Based on this result, Gracilaria crassa and Hypnea
concentrations of 25, 50 and 75 mg/10ml. Control valentia were selected for further studies.
was set up by 1 ml of methanol in 10 ml of water. Partial purification of antibacterial compound
Twenty five late third instar larvae were introduced by TLC
to each of the test concentration as well as control. Among the various solvent systems analyzed
The larval mortality was recorded after 24 h of in TLC, three well separated spots were observed in
exposure, during which no food was given to the Hexane: ethylacetate solvent system for Gracilaria
larvae. The lethal concentrations (LC50) were crassa and two well separated spots were seen for
calculated by probit analysis (Rajkumar et al., Hypnea valentia. RF values for Gracilaria crassa
2009). was calculated as 0.64, 0.78, and 0.84 and for
Hypnea valentia it was calculated as 0.62 and 0.67
RESULTS AND DISCUSSION respectively. The antimicrobial effect of bioactive
Screening of algae extract for antibacterial compound present in Dictyota acutiloba has been
activity purified by TLC to examine the compound for
The macroalgae from the Moroccan coast further analysis.
are potential sources of bioactive compounds for Antimicrobial activity of partially purified TLC
investigating natural antibiotics(Chiheb et al., fractions
2009).Different extracts of the brown algae Among the three different spots that were
Sargassum cinereum have different anti bioactive observed in Gracilaria crassa the third band named
properties such as antibacterial and antifungal G3 showed maximum inhibitory of 16mm while the
activity (Divya et al., 2011). Antibacterial activity Hypnea valentia showed inhibitory effects of 15
of selected algae extracts were given in table 1.In and 18mm.the results were presented in table-2.
388 Journal of Research in Biology (2011) 5: 385-392
Anandhan et al.,2011

Table .1.Antimicrobial activity of crude algae extract against selected pathogens

Table.2.Effect of partially purified compound against
Phytochemical analysis of partially purified
Bacillus subtilis antibacterial compound
Results of phytochemical analysis of
S. No Spots Zone on inhibition partially purifies antibacterial compound were
(mm) in diameter given in table- 3.Based on the results it was found
1 G1 - that the partially purified compound consists of
saponins. Similarly in a study, flavonoids and
2 G2 - tannins extracted from the Acacia albida and
3 G3 16 Pavetta crassipes which possess the antimicrobial
activity against MRSA (Aliyu et al.,
4 G4 15 2008).Phytochemical screening of C.decorticatum
5 G5 18 in methanolic and petroleum ether extracts revealed
the presence of saponins, phytosterols and

Journal of Research in Biology (2011) 5: 385-392 389
Anandhan et al.,2011

Table.3. Phytochemical properties of partially purified active compound

glycosides which showed a well documented REFERENCES
antimicrobial activity (Anbu Jeba Sunilson et al., Abdul Rahuman A, Geetha Gopalakrishnan, P.
2009). The phytochemical analysis of Gracilaria Venkatesan and Kannappan Geetha. 2008.
fergusonii revealed the presence of coumerins, Isolation and identification of mosquito larvicidal
phenols, quinines and steroids, which ultimately compound from Abutilon indicum (Linn.) Sweet.
may inhibit the microorganisms (Renuka Bai., Parasitol Res., 102:981-988.
2010).
Larvicidal bioassay Abdul Rahuman A., P.Venkatesan and Geetha
Insecticides of botanical origin may serve as Gopalakrishnan. 2008. Mosquito larvicidal
suitable alternative biocontrol techniques in the activity of oleic and linoleic acids isolated from
future (Kamaraj et al., 2009). An insecticide Citrullus colocynthis (Linn.) Schrad. Parasitol Res.,
containing azadirachtin, a neem tree (Azadirachta 103:1383-1390
indica) extract, was tested against mosquito larvae
in the Islamic Republic of Iran under laboratory and Aliyu AB, Musa AM, Abdullahi MS and
field conditions. (Vatandoost et al., 2004). Certain Oyewale AO. 2008. Phytochemical and
species of green algae in the order Chlorococcales antibacterial properties of Ludwigia suffruticosa
kill larvae primarily because they are indigestible (Willd.) Oliv. ex. O. Ktze (Onagraceae). Int. Jor. P.
(Gerald., 2007). The extracts of J. curcas and E. App. Scs., 2(4):1-5.
tirucalli were highly effective against the larvae of
A. aegypti (LC= 8.79 and 4.25 ppm) and against C. Anbu Jeba Sunilson J, Suraj R, Anandaraja
quinquefasciatus (LC =11.34 and5.52 ppm). The gopal K, Rejitha G, Vignesh M and Promwichit
LC values were 35.39, 256.77, 384.19, 703.76, and P. 2009. Preliminary phytochemicals analysis,
13.14 ppm against A. aegypti (Abdul Rahuman et elemental determination and antibacterial screening
al., 2008). In this study also, Gracilaria crassa and of Codium decorticatum- A marine green algae.
Hypnea valentia in methanolic extract have shown International journal of biochemistry 3(2):84-89.
good larvicidal activity with a LC50 of about 52.2
and 53.4 respectively. Anne Platt McGinn. 2002. Mosquitoes and DDT,
Thus, the present study discussed about the the toxic war against a global disease. World watch
marine algae as potential compound reservoirs Institute. May.
which not only act as antibacterial agents but also
used to control the mosquito larval population Antonio Gonzalez del Val, Gon zalo Platas,
which is a biggest growing threat. Angela Basilio, Angeles cabello, Julian
Gorrochatgui, Inmaculada suay, Fracisca
Vincente, Eduardo Portillo, Miguel Jimenez del
390 Journal of Research in Biology (2011) 5: 385-392
Anandhan et al.,2011

Rio,Guillermo Garcia Reina, Fernando Pelaez. Association Bulletin No. 7.
2001. Screening of antimicrobial activities in red,
green and brown macroalgae from Gran canaria James J. Sims, Mark S. Donell, John V. Leary
(Canary Islands, Spain).Int. Microbiol., 4:35-40. and George H. Lacy. 1975. Antimicrobial agents
from marine algae. Antimicrobial agents and
Aseer Manilal, Sugathan Sujith, George Seghal chemotherapy 320-32.
Kiran, Joseph Selvin, Chippu Shakir,
R a ma kr i s h na n G a nd hi ma t hi , a n d Jebakumar Solomon RD and Satheeja Santhi V.
Mamkoottathil Velayudhan Nataraja Panikkar. 2008. Purification of bioactive natural product
2009. Biopotentials of seaweeds collected from against human microbial pathogens from marine
southwest coast of India. Journal of Marine Science sea weed Dictyota acutiloba J. Ag. World J
and technology 17(1):67-73. Microbiol Biotechnol., 24:1747-1752.

Chau Van Minh, Phan Van Kiem and Ngyen Hai Kamaraj C, Bagavan A, Abdul Rahuman A,
Dang. 2005. Marine natural products and their Abduz Zahir A, Elango G and Pandiyan G.
potential application in the future.AJSTD. 22(4):297 2009. Larvicidal potential of medicinal plant
-311. extracts against Anopheles subpictus Grassi and
Culex tritaeniorhynchus Giles (Diptera: Culicidae).
Chiheb Ibtissam, Riadi Hassane, Martinez- Parasitol Res., 104:1163-1171.
Lopez Jose, Dominguez Seglar Jose, Francisco,
Gomez Vidal Jose Antonio, Bouziane Hassan Kasinathan thillairajasekar, Veeramuthu
and Kadiri Mohamed. 2009. Screening of Duraipandiyan, Pac hiappan Pe rumal,
antibacterial activity in marine green and brown Savaimuthu Ignacimuthu. 2009. Antimicrobial
macroalgae from the coast of Morocco. African activity of Trichodesmium erythraeum (microalga)
Journal of Biotechnology 8(7):1258-1262. from South east coast of Tamilnadu, India. IJIB. 10
Apr.
Cox S, Abu- Ghannam N and Gupta S. 2010. An
assessment of antioxidant and antimicrobial activity Nagi A. AL Haj, Nurmas I. Mashan, Mariana N.
of six species of edible Irish seaweeds. Shamasudin, Habsah Mohamad, Charles S.
International Food Research Journal 17:205-220. Vairappan and Zamberia Sekawi. 2010.
Antibacterial activity of marine source extracts
Divya CV, Devika V, Asha KRT and Bharat G. against multidrug resistance organisms. American
2011. Antimicrobial Screening of the Brown Algae journal of pharmacology and toxicology 5(2):95-
Sargassum cinereuma. Journal of Pharmacy 102.
Research 4(2):420-421.
Narisnh L Thakur, Archana N Thakur and
Fangming Kong, Raymond J. Andersen and Werner EG. Muller. 2005. Marine natural products
Theresa M. Allen. 1994. Ingamines A and B, new on drug discovery. Natural product radiance 4
cytotoxic alkaloids from the marine sponge (6):Nov-Dec.
Xestospongia ingens. The International Journal for
the Rapid Publication of Critical 50(21):6137- Prashantkumar P, Angadi SB and Vidyasagar
6144. GM. 2006. Antimicrobial activity of blue-green and
green algae. Indian journal of pharmaceutical
Faten M. Abou Elalla and Emad, Shalaby A. sciences 68(5):647-648.
2009. Antioxidant activity of extract and semi-
purified fractions of marine red macroalgae, Rajas uloc ha na P, D ha mot ha ra n R,
Gracilaria Verrucosa. Australian Journal of Basic Krishnamoorthy P, Murugesan S. 2009.
and Applied Sciences 3(4):3179-3185. Antibacterial activity of the extracts of marine red
and brown algae. Marsland Press. Journal of
Gerald G. Marten. 2007. Larvicidal Algae. American Science 5(3):20-25.
Reprinted from T.G. Floore (ed.), Biorational
Control of Mosquitoes, American Mosquito Control

Journal of Research in Biology (2011) 5: 385-392 391
Anandhan et al.,2011

Rajkumar S and A. Jebanesan. 2009. Larvicidal Vallinayagam KR, Arumugam, Ragupathi R,
and oviposition activity of Cassia obtusifolia Linn Raja Kannan G, T hiruma ra n and
(Family: Leguminosae) leaf extract against malarial Anantharaman P. 2009. Antibacterial Activity of
vector, Anopheles stephensi Liston (Diptera: Some Selected Seaweeds from Pudumadam Coastal
Culicidae). Parasitol Res., 104:337-340. Regions, Global Journal of Pharmacology 3(1):50-
52.
Renuka Bai N. 2010. Evaluation of Gracilaria
fergusonii for phytochemical analysis and Vatandoost H and Vaziri VM. 2004. Larvicidal
antibacterial activity. Plant archives 10(2):711- activity of a neem tree extract (Neemarin) against
713. mosquito larvae in the Islamic Republic of Iran.
East Mediterr Health J. Jul-Sep; 10(4-5):573-81.
Sreenivasan Sasidharan, Ibrahim Darah and
Mohd Kassim Mohd Jain Noordin. 2010. In Virendra K Dua, Akhilesh C Pandey, Kamaraju
vitroantimicrobial activity against Pseudomonas Raghavendra, Ashish Gupta, Trilochan Sharma
aeruginosa and acute oral toxicity of marine algae and Aditya P Dash. 2009. Larvicidal activity of
Gracilaria changii. New Biotechnology 27(4). neem oil (Azadirachta indica) formulation against
mosquitoes. Malaria Journal. Jul-Sep; 10(4-5):573-
Srinivasakumar KP and Rajashekhar M. 2009. 81.
In vitro studies on bactericidal activity and
sensitivity pattern of isolated marine microalgae Zheng Yi, Chen Yin-shan, Lu Hai-Sheng. 2001.
against selective human bacterial pathogens. Indian Screening for antibacterial and antifungal activities
Journal of Science and Technology 2(8). in some marine algae from the Fujian coast of
China with three different solvents. Chinese journal
Taskin E, Ozturk M, Taskin E and Kurt O. of Oceanology and Limnology 19(4):327-331.
2007. Antibacterial activities of some marine algae
from the Aegean Sea (Turkey).African Journal of
Biotechnology 6(24):2746-2751.

392 Journal of Research in Biology (2011) 5: 385-392
Journal of Research in Biology
An International Online Open Access
Original Research Paper
Publication group

Foliar spray of Earthworm urine to improve Amaranthus growth
performance and yield
Journal of Research in Biology

Authors: ABSTRACT:
1
Owa Stephen
Olugbemiga,
2
Ojediran John O and
3
Fadeyi Oluwatosin.
The effects of foliar spray of earthworm urine on plant growth parameters
and greengrocery values were studied on the leaf vegetable Amaranthus. The
Institution:
1. Department of Biological application caused 28% increase in plant height, 166% increase in plant girth taken at
Sciences, Landmark the ground level, 20% increase in the number of leaves per plant, 17% increase in
University, Omu-Aran, length of leaves, 15% in leaf breadth and about 49% in leaf area. It was also observed
Kwara State, Nigeria, that the stomatal diameters increased by about 19%. These results of foliar
2. Department of application indicate that earthworm urine contains some plant growth hormone that
Agricultural Engineering, can be gainfully used to increase not only the yield of the vegetable crop, but also
Landmark University, increase its market visual appeal with respect to the length of an Amaranthus stick,
Omu-Aran, Kwara State, the leaf-density per stick and the length and area of a leaf. On a commercial scale
Nigeria, earthworm urine application can be mechanized using a sprinkler.
3. Formerly, Department
of Plant Science and Applied
Zoology, Olabisi Onabanjo
University, Ogun State,
Nigeria.

Corresponding author: Keywords:
Owa Stephen Earthworm urine, horticulture, greengrocery, plant growth hormone,
Olugbemiga sprinkler application.

Web Address: Article Citation:
http://jresearchbiology.com/ Owa Stephen Olugbemiga, Ojediran John O and Fadeyi Oluwatosin.
Documents/RA0046.pdf. Foliar spray of Earthworm urine to improve Amaranthus growth performance and
yield.
Journal of research in Biology (2011) 5: 393-398

Dates:
Received: 08 Jun 2011 /Accepted: 21 Jun 2011 /Published: 28 Sep 2011

© Ficus Publishers.
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Olugbemiga et al.,2011

INTRODUCTION: protection from farming activity damages can lead
The importance of earthworms to the general to reduction of the quantities of inorganic fertilizers
health of crops and soil has been well documented. needed.
They affect the soil in many ways. Bayon and Binet Studying lowland rice agroecosystems Owa
2001 reported that earthworm surface casts affect et al. (2003b) showed that rice stands that have
soil erosion by runoff water and phosphorus earthworm casts associated with their bases grew
transfer in a temperate maize crop. Their casts more tillers, grew taller, had more leaves, more
improve the stabilization of organic matter in fine grains and bigger grain size, than others stands
soil fractions (McInerney, Little and Bolger 2001). lacking earthworm casts associated with their bases.
Their surface movement improves soil porosity and Ojediran 1990 reported that increasing irrigation
aeration (Mather and Christensen 1988). According frequency induced an increase in the shoot length
to Sharpley, Syers and Springett (1979) their and root growth of wheat. Both root mass and
surface casting improves transport of phosphorus length were reported to decrease significantly with
and nitrogen in surface runoff from pasture. water stress (Ojediran 1990, Snyman 2004).
One mechanism of nitrogen efficiency in Two major challenges confront expansion of
agricultural soils is due to release of nitrogen from earthworm urine irrigated farming: one is
herbage residues by earthworms (Ruz Jerez, Ball availability of water resource. Irrigation of
and Tillman 1988). According to Mackay and vegetables requires significant quantities of water
Kladivko (1985) earthworms improve the rate of of suitable quality. The second is water lifting and
breakdown of soybean and maize residues in the distribution device. One traditional method is the
soil. By competitive feeding, earthworms depress rope and bucket. An alternative traditional method
soil nematode populations (Yeates 1981). is the treadle pump which originated in Bangladesh
Earthworm burrowing activity reduces soil or motorized pumps.
compaction (Bostrom 1986). Soil damage due to The present study is aimed at checking if
opencast mining is sometimes rehabilitated by foliar application of earthworm urine can produce
earthworm transplant. (Stewart, Scullion, Salih and enhancement in the growth parameters and market
Al Bakri 1988). grocery values of the leaf vegetable crop
Owa, Moreyibi and Dedeke (2002) Amaranthus. An argument is also provided that the
demonstrated that wormcasting initiates thermal foliar application can be mechanized by use of a
and nutrient convection current in the soil. By that sprinkler. Such a mechanization can significantly
earthworms raise soil temperature and thereby improve the production and availability of this very
improve the physicochemical activities of crops important leaf vegetable.
(Owa et al 2004a).
When located at the base of a plant crop MATERIALS AND METHODS
their effect could amount to creating an isolating Earthworm urine was prepared by placing 1
environment (Owa et al. 2004b). Another positive kg live earthworms in 1 L earthworm saline for 1
effect on plant is that earthworms participate in hr. The decant was funnel filtered under suction.
dispersal of seeds (Decaens et al. 2003). According The filtrate was labeled as earthworm urine.
to Ayanlaja et al. (2001) leachate from earthworm Ninety (90) plastic plant pots were loaded
cast can break seed dormancy and promote radicle each with 1 kg top soil, set in an open space; and
growth. planted with viable seeds of Amaranthus. After
Earthworm affect soil microbial population. germination, the plants were thinned down to one
Their casts and compost improve soil microbial per pot. 30 pots randomly selected were reserved
activities and plant nutrient availability (Brown and properly labeled for earthworm urine treatment,
1995; Chaoni et al. 2003). Microbial biomass and another 30 for earthworm saline treatment and the
activities increase after the soil microbes have last 30 for water treatment.
passed through the gut of earthworms (Daniel and Each pot was watered with 500 ml water
Anderson 1992). between 6.30 am and 7 pm daily. Using a handheld
Studying earthworm populations and plastic sprayer, 30 cm3 of earthworm urine,
distribution in Nigerian soils Owa et al. (2003) earthworm saline and distilled water were daily
showed that earthworms make significant sprayed onto the leaves of each plant in the
contribution to the soil nitrogen, and that respective pots. The application was every morning.
application of earthworms in a farm, or their
394 Journal of Research in Biology (2011) 5: 393-398
Olugbemiga et al.,2011

On a regular weekly basis, the height of the
DISCUSSION
plant in each pot was measured from the ground Hitherto, the effects of earthworms on plants
level to the apical bud, using a meter rule. The have been viewed via their impacts on the soil. The
widest stem girth was measured at the ground level,present work shows that earthworm urine can affect
using a thread and plastic ruler. The lengths and plants more directly.
widest diameters of the lowest (and largest) three From agronomic considerations, the gain in
leaves on a plant were measured using a plastic height or length of a crop is a desirable effect
ruler. The total number of leaves on each plant wassimilar to the effect of organic and inorganic
recorded. At the end of the fifth week an additional
fertilizers. For a leaf vegetable, it shortens the time
factor, stomatal diameter, was measured and the taken for the crop to reach table size. It means
experiment was terminated. To measure the savings in time and cost of operations before the
stomatal diameter, nail vanish was applied to the crop is sold for a monetary gain. From the buyers’
lowest green leaf on a plant, in order to take an angle, the lengthening of the vegetable stick
impression of the stomata pores. The vanish was produces a visual advantage. In local
allowed to dry, carefully peeled off, placed on a greengroceries, buyers examine the length of the
labeled microslide and taken to the laboratory. sticks of the vegetable, and prefer those that are
Using a stage micrometer and eyepiece graticule, long.
the stomatal diameters were measured. From research angle, this result is similar to
The resulting data were analyzed using the the earlier results of Owa et al. (2002), Owa et al.
Statistical Package for Social Sciences (SPSS) (2003), Owa et al. (2004a, b) And it indicates that
Version 15. Analysis of variance, LSD and Duncan beyond just providing nitrogen and ammonia to
multiple range tests were performed on the data, inplants through the soil, earthworm urine provides
order to test the significance of the differences at p
some growth hormone.
= 0.05. That plant girth is greatly increased by
earthworm urine is beneficial in many ways. In the
RESULTS current drive to minimize artherogenesis and other
Compared to the controls, application of diet-related diseases, there is a growing emphasis
earthworm urine produces about 28% increase in on vegetarianism, especially the use of intake fibers
plant height, 20% in number of leaves per plant, to reduce dietary cholesterol absorption at the villi
166% in plant girth (Table 1), 17% in leaf length level. Among the local community, not only the
and about 40% in leaf area (Table 2). It also leaves of Amaranthus are shredded for cooking, but
produces an increase of about 19% in stomatal pore also the soft succulent stem. Thus, the application
diameter (Table 3). of earthworm urine could be indirectly contributing
Table 1: Effects of foliar application of earthworm urine on the height, number of leaves and stem girth of
Amaranthus
% Gain in
parameter
N Mean Std. Dev Minimum Maximum
relative to
control
Height of plant (cm) Earthworm urine 30 40.302a 6.5102 28.4 56.2 28.0502
Saline water 30 33.411b 5.7132 23.4 48.6 6.157594
Water (control) 30 31.473b 4.9797 21.8 41.1
Average 90 35.062 6.8585 21.8 56.2
No of leaves Earthworm urine 30 12.10a 2.845 7 16 20.19868
Saline water 30 10.70b 2.054 1 13 6.291391
Water (control) 30 10.07b 1.780 4 12
Average 90 10.96 2.403 1 16
Plant girth at ground Earthworm urine 30 3.627a .5836 2.5 5.1 166.4055
level (cm)
Saline water 30 1.660b .3001 1.3 2.2 21.93928
Water (control) 30 1.361c .0687 1.3 1.5
Average 90 2.216 1.0785 1.3 5.1
Note: Values labeled with same letter are not significant different, others with different letters are significantly different.

Journal of Research in Biology (2011) 5: 393-398 395
Olugbemiga et al.,2011

Table 2: Effects of foliar application of earthworm urine on leaf length, leaf width and leaf area of Amaranthus
% Gain
Std. Std. relative
N Mean Minimum Maximum
Deviation Error to
control
Leaf length
Earthworm urine 30 10.1150a 1.47177 .26871 7.08 14.08 17.11694
(cm)
Saline 30 9.5917a 1.53517 .28028 6.38 13.28 11.05751
Water 30 8.6367b 1.27177 .23219 6.20 11.98
Average 90 9.4478 1.54258 .16260 6.20 14.08
Leaf breath
Earthworm urine 30 5.9350a 3.04315 .55560 2.95 21.35 15.09373
(cm)
Saline 30 5.0192a .85872 .15678 3.30 6.78 -2.66645
Water 30 5.1567a 2.21831 .40501 3.50 16.33
Average 90 5.3703 2.24181 .23631 2.95 21.35
Leaf Area
Earthworm urine 30 20.4851a 12.08548 2.20650 6.95 78.03 39.83027
(cm**2)
Saline 30 16.3841a 5.24115 .95690 8.09 29.95 11.83741
Water 30 14.6499b 5.23739 .95621 8.25 37.37
Average 90 17.1730 8.45779 .89153 6.95 78.03
Note: Values labeled with same letter are not significant different, others with different letters are significantly
different.

significantly to higher fiber intake and thus But in addition, there is an esthetic premium
contributing to the battle against artherogenic attached to the length of a vegetable leaf. There is a
disease. Similarly amaranth oil is used in the social and psychological preference for Amaranthus
treatment of ischemic and other artherogenic sticks with longer leaves than for those with shorter
diseases (Czerwinski et al., 2004; Gonor et al. leaves, even when there is no known data that the
2006; Martirosyan et al. 2007). one is richer in nutrients than the other. Thus,
Earthworm urine increases the number of application of earthworm urine could significantly
leaves on a plant. To the local consumers the improve the premium value placed on Amaranthus
density of leaves on an Amaranthus stick is a major in a greengrocery.
buyer’s consideration. From dietary point of view The widening of stomatal diameter by
many of the nutrients that humans benefit from earthworm urine may be a factor to the higher
Amaranthus are derived from the leaves. These growth performance and productivity of the plants.
include, among others, vegetable oils, protein, It increases the rate of in- and out-diffusion
carbohydrates, macronutrients and micronutrients. processes, thus increasing the rate of foliar and
Vegetable leaf is one of the most important sources photosynthetic metabolism.
of dietary fiber. Thus, application of earthworm Beyond Amaranthus, the implication of the
urine can improve the nutritional value of effect of earthworm urine on plant growth and yield
Amaranthus for human consumption. performances is that the food basket can be

Table 3. Effects of foliar application of earthworm urine on leaf stomatal diameter of Amaranthus
Diameter of stomata (um)
% Gain
Std.
N Mean Std. Error Minimum Maximum relative to
Deviation
control
Earthworm urine 100 .006500a 0.00188 0.000188 0.0025 0.01 19.26606
Saline water 104 .005962b 0.001608 0.000158 0.0025 0.01 9.386027
Ordinary water 100 .005450c 0.00125 0.000125 0.0025 0.01
Average 304 0.00597 0.001651 9.47E-05 0.0025 0.01
Note: Values labeled with same letter are not significant different, others with different letters are significantly
different.

396 Journal of Research in Biology (2011) 5: 393-398
Olugbemiga et al.,2011

significantly improved by appropriate Lumbricus rubellus Hoffmeister. Soil Biology and
mechanization. Biochemistry 24:465-470.
A major constraint to increased irrigated
crop production is low water lifting and distribution Decaens T, Mariani L, Betancourt N and
capacity. Any pump supplying significantly greater Jimenez JJ. 2003. Seed Dispersion by Surface
flow rates relative to traditional rope and bucket Casting Activities of Earthworms in Colombian
system will increase irrigated surface areas and Grasslands. Acta Oecologica. International Journal
reduce irrigation labour time, resulting in increased of Ecology 24:175-185.
production (Hyman et al. 1995). Generally, a
proper irrigation interval increases the plant water Gonor KV, Pogozheva AV, Derbeneva SA,
stress tolerance and gives the vegetable leaves a Mal'tsev GIu, Trushina EN, Mustafina OK.
very appealing green colour look. 2006. The influence of a diet with including
Given the availability of land during the dry amaranth oil on antioxidant and immune status in
season and improved water lifting and distribution patients with ischemic heart disease and
technology, the use of earthworm urine will lead to hyperlipoproteidemia (in Russian). Vopr Pitan 75
larger irrigated surface areas and consequently, (6):30-3.
larger vegetable yield.
Hyman E, Lawrence E and Singh J. 1995. The
REFERENCES ATI/USAID Market Gardeners Project in Senegal.
Ayanlaja SA, Owa SO, Adigun MO, Senjobi BA Washington, D.C. Appropriate Technology
and Olaleye AO. 2001. Leachate from Earthworm International.
Castings Break Seed Dormancy and Preferentially
Promote Radicle Growth in Jute. HortScience 6(1). Mackay AD and Kladivko EJ. 1985. Earthworms
and rate of breakdown of soybean and maize
Bayon RCL and Binet F. 2001. Earthworm residues in soil. Soil Biol. Biochem. 17(6):851-857.
Surface Casts Affect Soil Erosion by Runoff Water
and Phosphorus Transfer in a Temperate Maize Martirosyan DM, Miroshnichenko LA,
Crop. Pedobiologia 45:430-442. Kulakova SN, Pogojeva AV, Zoloedov VI. 2007.
Amaranth oil application for coronary heart disease
Bostrom U. 1986. The effect of soil compaction on and hypertension. Lipids Health Dis 6:1.
earthworms (Lumbricidae) in a heavy clay soil.
Swedish J. Agric. Res. 16:137-141. Mather JG and Christensen O. 1988. Surface
movements of earthworms in agricultural land
Brown GG 1995. How do earthworms affect Pedobiologia 32:399-405.
microflora and faunal community diversity? Plant
and Soil 170:209-231. McInerney M, Little DJ and Bolger T. 2001.
Effect of Earthworm Cast Formation on the
Chaoni HI, Zibilske LM, & Ohno T 2003. Effects Stabilization of Organic Matter in Fine Soil
of Earthworm Casts and Compost on Soil Microbial Fractions. Eur. J. Soil. Biol. 37:251-254.
Activity and Plant Nutrient Availability. Soil
Biology & Biochemistry 35:295-302. Ojediran JO. 1990. Effect of Irrigation Frequency
on root growth, shoot length and crop-water use of
Czerwinski J, Bartnikowska E, Leontowicz H. wheat. An unpublished B.Sc project, Dept. of
2004. Oat (Avena sativa L.) and amaranth Agric. Eng., Univ. of Maiduguri.
(Amaranthus hypochondriacus) meals positively
affect plasma lipid profile in rats fed cholesterol- Owa SO, Moreyibi OH, Dedeke GA, Olojo FO
containing diets. J. Nutr. Biochem. 15(10):622-9. and Fashunwon OO. 2004a. Earthworm-created
Micro-environments Around Roots of Lowland
Daniel O. & Anderson JM 1992. Microbial Rice: Implication for Growth Performance. J. Sci.
biomass and activity in contrasting soil m a t e r i a l s Engr. Tech. 11(1):5261-5270.
after passage through the gut of the earthworm

Journal of Research in Biology (2011) 5: 393-398 397
Olugbemiga et al.,2011

Owa SO, Moreyibi OH, Morafa SOA and Smith (Eds.). Nitrogen Efficiency in Agricultural
Dedeke GA. 2004. Contributions of earthworms to Soils. (Publisher unknown). 355-370.
soil temperature and its physicochemical
implications on crops. J. Sci., Engr. and Tech. 11 Sharpley AN, Syers JK, Springett JA. 1979.
(1):5343-5350. Effect of surface casting earthworms on the
transport of phosphorus and nitrogen in surface
Owa SO, Dedeke GA, Morafa SOA and Yeye runoff from pasture. Soil Biology and Biochemistry
JA. 2003. Abundance of earthworms in Nigerian 11:459-461.
ecological zones: implications for sustaining
fertilizer-free soil fertility. African Zoology 38 Snyman HA. 2004. Effect of various water
(2):235-244. application strategies on root development of
opuntia ficus -indica and O. robusta. J. PACD. 35-
Owa SO, Moreyibi HO and Dedeke GA. 2002. 61.
Wormcasting as initiator of thermal and nutrient
convection current in the soil. African Journal of Stewart VI, Scullion J, Salih RO and Al Bakri
Science and Technology 3(1 & 2):6-7. KH. 1988. Earthworms and structure rehabilitation
in subsoils and in topsoils affected by opencast
Owa SO, Oyenusi AA, Joda AO, Morafa SOA mining for coal. Biol. Agric. Hort. 5:325-338.
and Yeye JA. 2003b. Effect of earthworm casting
on growth parameters of rice. African Zoology 38 Yeates GW 1981. Soil nematode populations
(2):229-233. depressed in the presence of earthworms.
Pedobiologia 22:191-195.
Ruz Jerez E, Ball PR and Tillman RW. 1988.
The role of earthworms in nitrogen release from
herbage residues. In: Jenkinson, D.S. and K.A.

398 Journal of Research in Biology (2011) 5: 393-398