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Original Research paper
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Seasonal Influences on the Distribution of Bacterial Pathogens and
Waterborne Diseases Transmission Potentials of Imo River, Nigeria.
Journal of Research in Biology

Authors: ABSTRACT:
Ihejirika CE1, Ogbulie JN2,
Nwabueze RN2, Orji JC2, The occurrence and population of pathogenic bacteria at certain points of
Ihejirika OC3, Adieze IE2, Imo River, were determined. Water samples were collected for two seasons at major
Azubike OC4 and Ibe IJ5.
points of human activities along the river and subjected to standard microbiological
and statistical analyses. Total heterotrophic bacterial count (2.9 x 10 9 – 3.7 x 103 CFU/
ml) and total coliform bacterial counts (9.0 x 106 – 2.5 x 102 CFU/ml) showed
Institution: significant variation at P<0.05 for the two seasons. Percentage occurrence of
Department of individual isolates across the sampling points showed the presence of Escherichia coli
Environmental Technology, (100%), Klebsiella spp. (71.0%), Shigella spp. (71.0%), Salmonella spp.(71.0%), Proteus
2 spp.(42.9%), Vibro spp. (42.9%), Pseudomonas spp. (42.9%), Staphylococcus spp.
Department of
Microbiology, (85.7%), Bacillus spp. (100%), Enterobacter spp. (57.1%), Citrobacter spp. (14.3%),
Department of Public Serratia spp.(14.3%) and Streptococcus spp.(14.3%). These organisms are of public
Health and health importance and imply that Imo River should be protected from pollution to
Department of avoid possible diseases outbreak and transmission.
Biotechnology, Federal
University of Technology,
Department of Biology and Keywords:
Microbiology, Federal Imo River, pathogenic bacteria, percentage occurrence, diseases outbreak
Polytechnic Nekede, Owerri. and transmission.

Corresponding author: Article Citation:
Ihejirika CE Ihejirika CE, Ogbulie JN, Nwabueze RN, Orji JC, Ihejirika OC, Adieze IE,
Azubike OC and Ibe IJ.
Seasonal Influences on the Distribution of Bacterial Pathogens and Waterborne
Diseases Transmission Potentials of Imo River, Nigeria.
Email: Journal of research in Biology (2011) 3: 163-172
Received: 02 Jun 2011 /Accepted: 21 Jun 2011 /Published: 07 Jul 2011

Web Address: © Ficus Publishers.
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Ihejirika et al.,2011

INTRODUCTION Description of Study Area
Imo river in Nigeria cuts across three eastern The study area of Imo River and is as shown
states and beyond, it serves as a source of water for in figure 1. Imo River is one of the major rivers in
domestic activities, agricultural irrigation, industrial the south-eastern Nigeria. It probably originates
and recreational activities for more than 4 million from Isiochi in Abia State and cuts across three
inhabitants. Many tributaries, domestic sewage, and states including Abia, Imo, and Rivers states. Imo
industrial effluents empty into Imo River causing River flows from the eastern-north to the eastern-
the river to serve as a reservoir of pathogenic south, emptying in the Atlantic Ocean. The river
organisms and toxic chemicals. serves as a source of water for domestic uses,
Microbial pathogens are ubiquitous in nature fishery, recreational activities, and agricultural
and are the second – leading cause of water body irrigation programs for more than 5 million people
impairment in the United States (USEPA, 2004), settling close to the water body. Apart from the
Nigeria and other developing countries were not left above listed uses, the river serves as a source of
out. Once microbial pathogens are in a stream, lake, sand for sand excavators, recipient of industrial
or estuary, they are capable of causing effluent discharges, dumping site for domestic
gastrointestinal, respiratory, skin, eye, ear, nose, wastes including sewage and industrial solid waste,
and throat diseases in humans (USEPA, 1986). and other rivers like Aba River emptying in Imo
Pathogenic diseases outbreaks following River. Some major human impacted points of the
recreational contact with pathogen – contaminated river include Ekenobizi, Udo, Owerrinta, Alulu,
surface waters have been well documented. For Owaza, Akwette, and Obigbo are highly notable.
instance, in 1982, an outbreak of gastrointestinal
illness occurred among New York City police and
firefighter scuba divers who swam in the Hudson
and East rivers (USEPA,1998). In 1998, a review
of 22 studies of recreational waters showed that the
indicator organisms correlate most closely with
enteric illness are fecal Enterococcus and
Streptococcus spp for fresh water and Escherichia
coli for fresh water only Streptococcus spp alone
for marine water (Pruss, 1998).
Microorganisms in surface waters may
originate from several sources (Schets et al., 2008).
It has been demonstrated that pathogenic
microorganisms enter surface waters through
discharges of raw and treated sewage and manure
runoff from agricultural land (van den Berge et al.,
2005; Lodder). The presence of waterborne
pathogens in bird feces has frequently been
reported. In Europe, Campylobacter jejuni has
been detected in gull (Larus spp.) feces in Northern
Ireland (Moore et al., 2002) and Sweden (Broman
et al., 2002). This information suggests that heavy
rainfall events may contribute to increased
concentrations of pathogens in Imo River.
Despite the awareness of sources possibly
contributing to surface water contamination, no
data were available on the seasonal water quality MATERIALS AND METHODS:
and distribution of pathogenic bacteria in Imo river, Sample collection:
Nigeria. This study was therefore aimed at Surface water samples were collected from 7
determining the presence and quantity of major human impacted points of Imo River.
waterborne bacterial pathogens at different points of Samples were collected in triplicates with the aid of
the Imo River. sterile 1 liter water sampling cans. Collected
samples were transported to the laboratory within 4
164 Journal of Research in Biology (2011) 3: 163-172
Ihejirika et al.,2011

hours. The samples were collected in two seasons – locations of the Imo River is as shown in Table
dry and rainy seasons for two years. The dry 1.The THBC during the dry season ranged from 3.6
season was between November and March while x 103 -1.23 x 106cfu/ml while the TCBC during the
the rainy season was between May and September. dry season ranged from 1.65 x 103 – 8.7 x 106 cfu/
Microbiological analysis ml. These results were above the limits of EPA
Sterilization of media was carried out by Maximum Contaminant Levels (MCLS) of
moist heat sterilization method using autoclave at <100cfu/ml in drinking water (USEPA, 2003).
1210C, 15psi for 15 minutes. Heat stable materials Table 2 shows the % occurrencea of
were sterilized using hot air oven at 1600C for 1 bacterial isolates from the different sampling point
hour as described by Cruickshank et al. (1982). or location of Imo River. A total of 14 organisms
Heat labile materials were aseptically rinsed with were isolated from the different locations. These
alcohol and distilled water. The water samples organisms include Escherichia coli (100%),
were aseptically subjected to 10 fold serial dilutions Klebsiella spp (71.0%), Shigella spp (71.0%),
to dilute the population of microorganism Salmonella spp (71.0%), Proteus spp (42.9%),
sufficiently in sterile blanks of 9ml peptone water Vibro spp(42.9%), Pseudomonas spp(85.7%),
and then plated to produce discrete colonies for Staphylococcus spp (85.7%), Bacillus spp
easy enumeration. The media used include Nutrient (100.0%), Enterobacter spp (57.1%), Citrobacter
agar, MacConkey agar, Eosin Methylene Blue agar, spp (14.3%), Serratia spp (14.3%) and
TCBS, and Salmonella – Shigella agar. All media Streptococcus spp (14.3%). This implied that
were prepared as directed by the manufacturer. The Escherichia coli and Bacillus spp were isolated
method was adopted for the inoculation of media. from all the 14 sampling points, Pseudomonas spp
Spread plates of appropriately diluted samples were and Staphylococcus spp isolated from 6 out of the 7
incubated at 370C for 24 hours for heterotrophic sampling points and so on.
bacterial count (THBC) while total coliform The % occurrenceb of total bacterial isolates
bacterial count (TCBC) were determined after at each sampling point showed Owerrinta (69.2%),
incubation at 450C for 24 hours in MacConkey Udo (61.5%), Alulu (61.5%), Akwette (38.4%),
agar. Identification of isolates was based on the Ekenobizi (76.9%), Owaza (53.8%), and Obigbo
scheme described by Cheesborough (1984). (53.8%).
The results were subjected to Analysis of Variance Table 3 shows the result of seasonal
(ANOVA) by using Statistical Program for Social variation of THBC and TCBC of Ekenobizi Point
Sciences (SPSS) and percentage occurrence. of Imo River. There were significant seasonal
variations (P<0.05) in THBC at P = 0.00 and T-
RESULTS AND DISCUSSION: 31.92. The mean THBC values were 1.2 x 106 cfu/
Results: ml (Dry) and 5.0 x 105 cfu/ml (Rainy). There were
The result of seasonal variation of Total no significant seasonal variations in TCBC at P =
Heterotrophic Bacterial Count (THBC) and Total 0.197 and T= 1.54. The mean TCBC values were
Coliform Bacterial Count (TCBC) at all sampling 8.7 x 105 cfu/ml (Dry) and 3.7 x 104 cfu/ml (Rainy).

cfu/ml cfu/ml DRY RAINY
4 4 3
Owerrinta 1.8 x 10 5.5 x 10 4.8 x10 2.0 x 104
Udo 2.0 x 105 2.9 x 109 1.9 x104 8.9 x 106
Alulu 8.6 x 103 1.3 x 107 5.0 x103 1.0 x 106
Akwette 4.1 x 104 5.6 x 106 1.0 x104 5.0 x 104
Ekenobizi 1.2 x106 5.0 x 105 8.7 x105 3.7 x 104
Owaza 3.7 x103 4.1 x 103 1.7 x103 2.5 x 102
Obigbo 4.5 x 104 2.9 x 106 1.9 x 104 3.0 x 103
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Ihejirika et al.,2011

Table 2: Percentage occurrence of microbial isolates at different sampling points of Imo River

a = % occurrence of individual isolate across the sampling points
b = % occurrence of total bacterial isolates at each sampling point
+ = Present and
- = absent.

Table 4 shows the result of seasonal Imo River. There were significant seasonal
variation of THBC and TCBC of Udo point of Imo variations in THBC at P = 0.00 and T = 85.99. The
River. There were significant seasonal variations in mean THBC values were 8.6 x103cfu/ml (Dry) and
THBC at P = 0.00 and T-99.98. The mean THBC 1.3 x 107 cfu/ml (Rainy). There were significant
values were 2.0 x 105 cfu/ml (Dry) and 2.0 x 109 seasonal variations in TCBC at P = 0.00 and T=
cfu/ml (Rainy). There were significant seasonal 32.70. The mean TCBC values were 5.0 x 103cfu/
variations in TCBC at P = 0.00 and T= 11.85. The ml (Dry) and 1.0 x 106cfu/ml (Rainy).
mean TCBC values were 1.9 x 104 cfu/ml (Dry) and Table 7 shows the result of seasonal
8.9 x 106 cfu/ml (Rainy). variation of THBC and TCBC of Owaza Point of
Table 5 shows the result of seasonal Imo River. There were significant seasonal
variation of THBC and TCBC of Owerrinta Point variations in THBC at P = 0.01 and T = 4.47. The
of Imo River. There were significant seasonal mean THBC values were 3.7 x103cfu/ml (Dry) and
variations in THBC at P = 0.00 and T = 32.04. The 4.1 x 103cfu/ml (Rainy). There were significant
mean THBC values were 1.8 x 104cfu/ml (Dry) and seasonal variations in TCBC at P = 0.00 and T=
5.5 x 104 cfu/ml (Rainy). There were significant 23.35. The mean TCBC values were 1.7 x 103cfu/
seasonal variations in TCBC at P = 0.00 and T= ml (Dry) and 2.5 x 102cfu/ml (Rainy).
60.21. The mean TCBC values were 4.8 x 103cfu/ Table 8 shows the result of seasonal
ml (Dry) and 2.0 x 104cfu/ml (Rainy). variation of THBC and TCBC of Obigbo Point of
Table 6 shows the result of seasonal Imo River. There were significant seasonal
variation of THBC and TCBC of Alulu Point of variations in THBC at P = 0.00 and T = 44.71. The
Table 3: Seasonal variation of THBC and TCBC of Ekenobizi Point of Imo River.

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Ihejirika et al.,2011

Table 4: Seasonal variation of THBC and TCBC of Udo Point of Imo River.

Table 5: Seasonal variation of THBC and TCBC of Owerrinta Point of Imo River.

Table 6: Seasonal variation of THBC and TCBC of Alulu Point of Imo River.

mean THBC values were 4.5 x104cfu/ml (Dry) and water (USEPA, 2003). The variation in the dry and
2.9 x 106cfu/ml (Rainy). There were significant rainy seasons might be due to heavy rainfalls
seasonal variations in TCBC at P = 0.00 and T= resulting to sewage overflow and surface runoff.
18.30. The mean TCBC values were 1.9 x 104cfu/ This was demonstrated by previous studies (Goyal
ml (Dry) and 3.0 x 103cfu/ml (Rainy) et al., 1977). These results suggest that heavy
Table 9 shows the result of seasonal rainfall events may contribute to Imo River
variation of THBC and TCBC of Akwette Point of contamination and may give rise to increased
Imo River. There were significant seasonal concentration of pathogens in the water.
variations in THBC at P = 0.00 and T 26.47. The The coliform test is a reliable indicator of
mean THBC values were 4.1 x104cfu/ml (Dry) and the possible presence of fecal contamination and is,
5.6 x 106cfu/ml (Rainy). There were significant consequently, correlated with pathogens. The
seasonal variations in TCBC at P = 0.00 and T= USEPA MCL is less than one coliform per 100ml
24.49. The mean TCBC values were 1.0 x 104cfu/ (USEPA, 2003).
ml (Dry) and 5.0 x 104cfu/ml (Rainy). The Total Heterotrophic Bacteria Count
(THBC) test also called “total count” or “plate
DISCUSSION: count”, provides an estimate of the total number of
The result of Total Heterotrophic Bacterial bacteria in a sample that will develop into colonies
Count (THBC) and Total Coliform Bacterial Count during a period of incubation in a nutrient. This test
(TCBC) as shown in table 1 were above the USEPA detects a broad group of bacteria including
Maximum Contaminant Levels (MCLS) in drinking pathogens, and opportunistic pathogens, but it does

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Ihejirika et al.,2011

Table 7: Seasonal variation of THBC and TCBC of Owaza Point of Imo River.

not pretend to report all of the bacteria in the water points. This result is supported by the works of
sample examined. High THBC may be an indicator Health Canada (2006) and Cabral (2010). E. coli is
of poor general biological quality of drinking water a coliform bacterium found exclusively in the
(USEPA, 2003). There might be presence of digestive tract of warm blooded animals, including
emerging waterborne pathogenic bacteria like humans. This might be responsible for the highest
Mycobacterium avium, Legionella spp., percentage occurrence (Swerdlow et al., 1992). As
Helicobacter spp., and Aeromonas hydrophyla in such E. coli is used in the drinking water industry as
Imo River that might not be isolated through the definitive indicator of recent fecal
conventional laboratory techniques. Health contamination of water.
agencies like the USEPA and World Health Citrobacter spp, Klebsiella spp, Salmonella,
Organization (WHO) have avoided setting Shigella, and Serratia spp are present in most
standards for plate counts possibly for lack of individuals although in low numbers, while Proteus
pathogenicity and great variation in density, spp and Enterobacter spp. are only present in
encountered (Dezuane, 1990). A recommended minority of humans (Wilson, 2005). Therefore,
MCL for human drinking water has not yet been these organisms are not suitable as indicators of
proposed, but the USEPA does recognize the water fecal pollution of the environment. This might be
quality deterioration implied by high plate counts. responsible for their <100% distribution in the
The upper limit for portable water is usually waters. This is supported by the work of Cabral
500cfu/ml. Dezuane (1990) says that water with (2010).While most strains of E. coli are non-
counts under 100cfu/ml should be considered pathogenic, some can cause serious diarrheal
“portable” and values 100 -500/ml is infections in human (Health Canada, 2006), urinary
“questionable”. Therefore Imo River samples have tract infections (Scheatz and Strockbin, 2005), and
questionable water quality. distribution of erythrocytes (Moe,
Among these organisms, the members of the 1997).Citrobacter spp (14.3%) is included in a
family Enterobacteriaceae include Escherichia number of pathogenic bacteria capable of causing
coli, Citrobacter spp., Klebsiella spp, Proteus spp, serious disease and being discharged into rivers
Enterobacter spp, Salmonella spp, Shigella spp, and (Donovan et al.,2008); has ability to produce an
Serratia spp (Prescott et al., 2005). As E. coli was enterotoxin and this become an intestinal pathogen
isolated from all the sampling points, it indicated in environments such as water, sewage, soil and
recent fecal contamination of the different sampling food (Frederisksen and Sogaard, 2003).

Table 8: Seasonal variation of THBC and TCBC of Obigbo Point of Imo River.

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Ihejirika et al.,2011

Table 9: Seasonal variation of THBC and TCBC of Akwette Point of Imo River.

The presence of Citrobacter spp. is The association of Shigella spp (71.0%) with
significant since the species - C. freundii can cause some points of Imo River is an implication of the
meningitis with high morbidity and mortality fecal contamination of the River. This is in
(Donovan et al., 2008). agreement with the reports and works of WHO
Klebsiella spp (71.0%) are ubiquitous in the (2008) and Kapperud et al., (1995). That Shigella
environment. They have been found in a variety of spp with <100% occurrence at sampling points of
environmental situations, such as soil, vegetation, Imo River might imply its presence at some points
or water, and they influences, many biochemical, but are viable and non-culturable. This argument is
and geochemical processes (Cabral, 2010). They supported by the report of Faruque et al. (2002).The
have been recovered from aquatic environments implication of the presence of Shigella spp in Imo
receiving industrial wastewaters, plant products, River samples is the risk of possible outbreak of
fresh vegetables, food with a high content sugars shigellosis. This was in agreement with the report
and acids, frozen orange juice concentrate, of Emch et al. (2008).
sugarcane waste and living trees (Grimont et al., Enterobacter spp (57.1%) might be an
2005). It is because Klebsiella spp. has high % implication of fecal contamination at Imo River.
occurrence (71.0%) in Imo River and especially its This was supported by the works of Grimont and
isolation from Owerrinta Point of Imo River that Grimont (2005). Apart from fecal contamination
receives effluents from paper mill industries. Enterobacter spp might have been introduced from
Klebsiella spp can cause human diseases, ranging other sources like soil, polluted water, and plants.
from asymptomatic colonization of the intestinal, The implication of this ubiquity is that Enterobacter
urinary, or respiratory tract to fatal septicemia. spp cannot be used as an indicator of fecal
Klebsiella are mostly considered nosocomial contamination of water bodies. The presence of
pathogens (Grimont et al., 2005). Enterobacter spp in some samples of Imo River
The presence of Salmonella spp in Imo implied possible risk of outbreak of nosocomial
River samples might be due to contamination from infections such as urinary tract infections and other
municipal sewage agricultural pollution, and storm healthcare- associated infections. This argument is
water runoffs. This argument is supported by the supported by the reports of Hirdron et al. (2008).
reports of WHO (2008) and Arvanitidov et al. Proteus spp (42.9%) is an enteric pathogen
(2005). Though Salmonella spp can survive in associated with the feces of animals including
water bodies, its presence and multiplication can be humans. Its low % occurrence (42.9%) might be
influenced by seasonal conditions, temperature, because it exists in minority of human feces. This is
humidity, and pH, which might be contributory to supported by the reports of Wilson (2005).
the <100% (71.1%) occurrence of Salmonella spp Serratia spp (14.3%) is another enteric
in Imo River water samples. This is supported by pathogen associated with feces of some humans. Its
the work of Le Minor (2003). Salmonella spp are lower % occurrence might be due to low presence
responsible for two types of salmonellosis: (1) in feces of humans and subsequently its lower
Typhoid and paratyphoid fever; (2) gastroenteritis availability as a water borne pathogen. This is
(Le Minor, 2003). This implies that controlled supported by the report of Cabral (2010).
water sewage systems, pasteurization of foods and Vibro spp (42.9%) might be present in some
personal hygiene will reduce the incidence of samples due to contamination from birds, frogs,
typhoid fever (Popoff et al., 2005) that might result fishes and shell fish present in aquatic
from the use of Imo River. environments. This argument is supported by the

Journal of Research in Biology (2011) 3: 163-172 169
Ihejirika et al.,2011

reports of Ali et al. (2001). They detected low for domestic and recreational activities.
distribution of Vibrio spp (42.9%) might be due to
environmental stress caused by adverse CONCLUSION:
environmental conditions making some of the cells Seasonal influences in addition to
viable but non-culturable, though retaining the anthropogenic activities contributed to the
potential for pathogenicity for significant periods of occurrence of water borne pathogens with high
time. This is in agreement with the report of Alam bacteria counts above established standards. The
et al. (2006) and Chaiyana et al. (2001). Vibrio spp River therefore is a source organism of public
especially V. cholerae is responsible for the disease health importance and should be protected from
cholera in humans (Cabral, 2010). human contamination and properly treated to avoid
Streptococcus spp (14.3%) were isolated in consequences.
one out of the seven locations and might be due to
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Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Microbial diversity and bioremediation of distilleries effluent
Journal of Research in Biology

Authors: ABSTRACT:
Ganapathy Selvam G1,
Baskaran R2 and
Mohan PM3.
The role of cyanobacteria in distilleries effluent was studied in Kanchipuram
Tamilnadu, India. Totally 12 species of cyanobacteria belonging to 6 genera falling
Department of under 4 families were identified. Six different genera of bacteria and 3 different
Microbiology, A.V.V.M, genera of fungi were isolated and identified. Among the cyanobacteria isolated,
Sri Pushpam College Nostoc muscorum was selected to treat the effluent. Distilleries effluent was the
(Autonomous), Thanjavur, potential source of cyanobacteria. Nostoc muscorum was found to be the most
Tamil Nadu, dominated genus in this effluent (heterocyst organism). The inoculation of Nostoc
India. muscorum resulted in removal of various chemicals as well as nutrients such as
nitrogen, ammonia and phosphorus from the effluent. It is concluded that Nostoc
2 and 3
Department of Ocean muscorum, a cyanobacteria found in the distilleries effluent, could be potentially
Studies and Marine Biology, employed for the treatment of distilleries effluent.
University, Port Blair,
Andaman and Nicobar Island
-744 103, India.

Corresponding author: Distillery effluent, Microbial diversity, Bioremediation, Physico-Chemical
Baskaran R. analysis.

Email: Article Citation: Ganapathy Selvam G, Baskaran R and Mohan PM.
Microbial diversity and bioremediation of distilleries effluent.
Journal of research in Biology (2011) 3: 153-162
Phone No:
03192213126 Dates:
Received: 22 Jun 2011 /Accepted: 02 Jul 2011 /Published: 07 Jul 2011
03192 261568
© Ficus Publishers.
Web Address: This Open Access article is governed by the Creative Commons Attribution License (http://, which gives permission for unrestricted use, non-
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Ganapathy Selvam et al.,2011

INTRODUCTION waste but also producing a variety of useful
Bioremediation is a pollution control byproducts from the biomass (Subramanian and
technology that uses biological systems to catalyse Shanmugasundaram, 1986).
the degradation or transformation of various toxic
chemicals to less harmful forms. Pollution of water MATERIALS AND METHODS
and land due to hazardous toxic wastes has become Isolation and identification of Cyanobacteria
a major global concern. The industrial, municipal Effluent was collected from the Distilleries,
and agricultural wastes which are legally or Padappai, Kanchipuram (TN), India where over
illegally discharged into the environment are diluted effluent has been released from the factory.
responsible for the environmental pollution (Lang Standard microbiological methods were followed
et al., 1999 and Bakare et al., 2003). Among the for isolation of Cyanobacteria from these samples.
major industries in India, distillery is one of those Algal samples were microscopically examined and
that contribute to water pollution. There are nearly plated on solid agar medium - BG11 (Rippka et al.,
290 distillery unit present in India, of which, 17 1979). The inoculated plates were incubated in
units are located in Tamilnadu. All these distillery culture room (temperature maintained at 25 ± 2oC
industries use molasses obtained from sugar fitted with cool white fluorescent tube emitting
industries as raw material and generate large 2500 lux for 18 hrs a day) and were regularly
amount effluents. Alcohol is separated by examined for the growth of cyanobacteria. Colonies
distillation and the residual liquor is discharged as appearing on solid medium were picked up and
effluent. This effluent called as spent wash, is hot, transferred to liquid medium. By repeated
pH is highly acidic, dark coloured nature. Estimated streaking, cultures were made unialgal and
quantity of distillery waste generated in India is maintained in BG11 liquid medium. Identification
8057 kilo litres per day. The waste is highly of algal forms was made with the help of keys
hazardous in nature because of very high COD and given by Desikachary (1959) and Geitler (1932).
BOD levels. Satyawali and Balakrishnan (2008) Isolation and identification of fungi
studied the molasses-based distilleries are one of Standard serial dilution method was used for
the most polluting industries generating large the isolation of fungus, 1ml of diluted sample was
volumes of high strength waste water. Different plated in petridishes containing Potato Dextrose
processes covering anaerobic, aerobic as well as Agar medium accumulated with Streptomycin
physico-chemical methods have been employed to sulfate 100µg/ml (PDA) to inhibit the bacterial
treat this effluent. Anaerobic treatment is the most growth. The fungus were purified and mounted
attractive primary treatment due to over 80% BOD over a clean slide, stained with lactophenol and
removal combined with energy recovery in the form cotton blue and observed under the microscope.
of biogas. Further treatment to reduce residual The fungus were identified by using standard
organic load and colour includes various: (i) manuals, such as Manual of soil fungi (Gillman,
biological methods employing different fungi, 1957), Dematiaceous Hyphomycetes (Ellis, 1971),
bacteria and algae, and (ii) Physico-chemical More Dematiaceous Hyphomycetes (Ellis, 1976),
methods such as adsorption, coagulation / Hyphomycetes (Subramanian, 1971).
precipitation, oxidation and membrane filtration. Isolation and identification of Bacteria
In recent years, urban people are facing Bacteria were isolated by standard method
many problems and water pollution is one among using nutrient agar. (Difco Manual, 1953). The
them. Environmentalist and government are looking purified bacterial cultures were used for
for efficient, cheap and lasting solutions to waste identification. The bacteria were identified based on
treatment and recycling. Physico-chemical methods colony characteristics, gram staining methods and
of waste water treatment are inevitably cost by various biochemical studies as given by Bergey
intensive and cannot be employed in all industries and Buchanan (1974).
especially in developing countries like India. Treatment of distilleries effluent by Nostoc
Hence, in recent years, the importance of biological muscorum
treatment systems has attracted the attention of Sterilized Erlenmayer flasks (250 ml) were
workers all over the world and has helped in used for the experiments. The initial physico-
developing relatively efficient, low cost waste chemical analysis of effluent was made following
treatment systems. Algal systems, more particularly the Standard Methods (APHA, 1975). The
the cyanobacteria, are not only useful in treating the following treatments were employed in order to
154 Journal of Research in Biology (2011) 3: 153-162
Ganapathy Selvam et al.,2011

study the interaction of Cyanobacterium with the for the estimation of biochemical compound
distilleries effluent. Effluent uninoculated was variation in effluent treated and control culture.
taken as, control for physicochemical analysis (C).
BG11 medium inoculated with Nostoc muscorum for RESULTS AND DISCUSSION
the estimation of growth (CO). Inoculation was Isolation of cyanobacteria:
made by adding 3.0 ml uniform suspension of In the present investigations totally 12
Nostoc muscorum separately (0.03 mg ml-1). The species (Fig-1 and Table -1) of cyanobacteria
experiment was conducted under controlled distributed in five genera falling under four
conditions (Temperature 25 ± 2oC with light different families were identified from the
intensities of 2500 lax provided form overhead cool distilleries effluent polluted habitat. Among the
with fluorescent tubes) for one month. Since the genera, Oscillatoria with six species followed by
growth of Nostoc muscorum was very slow in the Phormidium two species, Lyngbya, Microcystis,
effluent, a long duration was provided to get a Nostoc and Plectonema with single species each.
culture of exponential growth. The cultures were This is attributed to favorable conditions of
harvested on every 5th day by filtration through oxidizable organic matter, less DO and high
filter paper and washed repeatedly with distilled calcium content; (Table 4) an observation which
water. The filtered effluents (inoculated and supports Venkateswaralu (1969) who observed that
control) were used for physicochemical analysis. high orthophosphate levels favored the
Nostoc muscorum obtained both from the medium development of cyanobacterial bloom. Similar
and effluents were used for the estimation of observations were made in the present study with
growth (estimation of chlorophyll a). reference to various nutrients (Table-4).
Estimation of Chlorophyll ‘a’ Heterocystous cyanobacteria have not been
The cultures were centrifuged at 5,000 x g recorded in polluted waters rich in nitrogen
for 10 minutes. The pellets were washed with (Boominathan et al., 2007). In contrary to that
distilled water; suspended in 80 per cent methanol present study heterocystous cyanobacteria have
and vortexed thoroughly. Then the tubes were already been recorded and is because of the
covered with aluminium foil to prevent solvent presence of lower concentration of various forms of
evaporation and incubated in a water bath set at nitrogen effluent as suggested by Vijayakumar et
60oC for 1 hr, in dark with occasional shaking. al., (2007).
After 1 hr the contents were cooled and centrifuged Isolation of fungi and Bacteria:
at 5,000 x g for 5 minutes. The supernatant was In the present investigation, a total of three
saved and the above procedure was repeated twice different genera of fungus were isolated and
to ensure complete extraction of the pigment. The identified. Among the genera Aspergillus with four
pooled supernatant was made upto a known volume
with 80 per cent methanol (to compensate the
solvent loss during heating). The absorbency was
measured at 663 nm in Spectronic 20 against
methanol blank.
Physico-chemical Analysis of distilleries effluent
The physico-chemical properties of the
distillery effluent like pH, Carbondioxide, alkalinity
(Carbonate and Bicarbonate), Dissolved oxygen,
Biological Oxygen Demand, Chemical Oxygen
Demand (COD), Nitrate, Nitrite, Ammonia, total
phosphorus, inorganic phosphate, Organic
phosphate, Calcium, Magnesium and Chloride were
analyzed following the procedure of Apha (1975).
Biochemical analysis of Nostoc muscorum
Biochemical analysis of Nostoc muscorum
such as Carbohydrate (Dubois et al., 1956), Protein
(Lowry et al., 1951), aminoacid. (Jayaraman, 1981)
and Lipids (Sato and Murata, 1988), were analyzed
Fig-1 Microbial diversity of distillery effluent

Journal of Research in Biology (2011) 3: 153-162 155
Ganapathy Selvam et al.,2011

Table 1 Cyanobacteria from distilleries effluent
Name of cyanobacteria species
1. Microcystis aeruginosa Ktz
2. Oscillatoria acuta Bruhl et Biswas, Orth. Mut. Geitler
3. Oscillatoria earlei Gardner
4. Oscillatoria late-virens (Crovan) Gomont
5. Oscillatoria princeps Vaucher ex Gomont
6. Oscillatoria terebriformis Ag ex Gomont
7. Oscillatoria willei Gardner em.Drouet
8. Phormidium fragile (Meneghini) Gomont
9. Phormidium tenue (Menegh) Gomont
10. Lyngbya majuscula Harvey ex Gomont
Fig-2. Growth of Nostoc muscorum treated and control
11. Nostoc muscorum Ag ex Born. et Flah. conditions indicator species activity take part in the
SCYTONEMATACEAE degradation of organic matter (Gupta and Rao,
12. Plectonema notatum Schmidle 1980; and Vijayakumar et al., 2005).
Manoharan and Subramanian (1992 and
species dominant given in (Fig-1 and Table-2). 1993) found a rise in pH value upto 10th day in
Jaganathan (2006) isolated 21 species from oil various effluents inoculated with free BGA and
refinery effluent polluted habitat with Aspergillus after that it was declined. Similarly, Tang et al.
as the dominant genus. From the distilleries (1997) reported a rise in pH while tertiary waste
effluent, six different species of bacteria were water was treated with cyanobacteria. In the present
isolated (Fig-1 and Table-3). Pursuing the study, the initial pH of the effluent was 3.7 and it
literature, there has not been much work regarding rose till 6.7 after the effluent inoculated with Nostoc
the isolation and identification of bacteria from dye muscorum was observed for a period of 30 days
and other related effluent samples. Contrary to that, (Table-4).
Boominathan et al. (2007) isolated nine different There was no carbonate in the effluent, but,
species of bacteria were reported. Jain et al. (2001) fairly high level of free Co2 and bicarbonate was
isolated three different bacterial strains from the recorded. Gradual removal of both CO2 and
distillery sludge to treat the predigested distillery bicarbonate was observed from 5th day onwards. On
waste water. 30th day CO2 and bicarbonate was respectively 80
Bioremediation of distilleries effluent and 74 percent removal of CO2 and bicarbonate was
Now-a-days the use of algae, particularly noticed. The relative proportion of CO2 and HCO3
cyanobacteria for biomonitoring of pollution is depends on the pH of the medium. It was found that
being stressed. The isolated dominant taxa, HCO3 with 6 to 7 pH is predominant. Thus
encounted in the present study, Nostoc muscorum cyanobacteria may have competitive advantage
was selected for the treatment of distilleries effluent over chlorophytes since the former are capable of
(Fig-2). Many of the studies conducted in various assimilating HCO3 as source of inorganic carbon
laboratories have shown that under defined for photosynthesis (Colman, 1989). In general, the
removal of these carbon sources effectively by
Table 2 Cyanobacteria from distilleries effluent cyanobacteria, as expected, was observed in the
Sl. No. Name of fungal organisms present investigation.
1. Aspergillus flavus Link Chemical oxygen demand is generally
2. Aspergillus luchuensis Inui considered as a major indicator of organic pollution
3. Aspergillus niger Vantieghem in water (Dash and Mishra, 1999). The strength of
the waste water is determined by the level of BOD
4. Aspergillus terreus Thom
and COD. Inoculations of cyanobacteria reduced
5. Fusarium oxysporum Schlectenndhal the BOD and COD levels considerably when
6. Penicillium janthinellum Biourge compared to control (Table-4). More than 53 per
156 Journal of Research in Biology (2011) 3: 153-162
Ganapathy Selvam et al.,2011

Table 3.Biochemical characteristics of isolated bacteria from distilleries effluent
Sl. No. Name of the test E. coli E.aerogenes Lactobacillus P .aeruginosa P .vulgaris S.sonnei

1. Gram staining Gram –ve Gram –ve Gram –ve Gram –ve Gram –ve Gram –
Rods Rods Rods Rods Rords ve
2. Motility + + - + + -
3. Spore staining - - - - - -
4. Indole + - + - + -
5. Methyl red + - + - + +
6. Voges Proskauer - + - - - -
7. Citrate - + + + + -
8. Triple sugar iron + + + - + -
9. Gas production + + + - + -
10. H2S production - - - - + -
11. Catalase + + - + + +
12. Oxidase - - + + - -
13. Urease - - - - + -
14. Lysine + + - - - -
15. Phenyl alanine - - + - + -
16. Nitrate reduction + + - + + -
17. Carbohydrate
utilization test
18. Glucose + + + + + +
19. Maltose + + + - + +
20. Lactose + + + - - -
21. Mannose + + - - - +
22. Sucrose + + + - -

cent reduction of BOD and 68 per cent reduction of
COD were observed with both cyanobacteria. Use
of acclimatized algal cultures in considerably
reducing BOD and COD with different effluents
has been reported (Sharma et al., 2003 and
Vijayakumar et al., 2005).
Determination of the amounts of O2
dissolved in water at study sites is undoubtedly of
treatment importance since it is considered as one
of the best parameters in evaluating pollution stress
of aquatic habitats, unless it contains toxic
substances (Lester, 1975). As revealed from the
results obtained in the present investigation, the
initial DO content of distilleries effluent was very
low (1.2 mg1-1). From 5th – 10th day onwards a
gradual increase in DO was noticed and it was 1.2
and 2.1 effluent with Nostoc muscorum respectively
Fig- 3. Total carbohydrate, protein, amino acid and (Table-4). Increase in DO level when treated with
lipid content in Nostoc muscorum

Journal of Research in Biology (2011) 3: 153-162 157
Ganapathy Selvam et al.,2011

Table 4. Characteristics of treated and non treated distilleries effluent
Sl.No. Parameters Non Treated effluent (mg/ml) After 30 day Treated effluent (%)

1. Colour Dark Brown -
2. pH 6.7 6.7
3. Temperature 29°C 29°C
4. Free CO2 100 80
5. Carbonate Nil Nil
6. Bicarbonate 158 74.68
7. BOD 327 53.51
8. COD 232 68.53
9. Nitrate 583 52.83
10. Nitrite 115 66.08
11. Ammonia 423 58.86
12. Total phosphorus 59 55.93
13. Inorganic phosphorus 36 36.11
14. Organic phosphorus 23 86.93
15. Calcium 84.10 73.78
16. Magnesium 62.42 69.91
17. Chloride 179.9 48.36

Except pH and temperature all other parameters are in mg/l.

different cyanobacteria with different effluents has However, Nostoc muscorum was marginally better
already been reported by Vijayakumar et al. 2005 in removing all form of nitrogen. Suspended,
and Boominathan et al., 2007. This raise in DO cultivation of micro algae is one of the biological
level in effluents with cyanobacteria might be due process has been employed to eliminate residual
to oxygenic photosynthetic nature of cyanobacteria. inorganic nutrients as a tertiary treatment step from
The correlation between increase in DO and secondary treated effluents (Oswald, 1978). Studies
removal of BOD and COD observed in this study with Spirulina (Boominathan et al., 2007),
agree with observations of Kankal et al., (1987); Anabaena (Mallick and Rai, 1994), Oscillatoria
and Vijayakumar et al., (2005). (Manoharan and Subramanian, 1992 and 1993)
Suspended cultivation of microalgae is one concluded that cyanobacteria can efficiently
of the biological processes which have been eliminate inorganic nitrogen compounds from waste
employed to eliminate residual inorganic nutrients waters. In general, the removal of inorganic
as a tertiary treatment step from secondary treated nitrogen compounds by cyanbacteria is governed by
effluents (Prakasham and Ramakrishnan, 1998). growth conditions and physiological conditions of
Inorgornic compounds such as nitrite, nitrate, the state of the organism like pH, light, temperature,
ammonia and phosphate are the essential inoculum size and subtracted concentration.
requirements for the growth of cyanobacteria. They The capacity of cyanobacteria to remove
have high nutrient capabilities as they can large amount of phosphorus from industrial waste
accumulate inorganic phosphate and cynophycin water has been demonstrated by several workers
respectively (Fay, 1983). In the present (Monoharan and Subramanian (1992 and 1993),
investigation, in distilleries effluent, all form of Boominathan et al. (2007) and Vijayakumar (2005)
nitrogen were observed in appreciable quantities found a total or near total removal of all forms of
(Table-4) and cyanabacteria removed more than 55 phosphate by Oscillatoria and Aphanocapsa from
per cent of inorganic nitrogen from the effluent. different effluents. Tang et al., (1997) also reported
158 Journal of Research in Biology (2011) 3: 153-162
Ganapathy Selvam et al.,2011

a higher phosphate uptake, despite low biomass of treatment methods. However, in the present
cyanobacteria. Dash and Mishra (1999) observed investigation Nostoc was efficient by removing
100 per cent removal of phosphate from paper mill chloride from the sample (Table-4). Uma and
effluent while testing with Westiellopsis prolifica. Subramanian (1990) observed nearly 50 and 25 per
However in the present study more than 55 percent cent removal when ossein effluent which has very
removal of total phosphorous and 36 per cent high level of chloride was treated with Oscillatoria
removal inorganic phosphate were observed in and Aphanocapsa respectively. Manoharan and
effluent with Nostoc muscorum. The major Subramaniam (1992 and 1993) also observed more
phosphate reserve of cyanobacteria is than 40 percent removal of chloride from various
polyphosphate, which accumulates as discrete effluents by Oscillatoria sp. A similar observation
granules in the cytoplasm of the cell wall when attributing 50 percent chloride reduction under
phosphate is in excess (Fay, 1983). The intracellular laboratory conditions by O. brevis was also
accumulation of phosphate is energy dependent, reported in dye effluent (Vijayakumar et al., 2005),
being higher in the light than in the dark and oil refinery effluent (Boominathan et al., 2007) and
depends strictly on the pH of the cytoplasm. All the sugar mill effluent (Gopalakrishnan, 2007).
above findings confirm that cyanobacteria can Biochemical studies on Nostoc muscorum
absorb phosphate in excess amount as it is required Growth was measured in terms of
and this could be the reason for maximum removal chlorophyll ‘a’ as a biomass component. This was
of all forms of phosphate from the effluent. estimated both in BG11 medium (control) and
The total hardness is constituted by calcium effluent. Maximum growth of Nostoc was recorded
and magnesium. In the present study more than 68 in the control than in effluent inoculated (Fig.2). In
per cent removal of calcium and magnesium were recent years, scientists have been increasingly
observed in effluent with Nostoc. Uma and concentrated more on the influence of these systems
Subramaniam (1990) studied the effective use of on the removal of nutrients from the effluent but
cyanobacteria in ossein effluent which has high only a few have investigated the effect of effluents
levels of calcium. They found more than 50 per cent on the biochemistry of the cyanobacterial systems.
reduction of calcium within 4 days when the To develop suitable and efficient treatment system,
effluent was treated separately with Oscillatoria it is obligatory to understand the mutual influence
and Aphanocapsa. Similarly, Manoharan and and interactions between the effluents and the
Subramanian (1992 and 1993) reported the organisms, so that manipulation to improve the
reduction of calcium and magnesium in domestic treatment system becomes feasible and hence the
sewage, ossein and paper mill effluents by O. present investigation on the biochemistry of
pseudogeminata var. unigranulata. They observed effluent grown cyanobacteria was carried out.
more than 70 percent reduction of calcium and Distilleries effluent has decreased the total
magnesium with retention time of 15 days. Dash carbohydrate content of Nostoc muscorum to a
and Mishra (1999) observed 50 per cent reduction considerable level (Fig.3) when compared to BG11
of calcium in paper mill effluent by Westiellopsis medium (control). The reduction was around 16
(retention time of 15 days). On the other hand percent. Contrary to the present observation,
Vijayakumar et al. (2005) reported more than 90 increase in the carbohydrate level of cyanobacteria
percent removal of calcium and magnesium from with various effluents has been reported by many
dye effluent when treated with Oscillatoria sp. workers (Reddy et al., 1983 and Vijayakumar,
Although, calcium is undoubtedly required for 2005, 2007). Nitrogen limitation caused photo
cyanobacterial growth substantial reduction in assimilated carbon to be directed towards the
calcium and magnesium are known to be essential synthesis of carbohydrate instead of proteins and
for flocculation and would coflocculate (Richmond chlorophyll. This response is widely observed in
and Becker, 1986).) observed the excretion of many algal species (Turpin, 1991). Protein and
organic acids by microbes and especially chlorophyll decreases and carbohydrate increases
cyanobacteria and their capacity to solubilize by CO2 enrichment have been observed previously
magnesium in the waste water, which could explain in a number of species (Loehle, 1995). But in the
observed reduction. present investigation, considerable levels of all
Chlorides are generally considered as one of forms of inorganic nitrogen were observed; more
the major pollutants in the effluent which are over the carbohydrate content was also reduced by
difficult to be removed by conventional biological the effluent. There were no CO2 recorded in the
Journal of Research in Biology (2011) 3: 153-162 159
Ganapathy Selvam et al.,2011

effluent throughout the study period and hence this Bakare AA, Lateef A, Amuda OS and Afolabi
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162 Journal of Research in Biology (2011) 3: 153-162
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Standardized protocol for the in vitro culture of Artemisia annua L. – A
medicinal plant at high altitudes of Nilgiris, the Western Ghats.
Journal of Research in Biology

Authors: ABSTRACT:
Ganesan CM and
Paulsamy S.

A reliable protocol for callus induction and organogenesis, and successful
plantlet survivability through hardening were developed for leaf explants of the
Department of Botany, medicinal plant species, Artemisia annua L. MS medium containing the auxin, NAA at
Kongunadu Arts and Science 0.9mg/l is determined to be the optimum concentration for callus induction. Higher
College, Coimbatore shooting (20.67/callus) was performed in the medium supplemented with BAP and
641 029, Tamil Nadu, India. GA3 at 0.5 and 1.0 mg/l respectively while subculturing the callus. Maximum number
of roots (12.00 roots/shoot) was noted to be obtained in the medium containing IBA
at 0.9mg/l. The in vitro regenerated plantlets were successfully acclimatized (86%
survivability rate) in the hardening medium encomposing vermiculate, coir waste and
forest litter in the ratio of 1:1:1 by volume.
Corresponding author:
Paulsamy S

Keywords: In vitro culture, Medicinal plant, Artemisia annua, Nilgiris, Western Ghats, India.

Web Address: Article Citation:
Ganesan CM and Paulsamy S.
Standardized protocol for the in vitro culture of Artemisia annua L. – A medicinal plant
at high altitudes of Nilgiris, the Western Ghats.
Journal of research in Biology (2011) 3: 173-178

Received: 04 Jul 2011 /Accepted: 08 Jul 2011 /Published: 12 Jul 2011

© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://, which gives permission for unrestricted use, non-
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Ganesan et al.,2011

INTRODUCTION the percentage of shoot forming roots, roots per
Artemisia annua L. (Asteraceae) is a shoot length were assessed. Rooted shoots were
medicinal herb inhabiting at the high altitudes of thoroughly washed to remove the adhering gel and
Nilgiris, Western Ghats at open habitats. Presence planted in polythene bags containing different
of high content of bioactive alkaloids like hardening media and kept in greenhouse for
artemisinin in A.annua receives attention towards acclimatization. The pots were watered at one day
pharmacological industries as it is used for the interval and supplied with ½ strength MS salts,
treatment of malarial fever (Duke et al., 1987; Chen twice a week by spraying. The survival rate of
et al., 1991). As a result of over exploitation, the plantlets was recorded for one month after
species become lower in population size at the high transferring to polythene bags. Triplicates were
altitudes of Nilgiris (Paulsamy et al., 2008). maintained for all experiments.
Conventional method of propagation of this species
through seed was also not successful (Paulsamy, RESULTS AND DISCUSSION
2005). Hence, in vitro culture by employing tissue The number of days required for callus
culture technology has been attempted for this induction from the leaf explants of the study
species to enable bulk production. species, Artemisia annua is noted to be varied from
10 to 30 days according to the combinations and
MATERIALS AND METHODS concentrations of the growth regulators viz., BAP,
The leaf explants of Artemisia annua were NAA, 2,4-D, IBA and TDZ in the MS medium
collected from the healthy individuals at Nilgiris (Table 1). It may be explained that the specific
and washed thoroughly with tap water, then they growth hormones at appropriate concentrations can
were cut into small discs of 0.8cm diameter and play a major role to induce callus besides the other
then treated with a surfactant, tween 20 ( 5% w/v)
for 5 minutes. After repeated washes in double
distilled water, to eliminate the fungal
contamination, the explants were treated with
Carbendazim (50% w/v), fungicide (10%) for 15
minutes and rinsed with double distilled water for 2
or 3 times. To eliminate bacterial contamination
explants were also treated with 5% antibiotics
(ampicillin and rifampicin) for 30 minutes followed
by three rinses in sterile double distilled water.
Furthermore, surface sterilization was carried out
by dipping the explants in 0.1% HgCl2 for 3
minutes followed by 3-4 rinses in sterilized double
distilled water inside the Laminar air flow chamber.
Leaf discs were horizontally placed in Petri dishes
containing MS (Murashige and Skoog, 1962)
medium fortified with various combinations and
concentrations of different growth regulators viz.,
BAP, NAA, 2,4-D and TDZ for callus induction.
The pH of the medium was adjusted between 5.6
and 5.8 before autoclaving at 121oC for 20 min. Figure 1. Successful in vitro culture of Artemisia
The culture was incubated at a constant temperature annua from the leaf explants.
of 25+2oC with 14h photoperiod (3000 lux) and 8h a, Effective callus formation in MS medium
darkness. Callus from these primary cultures were containing 0.9 mg/l of NAA.
b, Higher response of callus for shoot formation in MS
transferred to MS medium containing different medium fortified with BAP at 2mg/l.
concentrations of BAP, IAA and GA3 for shoot c, High degree of multiple shoot formation while
induction. After the origin of multiple shoots, subculturing onto MS medium supplemented with
elongated shoots of 2 cm long were excised from BAP and GA3 at 2.7 and 0.9 mg/l respectively.
the culture and transferred to MS medium d, More pronounced root formation during
supplemented with different concentrations of IBA, subculturing onto MS medium containing 0.9mg/l of
IAA and NAA for root initiation. After two weeks, IBA.
174 Journal of Research in Biology (2011) 3: 173-178
Ganesan et al.,2011

factors (Ananthi et al., 2011). The amount of leaf endogenous level of growth regulators as observed
explant responding for callus formation was in many other plants (Farternale et al., 2002;
ranging between 10 and 84% (Table 1). MS Senthilkumar and Paulsamy, 2010b). It was noted
medium fortified with NAA at 0.9 mg/l initiated that the NAA alone or in combination with TDZ
98.66% of leaf discs for callus formation (Fig. 1a) generally have the efficiency of initiation at high
followed by 0.7mg/l of NAA initiated 84% of leaf percentage of (>50%) leaf explant for callus
discs for callusing and 0.5mg/l of NAA and TDZ formation. It indicates the higher requirement of
each initiated 73% of discs for callusing. The other certain auxins like NAA alone or in combination
combinations and concentrations of growth with low quantity of cytokinins like TDZ for callus
hormones in the medium initiated around only 20 to formation of the study species, A. annua.
50% of leaf discs of A.annua for callus formation. Karappusamy and Pullaiah (2007) for the species,
Baskaran and Jayabalan (2005) explained that the Bupleurum distichophyllum and Senthilkumar and
differential response of same or different explants Paulsamy (2010a) for the species, Ageratum
for callus formation could be due to the nature of conyzoides also have reported effective callus
tissue, degree of totipotency and composition of formation from the leaf explants in the medium
medium with respect to micronutrients and containing high quantity of NAA. Mariani et al.,
hormones. Further it is explained that the variation (2011) reported the requirement of the cytokinin
in response of discs in terms of callus initiation may like compounds, TDZ for effective callus formation
be due to the variation in distribution of in the ornamental plant, Aglaonema sp. The colour
Table 1. Effect of different concentrations of growth regulators on per cent callus induction from leaf, node
and intermodal explants of the species, Artemisia annua.
Days required for
Growth regulator Callus formation Colour of the
callus formation after
(mg/l) (%) callus
BAP NAA 2,4-D IBA TDZ Leaf explant Leaf explant Leaf explant
0.5 0 0 0 0 10 10.00±2.00a G
1.0 0 0 0 0 12 13.66±1.52a G
1.5 0 0 0 0 14 18.00±2.00b G
2.5 0 0 0 0 16 21.33±1.53bc G
3.0 0 0 0 0 18 23.33±1.58c G
0 0.1 0 0 0 18 25.00±2.00c G
0 0.3 0 0.5 0 21 29.00±2.00cd G
0 0.5 0 1.0 0 20 30.00±1.00d DG
0 0.7 0 1.5 0 23 49.00±2.00d DG
0 1.0 0 2.0 0 22 57.33±2.51e DG
0.5 0 0.5 0 0 18 39.00±1.00f G
1.0 0 1.0 0 0 17 44.33±2.08d DG
1.5 0 1.5 0 0 15 46.66±1.52d DG
2.0 0 2.5 0 0 21 49.33±2.08d LG
2.5 0 3.0 0 0 19 51.33±2.08d LG
3.0 0 3.5 0 0 23 55.00±1.00e B
0 0.1 0 0 0 14 39.66±1.53f LG
0 0.3 0 0 0 18 49.66±1.55d LG
0 0.5 0 0 0 20 46.66±1.59d B
0 0.7 0 0 0 21 84.00±4.35g LB
0 0.9 0 0 0 30 98.66±2.51h LB
0 0.5 0 0 0.1 22 54.33±1.58e DB
0 0.5 0 0 0.2 19 54.66±0.57e DB
0 0.5 0 0 0.3 17 56.66±2.08e DB
0 0.5 0 0 0.4 25 58.00±3.00e DB
0 0.5 0 0 0.5 23 73.00±4.00i LB
G-Green, DG- Dark green, LG- Light green, B-Brown, DB-Dark brown, LB- Light brown
Means in column followed by different letter(s) are significantly different at 5% level according to DMRT.

Journal of Research in Biology (2011) 3: 173-178 175
Ganesan et al.,2011

of the calli was showing wide degree like green, species (Vijaykumari et al., 2001; Roy et al., 2008;
dark green, light green, brown, dark brown and Senthilkumr and Paulsamy, 2010a; Sunder and
light brown according to the combinations and Jawahar, 2011).
concentrations of the growth regulators in the MS The rooting attributes of A.annua while
medium (Table 1). subculturing the secondary explant shoots were
The results of the subculturing experiments well pronounced in the MS medium supplemented
by using the secondary explant, leaf derived callus with the auxin, IBA alone at higher concentrations
showed that the cytokinin, BAP alone in higher from 0.5 to 0.9 mg/l (Table 3). The IBA
concentration (>1.5mg/l) (Fig. 1b) or BAP in concentration at 0.9 mg/l initiated 85% shoots for
combination with GA3 have enhanced the response root formation (Fig. 1d) followed by 0.7mg/l
of calli for shoot formation by 98 and 90% initiated 80% and 0.5 mg/l initiated 71% shoots for
respectively (Table 2). In addition, greater number root formation. The number of roots per shoot were
of 20 shoots/callus was also noted to be produced also observed to be higher (12 roots/shoot) in the
while subculturing the calli on MS medium with MS medium containing 0.9mg/l IBA for the study
BAP and GA3 at 2.7 and 0.9 mg/l respectively species, A.annua. Similarly, the root length was
(Table 2) (Fig. 1c). However, the higher shoot greater (5.1cm) during the subculturing of in vitro
length of 10cm was achieved in the MS medium cultured shoots for roots on MS medium with IBA
fortified with BAP alone at 2 mg/l (Table 2). All at 0.9 mg/l. All these facts showed that the auxin,
these facts indicate that the cytokinin, BAP is the IBA is the most required growth regulator for
most essential growth regulator for effective shooting characters of the study species, A.annua.
shooting of the study species, A.annua. It is of It agrees with the concept that auxins are the plant
common fact that cytokinin is the major growth hormones endogenously or exogenously inducing
hormone involved in shoot formation in many plant root formation in majority of plant species (Van

Table 2. Effect of different concentrations of growth regulators on shoot initiation, shoot number and shoot
length after subculturing the leaf derived callus of the species, Artemisia annua.
Growth regulator (mg/l)
Culture response (%) No. of shoots/callus Shoot length (cm)
0.5 0.0 0.0 57.33±2.08ag 08.66±1.52a 07.57±0.35a
b a
1.0 0.0 0.0 65.66±3.51 09.00±2.00 07.93±0.57a
c b
1.5 0.0 0.0 85.33±3.05 12.00±1.00 08.83±0.74a
d b
2.0 0.0 0.0 98.00±2.00 14.00±1.00 10.20±0.26b
0.5 0.2 0.0 20.33±3.05e 06.33±1.53a 04.80±0.30c
e a
1.0 0.4 0.0 29.66±1.53 07.33±1.53 06.50±0.20ca
af a
1.5 0.6 0.0 48.33±2.08 09.67±1.54 07.00±0.20a
b b
2.0 0.8 0.0 62.33±4.51 14.00±2.00 07.37±0.15a
d cd
3.0 1.0 0.0 93.00±2.00 17.66±1.56 08.50±0.36a
f b
0.3 0.0 0.1 45.00±2.00 12.33±1.53 05.03±0.25c
0.6 0.0 0.2 50.33±3.21f 15.00±2.00bd 05.13±0.15c
a bd
0.9 0.0 0.3 54.00±3.00 15.67±1.53 05.23±0.15c
a bd
1.2 0.0 0.4 55.33±2.08 16.67±1.53 05.20±0.10c
a cd
1.5 0.0 0.5 55.33±1.53 17.00±2.00 05.30±0.10c
g cd
1.8 0.0 0.6 59.67±3.05 18.00±2.00 05.67±0.15c
2.1 0.0 0.7 70.33±1.53b 19.67±1.57cd 05.90±0.20c
c cd
2.4 0.0 0.8 80.00±1.00 19.00±2.00 05.90±0.10c
e cd
2.7 0.0 0.9 90.00±2.00 20.33±2.08 06.67±0.15ca
e cd
0.5 0.0 1.0 94.67±2.08 20.67±1.58 07.10±0.20a
af a
1.0 0.2 1.0 46.33±2.52 07.00±2.00 02.87±0.20d
1.5 0.4 1.0 50.67±2.52f 06.67±1.53a 02.93±0.15d
f a
2.0 0.6 1.0 51.33±2.52 06.00±1.00 03.00±0.10dc
g b
2.5 0.8 1.0 59.33±2.08 07.00±1.00 03.47±0.25dc
b a
3.0 1.0 1.0 74.00±2.00 11.33±1.53 04.70±0.20c
Means in columns followed by different letter(s) are significantly different at 5% level according to DMRT.
176 Journal of Research in Biology (2011) 3: 173-178
Ganesan et al.,2011
Table 3. Effect of different concentrations of growth regulators on root number, rooting percentage and
root length after subculturing the leaf calli derived shoots of the species, Artemisia annua.
Growth regulator (mg/l)
Shoots rooted (%) No. of roots/shoot Root length (cm)
0.1 0.0 0.1 19.00±1.00a 05.00±0.81a 2.10±0.20a
a b
0.2 0.0 0.2 20.66±1.52 07.00±0.82 2.70±0.10ab
0.3 0.0 0.3 22.67±2.51a 08.00±0.85b 3.03±0.15b
b c
0.4 0.0 0.4 27.33±1.57 10.66±1.24 3.73±0.16b
0.5 0.0 0.5 29.00±1.00b 08.66±1.26b 2.80±1.90ab
a b
0.0 0.1 0.1 22.33±1.52 07.33±1.27 3.46±0.15b
0.0 0.2 0.3 26.00±1.00b 10.00±2.44c 3.53±0.15b
0.0 0.3 0.5 29.67±1.54b 09.33±1.21c 3.63±0.15b
ce b
0.0 0.4 0.7 40.00±1.00 07.00±1.64 3.80±0.10b
0.0 0.5 0.9 46.00±1.01d 09.00±1.63c 4.06±0.17b
ce cd
0.0 0.1 0.0 39.00±1.05 10.67±1.28 2.17±0.21a
0.0 0.2 0.0 41.67±2.08ce 08.67±1.23b 2.70±0.20ab
ce c
0.0 0.3 0.0 42.67±1.54 10.00±1.63 3.00±0.13b
0.0 0.4 0.0 44.68±1.52df 09.00±2.16c 2.80±0.10ab
f b
0.0 0.5 0.0 48.33±1.27 08.67±1.25 3.13±0.25b
0.1 0.0 0.0 50.66±2.51f 07.00±1.63b 3.20±0.27b
g c
0.3 0.0 0.0 60.00±1.00 09.66±1.26 4.03±0.15bd
0.5 0.0 0.0 71.00±2.00h 11.66±1.27cd 4.43±0.31bd
0.7 0.0 0.0 80.00±1.00i 10.33±1.20cd 4.80±0.20d
j d
0.9 0.0 0.0 85.00±1.00 12.00±0.81 5.10±0.10d
1.0 0.0 0.1 41.66±1.52ce 11.33±1.27cd 3.26±0.15b
f c
1.0 0.0 0.2 47.00±2.00 09.00±1.63 3.60±0.20b
1.0 0.0 0.3 50.66±2.08f 07.33±1.25b 3.73±0.20b
g a
1.0 0.0 0.4 56.00±1.00 05.00±0.81 3.73±0.15b
1.0 0.0 0.5 60.00±1.00g 06.00±0.83a 3.90±0.10b
Means in columns followed by different letter(s) are significantly different at 5% level according to DMRT.

Eck and Kitto, 1992). Similar kind of findings of The present paper describes a prime and
effective root formation by the influence of various easy-to-use protocol for large scale production of
types of auxins in many plant species have been plantlets of A.annua through leaf culture and the
reported elsewhere (Mallikadevi and Paulsamy, method is useful for the ex situ conservation of this
2009; Mahesh et al., 2010; Loc et al., 2011; species as well. In addition, the findings of the
Mungole et al., 2011; Rajput et al., 2011). present investigation provide a baseline data for
The hardening experiments showed that high further research in this species.
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178 Journal of Research in Biology (2011) 3: 173-178
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

DNA barcoding, phylogenetic relationships and speciation of
Genus: Plectropomus in Andaman coast
Journal of Research in Biology

Authors: ABSTRACT:
Mohan¹PM, Dhivya¹P,
DNA barcode technique has been recently promoted as a method for both
Chaaithanya²IK and
assigning specimens to known species and for discovering new and cryptic species.
The present study carried out for the barcode sequences for identification of the
Plectropomus leopardus. The results of shorter sequence 113bp of Plectropomus
Institution: leopardus is effective to identify a specimen and confirm up to a species level. This
1. Department of Ocean sequence also exhibited deep interspecific divergences that allowed for efficient
Studies and Marine Biology, discrimination among the Grouper species. This mini barcode sequence technique
Pondicherry University, might be used efficiently to distinguish Plectropomus leopardus with minimal
Andaman and Nicobar expenditure and efforts. These results also support that formalin fixation samples can
Islands, India. also be used for DNA molecular taxonomy.
2. Regional Medical
Research centre (ICMR),
Andaman and Nicobar
Islands, India.

Corresponding author:
Dr. PM. Mohan, Ph.D., DNA barcode, Serranidae, Phylogenetic and Andaman Sea.

Email: Article Citation:, SachithanandamV, Mohan PM, Dhivya P, Muruganandam N, Baskaran R,
Chaaithanya IK and Vijayachari P.
DNA barcoding, phylogenetic relationships and speciation of Genus: Plectropomus in
Phone No: Andaman coast
+91-9434283292 Journal of research in Biology (2011) 3: 179-183

Web Address: Received: 04 Jul 2011 /Accepted: 08 Jul 2011 /Published: 18 Jul 2011
© Ficus Publishers.
This Open Access article is governed by the Creative Commons Attribution License (http://, which gives permission for unrestricted use, non-
commercial, distribution, and reproduction in all medium, provided the original work is properly

179-183 | JRB | 2011 | Vol 1 | No 3
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INTRODUCTION The PCR cycling parameters were as
Pisces phylum is a highly diverse group follows, in 95°C for 5min the Taq Polymerase were
among the vertebrate and exhibit a deep phenotypic initially denatured followed by 40 cycles of
changes during development; its identification and denaturation for 30sec at 95°C, annealing at 58°C
confirmation needs a modern tool. Among all the for 60 sec and extension at 72°C for 60 sec, After
methods DNA barcode is an efficient method for the completion of 40 cycles a final extension step of
species-level identification using an array of species 7mints at 72°C was performed. The PCR products
specific molecular tags derived from 59 region of were tested in 1% Agarose gel, visualized and
the mitochondrial cytochrome c oxidase I (mt COI) photographed using Gel Doc System.
gene (ward et al., 2005). Twenty-eight species have Nucleotide sequencing was performed using
been reported under Epinephelus, from the east and the Sanger method (Sanger et al., 1977) modified
west coasts of India and the islands of by Chen and Seebug (Chen, et al., 1985).
Lakshadweep in the Arabian Sea and Andaman and Sequencing was performed using BigDye
Nicobar in the Bay of Bengal (James et al., 1996). Terminator Cycle Sequencing kit, following
Generally groupers are identified by their colour manufacturer’s instructions (Applied Biosystems,
patterns or a suite of morphologic characters like Foster City, CA, USA). The sequencing was done
body configuration, size, number of body parts etc. both in the forward and reverse directions.
Though generally the colour patterns in medium- Sequence Analysis
sized fishes are distinctive enough to identify The DNA sequences of phenotypically
different species, one need to be aware of identified fishes were assembled using the SeqMan
intraspecific variations in colour patterns of II version 5.03 (DNASTAR) and aligned using
juveniles, which may be completely different from Clustal W pair wise and multiple alignment was
the adults of the same species (Heemstra and completed for phylogenetic identification.
Randall, 1993). Hence identification based on Molecular evolutionary analyses were conducted
morphology in groupers has to be supported by using MEGA version 4 (Tamura et al., 2007).
other techniques including DNA barcoding.
Consequently the phylogenetic based study related RESULT
to serrianidae fishes are very less reported in DNA sequences were submitted to GenBank
Andaman and Nicobar Islands. (PubMed) and their accession number is JF414594
(Table.1). Out of 651 – 655bp basic taxonomic
MATERIALS AND METHODS sequences length, the present study exhibit the
Sample collection and preservation sequences of 113bp for Plectropomus leopardus.
Family Serrianidae (Grouper) were collected All assemblages of conspecific individuals had boot
from local fish landing centre at Port Blair, strap support of very vast species divergences
Andaman. Collected samples were identified compared with other same species DNA sequences
morphometrically with FAO sheets (Heemstra and collected from NCBI genbank and algorithms used
Randall, 1993) and stored in formalin. The piece of in MEGA 4. The overall average within species
muscle collected above the lateral line was stored at K2P distances is 0.21618, with very less in
-20°C for DNA extraction. EU595233 Plectropomus leopardus, for DQ
DNA extraction and PCR reaction 101270 Plectropomus leopardus. While GU
Total DNA extracted from 0.25g of tissue by 674047 perciformes species with 52% were slightly
the standard proteinase-K/ phenol-chloroform- showing higher distances. The congeneric distances
isoamyl alcohol-ethanol method (Sambrook et al., was 34% which was higher than conspecific
1989). An approximately 650bp section of the distances of species compared with Andaman
mitochondrial (mt) DNA genome from the COI Plectropomus loepardus distance and its range is 10
gene was amplified using published universal - 11%.
primer of two set (Ward et al., 2005).
The polymerase chain reaction (PCR) DISCUSSION AND CONCLUSIONS
components per 50ml reaction were as follows : The sequence results of DNA barcode for
PCR buffer (10X), MgCl2 (25mM), dNTP (10mm), Plectropomus leopardus revealed the potential
Forward Primer (30ng/ml), Reverse Primer (30ng/ ability of mini-barcodes to discriminate among
mL) 1ml, Taq polymerase (3U), ultra pure water, species. While mini-barcodes produced divergence
DNA (100ng/ml). values comparable to full-length barcodes in both
180 Journal of Research in Biology (2011) 3: 179-183
Sachithanandam et al.,2011

Table 1 DNA Sequences details of Plectropomus leopardus in GenBank files version
Definition Locus Plectropomu leopardus COI gene CDS
Accession Number JF414594
Submitted Date VRT 08-APR-2011
Classification Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Actinopterygii; Neopterygii; Teleostei; Euteleostei; Neoteleostei;
Acanthomorpha; Acanthopterygii; Percomorpha; Perciformes; Percoidei;
Serranidae; Epinephelinae; Plectropomus.
Alignment of Partial Sequence of >1 tcattaacat gaaaccccct gctatctctc aataccagac ccccctattc gtatgagcag tcctaattac
COI Gene 113bp. Plectropomus tgcagttctt cttctcctat cactacctgt tctagctgct gga.
Protein_ID, ="AEB31228"

data sets, they were somewhat less effective in The study has successfully proved the utility
discriminating among the species in large of COI divergences in identifying all the
assemblages (e.g. 204 species of Australian fishes). Plectropomus leopardus fishes in Andaman Sea
However, most applications of mini barcodes will with minimum base pair (113). The present
not involve cases that seek to place a specimen analysis was not meant to be exhaustive, but to
among all known species, but rather within a small highlight the most important feature of mt COI
assemblage. In the species of Plectropomus sequences in distinguishing closely related species
leopardus, mini-barcodes were positioned and intra-specific distances which prove beyond
specifically to discriminate this species pair, and doubt. Further studies involving other family and
their relatively short lengths took into account the groups of marine fishes of the area and also by
old age of the specimens. In addition, the fact that increasing the sample size in future studies will
the forward primers in the two mini-barcodes were clarify the issues.
designed specifically for this species complex
might have positively influenced the chance of ACKNOWLEDGMENT
amplifying potentially degraded DNA. We are thankful to the authorities who
Interestingly, a similar sized fragment from the provided facility, to carry out this work in
other end of the barcode region would have Pondicherry University and Regional Medical
achieved identification. Full-length barcode Research Centre (ICMR), Port Blair.
sequences can be easily and cheaply obtained from
recently collected tissue or from those preserved for REFERENCE
DNA extraction (Hajibabaei et al. 2005). Chen EY, Seeburg PH. 1985. Supercoil
The resultant records provide a ‘gold sequencing: a fast and simple method for
standard’ with high confidence for species sequencing plasmid DNA. DNA 2:165-170.
discrimination within large species pools (Hebert et
al. 2003a, 2003b; Smith et al. 2005, 2006; Ward et Fakhrai-Rad H, Pourmand N, Ronaghi M. 2002.
al . 2005; Hajibabaei et al. 2006). This study Pyrosequencing: an accurate detection platform for
confirms that short barcode sequences (113bp) are single nucleotide polymorphisms.
also valuable for the identification of specimens Human Mutation 19:479-485.
from selected narrow taxonomic arrays, such as
comparing a newly collected specimen. Similarly, it Hajibabaei M, Gregory A, Singer C, Hickey DA.
may well be possible to employ mini-barcodes for 2006. Benchmarking DNA Barcoding: an
the identification of formalin-fixed samples, which assessment using available primate sequences.
often contain highly fragmented DNA (Schander & Genome 49:851-854. Doi:10.1139/G06-025.
Kenneth 2003). Mini-barcodes may also provide the
option to employ alternative sequencing methods, Hajibabaei H, DeWard JR, Ivanova NV,
such as pyrosequencing (Fakhrai-Rad et al. 2002), Ratnasingham S, Dooh RT, Kirk SL, Mackie
that yield only short sequences, but offer lower PM, Hebert PDN. 2005. Critical factors for
costs and higher throughput than standard assembling a high volume of DNA barcodes. Phil.
approaches. Trans. R. Soc. B. 10:1-9.

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Hebert PDN, Cywinska A, Ball SL, DeWard JR. Sanger F, Nicklen S, Coulson AR. 1977. DNA
2003a. Biological identifications through DNA Sequencing with chain terminating inhibitors. Proc.
barcodes. Proc. R. Soc. Lon. B. 270: 313-321. Doi Natl. Acad. Sci. USA. doi:10.1073/pnas.74.12.5463.
10. 1098/rspb. 2002.2218. 74:5463-5467.

Hebert PDN, Ratnasingham S, DeWard JR. Schander C, Kenneth HM. 2003. DNA, PCR and
2003b. Barcoding animal life: cytochrome c formalinized animal tissue - a short review and
oxidase subunit1 divergences among closely related protocols. Organisms Diversity and Evolution
species. Pro. Royal Soc. Lon. B. 270:S96-S99. 3:195-205.

Heemstra PC, and Randall JE. 1993. Groupers of Smith MA, Fisher BL, Hebert PDN. 2005. DNA
the World. FAO Fisheries Synopsis. 16:1-125. barcoding for effective biodiversity assessment of a
hyperdiverse arthropod group: the ants of
James PSBR, Murthy VS, Nammalwar P. 1996. Madagascar. Phil. Trans. R. Soc. Lon. B., 360:1825-
Groupers and snappers of India: biology and 1834.
exploitation. In: Biology, Fisheries and Culture of
Tropical Groupers and Snappers, Arreguin-Sanchez Tamura K, Dudley J, Nei M, Kumar S. 2007.
F, Munro JL, Balgos MC, Pauly D. (Eds.). ICLARM MEGA4: Molecular Evolutionary Genetics
Conf. Proc. 48:106-136. Analysis (MEGA) Software Version 4.0. Molecular
Biology and Evolution, 24:1596-1599.
Sambrook J, Fritsch EF, Maniatus T. 1989.
Molecular cloning: a laboratory manual, 2nd Ward RD, Zemlak TS, Innes BH, Last PR,
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Spring harbour, NY.13. fish species, Phil. Trans. R. Soc. Lon. B., 360:1847-

182 Journal of Research in Biology (2011) 3: 179-183
Sachithanandam et al.,2011

Fig. 1. Neighbor-joining trees based on the mtDNA COI gene nucleotide sequences of Plectropomus species
analyzed. Numbers at nodes are bootstrap values based on 1000 replicates. The scale bar represents an interval
of Tamura-Nei genetic distance for Plectropomus leopardus.

57 DQ107920.1 Plectropomus leopardus
51 DQ107911.1 Plectropomus maculatus
93 DQ107912.1 Plectropomus maculatus
FJ583869.1 Plectropomus maculatus
70 FJ583869.1| Plectropomus maculatus
DQ107921.1 Plectropomus leopardus

62 EU595233.1| Plectropomus leopardus
ANDAMAN Plectropomus leopardus
31 DQ101270.1 Plectropomus leopardus
DQ108065.1 Lampris immaculatus
EF143386.1 Niphon spinosus
99 GQ329867.1 Lutjanus cyanopterus
46 GU440386.1 Lutjanus novemfasciatus
EU502685.1 Lutjanus argentimaculatus
AP006000.1 Lutjanus rivulatus
EU600138.1 Lutjanus kasmira
41 77
EU600135.1 Lutjanus bengalensis
EF609399.1 Lutjanus quinquelineatus
28 EU502667.1 Lutjanus fulvus

EF607554.1 Seriola dumerili
99 FJ237809.1 Lutjanus lutjanus
EU600141.1 Epinephelus fario
99 FJ237765.1 Epinephelus bleekeri
33 EU600147.1 Epinephelus akaara
HQ174862.1 Epinephelus fuscoguttatus
FJ583012.1 Cephalopholis urodeta
DQ107868.1 Epinephelus areolatus
DQ107886.1 Epinephelus multinotatus
FJ237772.1 Epinephelus spilotoceps
33 6 EF609352.1 Epinephelus undulosus
EF60922.1 Epinephelus longispinis
18 FJ237734.1 Epinephelus amblycephalus
FJ583397.1 Epinephelus ongus
99 EU266383.1 Epinephelus moara
7 FJ594964.1 Epinephelus bruneus

GU673644.1 Perciformes sp.
EF609518.1 Epinephelus diacanthus
FJ583396.1 Epinephelus adscensionis
EU595118.1 Epinephelus sexfasciatus

0.12 0.10 0.08 0.06 0.04 0.02 0.00

Journal of Research in Biology (2011) 3: 179-183 183
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

The comparative analysis of the Optical Density for MRET activated water and non-
activated water in visible, ultraviolet, and infrared regions of the spectrum
Journal of Research in Biology

Authors: ABSTRACT:
Igor Smirnov MRET activated water is produced with the help of the USA patented
Molecular Resonance Effect Technology (MRET). The anomalous electrodynamic
characteristics (dielectric permittivity and electrical conductivity) of MRET water
subjected to applied electromagnetic field in the area of very low range of frequencies
and the anomalous viscosity of MRET water subjected to very low tangential pressure
Affiliation: Global have provided some evidence for the long range dynamic polarized-oriented
Quantech, Inc. Encinitas, multilayer structuring of such activated water .
California, USA. The objective of this article is to demonstrate the relatively high, long range
dynamic structuring of water molecules in activated water produced with the help of
the MRET activation process. To achieve this goal there were conducted a number of
the comparative analysis of the optical density for MRET activated water and non-
activated water in visible, ultraviolet (UV), and infrared (IR) regions of the spectrum.
Corresponding author: This analysis confirms that MRET activation process has the tendency to force water
Igor Smirnov molecules to form large size clusters in the volume of water.
Such findings provide some evidence that MRET activation leads to the
formation of polarized-oriented multilayer structuring of water molecules. The
correlation analysis of the scattered laser emission also provides the unambiguous
confirmation of the presence of the stable water memory phenomenon. The duration
Email: of water memory phenomenon was observed for many hours and days in this
particular experiment. The water memory phenomenon can be interpreted as the
existence of stable super-molecular clusters in the volume of activated water.

Web Address: Keywords: optical density, electrodynamics characteristics, viscosity, polarized-oriented.

Article Citation:
Igor Smirnov.
The comparative analysis of the Optical Density for MRET activated water and non-
activated water in visible, ultraviolet, and infrared regions of the spectrum.
Journal of research in Biology (2011) 3: 184-190

Received: 21 Jun 2011 /Accepted: 24 Jun 2011 /Published: 18 Jul 2011

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

184-190 | JRB | 2011 | Vol 1 | No 3
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Submit Your Manuscript
An International Open Access Online
Research Journal
Smirnov et al.,2011

INTRODUCTION of MRET activated water subjected to applied
MRET activated water is produced with the tangential pressure in the area of very low
help of the USA patented Molecular Resonance magnitudes provide some evidence regarding
Effect Technology (MRET). The MRET water polarized-oriented multilayer structuring of MRET
activator is the stationary source of a subtle, low activated water and the possible effect of MRET
frequency, resonant electromagnetic field with a water on the proper functioning of cells in
composite structure. The origin of low frequency biological systems.
composite electromagnetic field is the intensive The fundamental biophysical theories
electrical activity inside nano-circles formed by revealed the scientific paradigm regarding polarized
linear molecular groups of MRET polymer -oriented multilayer structuring of cell water in
compound (volumetric fractal geometry matrix) biological systems. The suggested model of
when the polymeric body is exposed to external polarized-oriented multilayer structuring of cell
electromagnetic fields of specific frequency and water due to the interaction of water dipoles with
wavelength. Research conducted at Moscow State pervasive matrix of fully extended proteins
University, Russia provides evidence that distilled constitutes the basis for the cellular transduction
water after MRET activation process posses mechanism [Drost-Hansen at el., 1991]. Based on
anomalous physical properties (dielectric this scientific approach, the similarity of molecular
permittivity, electrical conductivity and viscosity) formations of cell water and MRET activated water
compared to distilled non-activated water from the can contribute to their compatibility, easy
same source. bioavailability and assimilation of MRET activated
The electrical conductivity of MRET water, as well as to the enhancement of cellular
activated water at 20°C in the range of frequencies functions in biological systems.
0.1Hz – 100 kHz decreased by 77–90% in 30 The objective of this article is to
minutes activated water and by 66–70% in 60 demonstrate the relatively high, long range dynamic
minutes activated water compared to non-activated structuring of water molecules in activated water
distilled water respectively. The dielectric produced with the help of the MRET activation
permittivity in the very low frequency range 0.1– process. To achieve this goal there were conducted
1000 Hz decreased by 80–90%, and in the range of a number of the comparative analysis of the optical
frequencies 1–100 kHz and decreased by 18% in 30 density for MRET activated water and non-
minutes activated water; a decrease by 70–85% was activated water in visible, ultraviolet (UV), and
observed in the range 0.1–1000 Hz in 60 minutes infrared (IR) regions of the spectrum.
activated water compared to non-activated water
[Smirnov, 2008]. MATERIALS OF METHODS:
The anomalous low viscosity of MRET The finding of the sharp increase of the
activated water in the area of very low magnitudes absorption of the UV emission in the region of
of tangential pressure applied to the water was wavelengths shorter than 190 nm is related to
discovered during another experiment. The several mechanisms. The main mechanism in the
experiment revealed that at a very low velocity of region of the near-UV emission is conditioned by
motion of water (tangential pressure in the range of transitions in the electron subsystem of a H2O
0.004–0.005 Pa, temperature 20°C), the viscosity of molecule which lead to the dissociation of this
MRET water activated for 60 minutes decreased by molecule and the formation of the ground (non-
200–250 times compared to non-activated water excited) state of the system H + OH. Under the
from the same source. The most significant action of the emission with shorter wavelengths, the
phenomenon of anomalous low viscosity of following becomes essential: the processes leading
activated water; a decrease by 300–500 times, was to the dissociation of molecules of water with the
observed for water activated for 30 minutes formation of excited molecules OH∗ and radicals H,
[Smirnov, 2007]. as well as the radiolysis products H2 and O. Fig. 1
The anomalous behavior of the presents the results of measurements of the optical
electrodynamics characteristics (dielectric density in the region of near-UV emission for non-
permittivity and electrical conductivity) of MRET activated water and for MRET water activated for
activated water subjected to applied 0.5 hour.
electromagnetic field in the area of a very low The same fraction of MRET activated water
range of frequencies, and the anomalous viscosity (the duration of activation was equal to 30 min) was
185 Journal of Research in Biology (2011) 3: 184-190
Smirnov et al.,2011

Fig. 1 Optical density of non-activated water (1) and Fig. 2 IR-spectra of non-activated water (upper
MRET water activated for 30 min (2) versus the curve) and activated water that was studied in 5
wavelength in the visible and UV ranges. Both plots hours after the activation (middle curve) and in 1
visually coincide with each other [Vysotskii et al., 2009]. hour (lower curve) [Vysotskii et al., 2009].

studied by the method of infrared Fourier- conducted on four different samples of the initial
spectroscopy with the help of a device “Nexus” non-activated water (see Fig.5):
produced by the firm “Thermo Nicolet”. The 1. Natural Mineral Water “AVIAN” (produced in
studies were performed in the range of wave France);
numbers from 50 to 4000 cm−1 (Fig.3). 2. FreshWater Life “JINBO/Seok Su” (produced
MRET activated water was also studied by in the Republic of Korea);
Raman scattering spectroscopy. The Raman 3. Natural Mineral Water “HAITAI/Gangwondo/
scattering spectra for non-activated water and for Pyeong Chang” (produced in the Republic of
MRET activated water was studied in 24 hours after Korea);
the completion of the activation and are presented 4. Natural Mineral Water “JEJU/Sa Da
in Fig. 4. The studies were performed with the same Soo” (produced in Iceland).
fraction of water (activation for 30 min). The
spectra determine the Raman scattering RESULTS:
characteristics in the region from 1 cm−1 to 4000 Visually, both plots in Fig.1 coincide with
cm−1. each other. This corresponds to that almost no
With the purpose to discover the presence of difference between the spectra of the initial non-
structural elements in the water bulk there carried activated water and activated one observed in the
out an additional study in the optical characteristics visible and UV regions of the absorption spectrum
of MRET activated water with the help of the laser in the limits of accuracy of the method (0.5%),
correlation analyzer AKVA-01. The principle of except for a small increase in the absorption, at
action of this device is based on the analysis of the most 1–2%, in the UV region of the spectrum at
characteristics of a mutual temporal coherence wavelengths near λ = 190–210 nm.
function of the laser emission scattered in the This result differs basically from the above
volume of the studied water in the direction mentioned very essential changes in the
perpendicular to the initial laser beam. There was electrodynamics characteristics of activated water
used the emission with a wavelength of λ = 0.63μm in the region of low frequencies. It shows that the
generated by a He–Ne laser was used in the process of activation by means of MRET activation
analysis. The longitudinal coherence length of such does not lead to a significant change of the electron
an emission prior to its scattering is very large and subsystem of atoms and involves only structural
exceeds hundreds of meters. The width of the and juxtaposition changes of the water molecular
spectrum of this emission is very small [Vysotskii system.
et al., 2009]. The results of the study in various regions of
The laser spectroscopy measurements were IR-spectra are presented in Figs. 2. This diagram

Journal of Research in Biology (2011) 3: 184-190 186
Smirnov et al.,2011

Fig. 3 IR-spectrum of non-activated water (upper Fig. 4 Raman scattering spectra of non-activated (1)
curve) and water activated for 30 min (lower curve). and activated (2)water. Fragments of the spectra (1a)
The spectrum was studied in 1 hour after the and (2a) correspond to the increase in the scale by 10
activation [Vysotskii et al., 2009]. times. All spectra were measured in 24 hours after
the activation [Vysotskii et al., 2009].
gives the IR-spectra in the range of 50 – 550 cm−1
for the initial non-activated water and MRET water cm−1. This result is presented in Fig. 3 for non-
activated for 30 min. The study was carried out activated water and water activated for 30 min (the
with three samples of water derived from the spectrum of activated water was studied in 1 hour
identical distilled water: after the activation).
 Initial distilled water; MRET activated water was also studied by
 MRET activated water stored after the Raman scattering spectroscopy. It is seen from the
activation prior to the measurement for 30 min data presented in Fig. 4 that MRET activation of
at room temperature; water, it leads to a small increase of the Raman
 MRET activated water which was stored after scattering amplitude in the whole range from 1 cm−1
the activation prior to the measurement for 5 to 4000 cm−1.
hours at room temperature. The optical characteristics of MRET
Such study allows us to determine the activated water was additionally studied with the
dependence of the influence of the activation on the help of laser spectroscopy.
IR-spectrum of water, as well as the influence of the As a quantitative characteristic there was used the
storage duration of this water on its properties. It is integral index of the molecular dynamics of water
seen from Fig. 2 that a decrease in the absorption W which is inversely proportional to the coefficient
of water (a decrease of the imaginary part of the of diffusion D of the scattering objects including
dielectric permittivity) in the long wave part of the the normalizing factor (in order to make W
IR- occurs in the process of MRET activation. This dimensionless).
decrease exceeds 10% for the range of wave The parameters of the scattered emission
numbers less than 300 cm−1. depend on the motion of scattering molecules of
With increase of the wave number, the water and the ensembles of these molecules. Due to
absolute value of a change in the coefficient of the presence of the fluctuation (diffusive)
absorption remains approximately constant, and the mechanical, motion of the scattering objects, the
relative change decreases, respectively. With phase of the scattered emission continuously
increase of the duration of storage of activated fluctuates, which decreases very sharply the
water, the discovered change in the optical density coherence length of the scattered laser field. A
in the IR-range gradually decreases. change in the coherence length of the emission and
The effect of a small decrease in the optical the associated change in the spectrum of the
density of MRET activated water is also observed emission are those parameters of which the
in the region of great wave numbers 1000–4000 measurement allows us to determine the
187 Journal of Research in Biology (2011) 3: 184-190
Smirnov et al.,2011

activation). The characteristics of water sample #3
after the completion of all actions and all
measurements turned out to be equal to the initial
values of the index W0 which was determined prior
to the beginning of the cycle of measurements
[Vysotskii at el., 2009].

The obtained experimental data provides
evidence that the specific features of the spectrum
of MRET activated water in IR, visible, and UV
ranges preserve for a long time after the completion
of the activation, though the very changes in the
spectrum in these ranges turned out to be a small
Fig. 5 Integral index W of the molecular dynamics ones. Such changes in the optical properties of
of activated water of different types versus time. The MRET activated water which are small in
numbers stand for the types of water mentioned magnitude but with the long time of their
above in the text [Vysotskii et al., 2009].
preservation can be satisfactorily substantiated if we
characteristics of the motion of scattering objects consider that they are related to a change in the
(the coefficient of diffusion) and, hence, the mass properties of a relatively small number of separate
and size of these objects. The coherence length can molecules of water isolated from the external action
be found with the help of the autocorrelation and corresponding to the hindered relaxation. Based
function (1.1). on the proposed model of the memory of water, we
It is seen (Fig.5) that the index W was can refer such changes to the molecules of water
changed comparatively slightly (except for water being in the volume of clathrate microcavities
sample #3) for the time of the first activation [Vysotskii at el., 2005].
process. Thereafter for 24 hours (with an interval of It was proposed earlier that there exists a
two hours) there was measured the index W for all strong repulsive electrostatic field in such
types of water. It is seen that, for this time interval, microcavities. For this reason, the internal “walls”
there occurred a systematic increase of the index W of microcavities possess the hydrophobic properties
for all types of water except for water #3. In this and do not form hydrogen bonds with molecules of
case, a change in W was accompanied by very water placed in the volume of microcavities. These
strong oscillations for the water samples #2 and #3. molecules, due to the absence of a hydrogen bond
At the same time, the index W for all the remaining with molecules of water forming the “walls” of
types of water was changed as a monotonously microcavities, have a changed configuration of the
increasing function of time. There was performed electron shell (it corresponds to a free H2O
the second activation of water in 24 hours after the molecule, rather than that of a bound molecule) and,
first activation with the same duration of one hour. respectively, some different optical properties
The results of this activation also turned out [Vysotskii at el., 2009].
ambiguous. After the activation, there was observed The process of MRET activation renders a
a very sharp increase of the index W for water strong influence on the dynamics of the mechanical
sample #4. At the same time, this index was fluctuation motion of stable global structural
practically constant for the remaining samples of elements in the water bulk. The formation and the
water. The measurements after the second long-term existence of such structural elements
activation were performed for 42 hours (At first, depend on the activation of water and confirm the
three measurements were carried out with an possibility for the memory of water to exist. To find
interval of two hours and then the other three such structural formation in the body of MRET
measurements with an interval of 12 hours). After activated water there was used the laser
the completion of the whole cycle of measurements, spectroscopy method. The study of the correlation
the values of the index W for all samples of water spectrum allows us to determine the mean
(except for water sample #3) turned out to be characteristics of the motion of scattering objects in
exceeding the initial value W0 (prior to the first the water bulk and, on this basis, to perform the

Journal of Research in Biology (2011) 3: 184-190 188
Smirnov et al.,2011

qualitative analysis of the spatial structure of water. size of a stiffly bound molecular complex which is
In addition, such studies allow one to determine the present in the water bulk and scatters the laser
dependence of these characteristics on the emission. There was performed the following
activation of water. procedure and the sequence of studies that total
In order to determine the regularities of the duration was equal to 71 hours. First of all there
process of scattering, it is necessary to determine was carried out three measurements of the index W
those quantities which are not random and describe for 3 hours for each of the studied types of water.
unambiguously the scattering. The deterministic The purpose of these measurements were related to
(nonrandom) characteristics of the scattered the determination of the stability of a measuring
emission can be found if the autocorrelation unit. The results of this “testing period” correspond
function of field is known: to the first three points on all the plots presented in
Fig. 5. The very small variance of the index W
confirms a high degree of stability, a small value of
the apparatus error, and the error related to the
procedure of measurements. It is obvious that the
differences of the indexes of molecular dynamics W
for different types of water prior to the activation
are related to a great extent to the differences of
(1.1) their salt composition. This is explained by the fact
Where: E0 is the amplitude of an incident wave; that the ions dissolved in water influence the mass
and size of molecular complexes in water and,
is the scattering amplitude of the
hence, the coefficient of diffusion of these
complexes and the spectrum of the scattered laser
emission. It is possible to assume that such an
is the wave vector scattered by an angle ; influence can be realized in at least two ways: on
R is the distance from the region center in the water the one hand, the dissolved ions break the chemical
bulk, where the objects scattering the light are homogeneity of water and therefore affect the
positioned; formation of molecular complexes formed from
N is a number of scattering objects in the volume molecules of water; on the other hand, the union of
of water; dissolved ions with these complexes changes the
E is the amplitude of the scattered electric field density and the mass of the latter. It is obvious that
intensity; both mechanisms lead to the essential influence of
D is the diffusion coefficient of scattering objects; dissolved salts on the spectrum of the scattered
The spectrum of the scattered emission is emission, which was observed in the study of
calculated with the help of the Wiener –Khinchin various samples of water.
formula: After the completion of the whole cycle of
measurements, the values of the index W for all
samples of water (except for water sample #3)
turned out to be significantly exceeding the initial
value W0 (prior to the first activation) that allows to
assume the formation of the water molecular super-
clusters in the volume of water after MRET
activation process.
(1.2) The results of the optical study of MRET
It is seen from formula (1.2) that the activated water in UV, IR spectrum, and Raman
spectrum of the scattered emission depends on the scattering spectra also provide evidence of the
coefficient of diffusion D defining the mean stable polarized-oriented multilayer molecular
characteristics of the motion of scattering objects. formation in activated water. It shows that the
This coefficient, in turn, depends on the size and process of activation does not lead to a significant
mass of these objects. change of the electron subsystem of atoms and
An increase of the index W corresponds to a involves only structural and juxtaposition changes
decrease of the coefficient of diffusion and of the water molecular system. The confirmation of
characterizes uniquely the increase of the mass and such molecular formations can explain the
189 Journal of Research in Biology (2011) 3: 184-190
Smirnov et al.,2011

phenomenon of anomalous viscosity and REFERENCES:
electrodynamic characteristics of MRET activated Drost-Hansen W and Singleton JL. 1991. “Our
water [Smirnov 2007, 2008]. aqueous heritage: evidence for vicinal water in
cells”, Fundamentals of Medical Cell Biology, JAJ,
There can be made the following
conclusions based on the experimental results and Smirnov IV. 2008. “The Anomalous
analysis of measurements: Electrodynamic Characteristics and Polarized-
There was found both a very significant Oriented Multilayer Molecular Structure of MRET-
increase of the degree of correlation of the scattered Activated Water” International Journal of
emission and a decrease of the coefficient of Nanoscience, World Scientific Publishing. Vol.7
diffusion of scattering complexes in water that (4) and (5):1-5.
testifies unambiguously the formation of very large
and stable clusters in water after MRET activation. Smirnov IV. 2007. “The Anomalous Low
The results of the comparative optical study of Viscosity and Polarized-Oriented Multilayer
MRET activated water and non-activated one in Structure of MRET Activated Water” Explore
UV, IR spectrum, and Raman scattering spectra Magazine, USA. Vol.16 (4):37-39.
show that the process of MRET activation does not
lead to a significant change of the electron Vysotskii VI, Kornilova AA, Smirnov IV. 2009.
subsystem of atoms and involves only structural “Applied Biophysics of Activated Water: The
changes of the water molecular system. Physical properties, Biological Effects and Medical
The processes of formation and change of Applications of MRET Activated Water”, World
these molecular clusters did not stop after the Scientific Publishing, Singapore.
termination of the activation but continue for many
hours and days. Such an effect can occur in the case Vysotskii VI, Smirnov IV, Kornilova AA. 2005.
where the activation of water induces the change of “Introduction to the Biophysics of Activaterd
the properties of separate water molecules and Water”, Universal Publishers, USA.
strongly-bound molecular groups in a way that their
further joining in great clusters becomes energy-
gained. The process of formation of stable clusters
in the volume of water depends significantly on the
salt composition of water. In this case, different
types of mineral water are characterized by
different dependences of the parameter W on the
time interval after the activation.
Such findings provide evidence that MRET
activation may lead to the formation of polarized-
oriented multilayer structuring of water molecules.
The correlation analysis of the scattered laser
emission also provides the unambiguous
confirmation of the assumption about the presence
of the stable water memory, the duration of
existence of which can be equal to many hours and
days. This memory can be interpreted as the
existence of stable super-molecular clusters in the
volume of water.

I deeply acknowledge Prof. Vysotskii V. I.
and Kornilova A.A., Ph.D. Moscow State
University, Russia, and Helios Alpha (USA) for
their help in my research work.

Journal of Research in Biology (2011) 3: 184-190 190
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Study of morphomatric biology of cotton Pygmy-Goose Nettapus
coromandelianus coromandelianus Gmelin
Journal of Research in Biology

Authors: ABSTRACT:
Upadhyaya S1 and
Saikia2 PK. Morphomatric of Cotton Pygmy-goose, Nettapus coromandelianus
coromandelianus was studied during 2006 to 2008. The males are comparatively
bigger in size than the females. The average weight was found to be 226.50 gm and
219.50 gm for male and female respectively. The primary (wing) feather arrangement
1. Deptt. of Zoology, in male was found to be P1 < P11 < P10 < P9 < P8 < P7 < P2 < P6 <P5 < P3< P4. The
Tyagbir Hem Baruah females have a more or less similar arrangement except the P2 and P6 where P6<P2.
College, Karchantola, The mean length of the middle toe in male was found to be 34.32+0.194 mm; where
Dist.-Sonitpur (Assam), as in female the same remains 0.5 mm shorter (+0.163). The wing expansion was
India. PIN-784189. ranged between 424 mm to 426 mm in both male and female, but with slight variation
2. Department of Zoology, in mean value (male- 425.17+0.753 mm; female- 425.53+0.816 mm). Since no
Gauhati University, Assam morphomatric studies has been done so far on this species, the present paper was
India. hypothesized to the study of morphomatric variation in various aspects of Cotton
Pygmy-goose indicating the relation of wings, hind-limbs, head neck, beak, tarsus,
different types of toes and tail in respect to the habitat utilization and ecology of the
Corresponding author:
Dr. Sanjib Upadhyaya

Email: Keywords: Eco-morphology, middle toe, tarsus and wing expansion.

Article Citation:
Web Address: Upadhyaya S and Saikia PK.
Study of morphomatric biology of cotton Pygmy-Goose Nettapus coromandelianus
coromandelianus Gmelin.
Journal of research in Biology (2011) 3: 191-201

Received: 27 Jun 2011 /Accepted: 08 Jul 2011 /Published: 18 Jul 2011

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

191-201 | JRB | 2011 | Vol 1 | No 3
Journal of Research in biology
Submit Your Manuscript
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Research Journal
Upadhyaya et al.,2011

INTRODUCTION The non-protected areas of Sonitpur district
Avian morphology was a major focus of of Assam (India) were selected for the
interest within avian biology during the last century morphomatric study of Cotton Pygmy-goose. The
and the first decades of this century. The study of Sonitpur District of Assam, with an area of about
morphology provides the data to understand the 5,25,520 hectares, is located in between 9302/80// E
evolutionary as well as ecological questions. to 930 57/1// E longitude and 260 22/1// N to 26 0
Though the morphology of various birds including 42 / 2// N latitude. The district is bounded by
the anatid have been studied by various Hawajan tributary in the east, Pachnoi tributary in
ornithologists the morphology of the Cotton Pygmy the west, the mighty river Brahmaputra in the south
-goose, Nettapus coromandelianus and the state Arunachal Pradesh (previously North
coromandelianus Gmelin still awaiting a detailed Eastern Frontier Area or NEFA) in the north.
study. This species is very poorly described for Physio-graphically, major parts of the district are
which it was considered as bird of Least Concern plain area with a number of tributaries like Pachnoi,
(Birdlife International 2004). Though there is no Mora-Bhoroli, Jia-Bhoroli, Ghiladhari, Buriganga,
extensive research have been done so far in this Borgang, Buroi, and Satrang arose from the hills of
bird, the works of Ali and Ripley (1983), Whistler Arunachal Pradesh and joins with the river
(1986) and Ali (2002) are quite remarkable in this Brahmaputra. Physically, a massive part of its area
regard. More importantly, theoretical analyses of is covered with evergreen and semi-evergreen type
questions relating to evolutionary morphology were of forests accounting about 20.52% of its area.
published during the 1950’s. These includes the
study on pre-adaptation (Bock 1959), and METHODOLOGY
adaptation (Bock and von Wahlert 1965, Bock The morphomatric of Nettapus c.
1980). There lies a seasonal variation in weight, coromandelianus Gmelin was studied as per
body measurements and condition of free-living methods explained by Bibby et al. (1992) and
Teal (Fox et al. 1992). Ecologists were analyzing a Balachandran (2002). The birds were collected
series of questions on the concept of the niche, from different parts of the Sonitpur district. Only
habitat partition, community structure, diversity living or naturally died birds were selected for the
within the taxa etc., and uses simple morphological study. Living birds were released after observation.
measures as tools. The body temperature was recorded with the help of
The N. c. coromandelianus Gmelin is the a Centigrade thermometer from the living birds at
smallest of our wild ducks (Anon. 1965) with a the field. The body weight was measured with the
length of about 13-inches (Ali 2002, Whistler help of a spring balance (capacity 500 gm). The
1986). The breeding male has a blackish brown body length, wing length and wing expansion were
crown and back face, neck and under parts white, measured with the help of a meter scale (least count
with prominent black collar round base of the neck, 0.1 mm). The length of bill, neck, tarsus, middle
and white wing-bar (Ali and Ripley 1983). The toe, wing feathers and rectrices were measured with
male has broad white band across the wings the help of a pointer and slide calipers (0.001 mm
(Grimmett 1999) but during winter it loses its accuracy).
collar and resembles the female except for the white The data so obtained were arranged and
wing bar and some of the green gloss on the upper mean, standard deviation and co-efficient of
plumage and wings. The female don’t have a correlation (Relief 1965) were calculated to detect
colorful body. The females have a brown top of the the relationship amongst various aspects of their
head and a line through the eye, the rest of the head body parts using computer software package MS-
and neck being speckled with the brown marks. excel.
Upper parts of the body wings and tail are brown in
color (Whistler 1986). The present paper deals with RESULTS
the study of morphomatric variation in various a. Body weight (BoW)
aspects of Cotton Pygmy-geese viz. wings, hind- The mean BoW (irrespective of sex) of the
limbs, head, toes, neck, beak or bill, tail, etc. and Cotton Pygmy-goose was found to be 224.1
correlation between different parameters in male gm+13.5SD (range = 180.65 to 260.0 gm, n =42,
and female so as to explore its relation with the Table 5.1, Figure 5.1). The mean BoW of male
wetland environment. goose was estimated to 221.2 gm +16.4SD (range =
180.65 to 260.0 gm, n =27), whereas in female the
192 Journal of Research in Biology (2011) 3: 191-201
Upadhyaya et al.,2011

Table 1. Morphomatric variation in male and female Cotton Pygmy-goose (n =42)
Parameters Range (male) Mean +SD Range (female) Mean +SD
1. BoW (gm) 180.6* - 260.0 221.2 +16.4 215.5 - 236.34 227.0 +6.9
2. BoL (mm)BT to TT 332.9 - 336.5 335.3 +1.1 350.6 - 353.4 351.5 +1.7
3. BoL (mm) N to A 143.3-147.0 145.7 +1.1 162.0 - 165.0 163.5 +1.2
4. NL (mm) 42.0 - 43.5 42.6 +0.4 40.0 - 42.1 41.4 +0.7
5. NC (mm) 86.7 - 87.2 87.0 +0.2 86.5 - 87.0 86.8 +0.2
6. BL (mm) BT to F 26.2 - 26.9 26.6 +0.2 26.2 - 26.8 26.5 +0.2
7. BL (mm) BT to FC 22.4 - 23.2 22.8 +0.3 22.3 - 22.9 22.7 +0.3
8. BD (mm) 15.1 - 15.6 15.4 +0.2 15.1 - 15.4 15.3 +0.1
9. BW (mm) 12.0 - 12.7 12.3 +0.2 11.5 - 12.0 11.8 +0.2
10. HC (mm) 93.6 - 94.2 93.9 +0.2 92.7 - 93.2 92.9 +0.2
11. TL (mm) 28.9 - 29.3 29.1 +0.1 28.9 - 29.2 29.1 +0.1
12. HTL (mm) 8.0 - 8.3 8.2 +0.1 7.9 - 8.2 8.1 +0.1
13. ITL (mm) 31.4 - 32.0 31.8 +0.2 31.5 - 31.8 31.6 +0.1
14. MTL (mm) 34.0 - 34.5 34.3 +0.2 34.1 - 34.5 34.3 +0.2
15. OTL (mm) 33.0 - 33.5 33.3 +0.2 33.0 - 33.5 33.3 +0.2
16. IWD (mm) 19.9- 20.6 20.2 +0.2 19.0 - 20.6 20.2 +0.2
17. OWD (mm) 16.2 - 16.8 16.5 +0.3 16.2 - 16.8 16.4 +0.2
18. WS (mm) 424.0 - 426.0 425.3 +0.7 424.0 - 426.0 425.3 +0.7
19. WL (mm) 168.0 - 171.0 169.3 +0.9 168.0 - 171.0 169.1 +0.9
20. PWF (mm) 107.1 - 107.3 107.2 +0.07 101.1 -101.4 101.2 +0.1
21. SWF (mm) 85.5 - 85.6 85.6 +0.06 80.6 - 80.7 80.7 +0.05
22. SFL (mm) 41.9 - 42.6 42.2 +0.3 41.7 - 42.1 41.9 +0.1
23. TaL (mm) 63.4 - 64.7 64.03 +0.5 64.1 - 64.8 64.53+0.2
24. TFL (mm) 73.36 - 73.46 73.42 +0.04 74.9 - 75.06 75.01+0.1
*Found after three days captivity in a poacher’s resident (see text for explanation)

same found to be 227.0 gm + 6.8SD (range= 215.5 =332.9 to 336.5 mm, n =27), whereas the mean
to 236.34 gm, n =15). A greater BoW (in either sex) BoL (BT to TT) measured in female was 351.5 +1.6
was found during the monsoon season with a mean mm (range =350.6 to 353.4 mm, n =15). Seasonal
of 225.6 gm +20.4SD (male: mean 222.1 gm variation was observed in BoL (BT to TT) of goose
+22.2SD, range =180.65 to 260.0 gm, n =13; during the study period. In winter season, a greater
female: mean 229.08 gm +18.6SD, range =221.7 to mean BoL (BT to TT) was observed in female with
234.5 gm, n =6). 351.2 mm +1.5SD (range =348.8 to 353.2 mm, n
b. Body length (BoL) =9) than the male with a mean BoL (BT to TT) of
The mean BoL (BT to TT) of Cotton Pygmy 334.8 mm +1.2SD (range =332.7 to 336.1 mm, n
-goose, irrespective of sex was 335.3 mm + 1.1SD =14), while the case is reversed during monsoon
(range =332.9 to 353.4 mm, n =42, Table 5.1, (Female, mean: 351.8 mm +1.7SD, range: 349.1 to
Figure 5.1). In male goose, the mean BoL (BT to 353.4 mm, n =6; Male, mean: 335.8 mm +0.7SD,
TT) measured was 335.3 mm +1.1SD (range range: 333.9 to 336.5 mm, n =13). The mean BoL
(N to A) of the goose was found to be 154.7 mm +
1.1SD (range =143.5 to 165.0 mm, n =42, Table
5.1, Figure 5.1) irrespective of sex and seasons. The
mean BoL (N to A) was found higher in female
with a mean length 163.6 mm +1.2SD (range
=162.0 to 165.0 mm; n =15) than the male which
has a mean BoL (N to A) of 145.7 mm + 1.1SD, n
=27) irrespective of the seasons (Table 5.1). The
male and females were found with a greater BoL (N
to A) during monsoon then the winter (male
(a) (b) monsoon: mean BoL 146.0 mm +1.1SD, range
Plate 1 Primary wing feathers of (a) male & (b) =143.3 to 147.0 mm, n =13; winter: mean BoL
female CPG (from inner to outer)

Journal of Research in Biology (2011) 2: 3: 191-201 193
Upadhyaya et al.,2011

145.4 mm +0.9SD, n =14; female winter: 163.5 mm female (male: mean 15.4 mm+0.2SD, n =13;
+1.3SD, n =9 & monsoon: 163.9 mm +1.1SD, female: mean 15.3mm +0.1SD, n =21) during the
range =162.7 to 165.0 mm, n =6). monsoon season of a year.
c. Bill length (BL) e. Bill width (BW)
The mean BL (BT to F) of the Cotton The overall mean BW (at culmen) in Cotton
Pygmy-goose was found to be 26.5 mm +0.2SD Pygmy-goose was found to be 12.03 mm +0.2SD
(range =26.2 to 26.9 mm, n =42, Table 5.1, Figure (range 11.5 to 12. 7 mm, n = 42, Table 5.1, Figure
5.1) and BL (BT to F) between 22.3 mm to 23.2 5.1). The BW of the male goose was found to be
mm (mean 22.7 mm +0.3SD, n =12) with p >0.05 12.3 mm +0.2SD (range =12.0 to 12.7 mm, n =27),
(Students’ t-test) in both the cases irrespective of whereas in females the same found to be 11.8 mm
sex and seasons of a year. The male goose was of +0.2SD (range =11.5 to 12.0 mm, n =15). The BW
greater BL (BT to F) than the females with mean (at culmen) in case of male goose during monsoon
length 26.6 mm +0.2SD (range 26.2 to 26.9 mm, n was found to be 12.3 mm +0.2SD (range =12.0 to
=27) and 26.5 mm +0.2SD (range 26.2 to 26.8 mm, 12.5 mm; n =13), whereas the mean BW remains
n =15) respectively. The BL (BT to F) of the goose at12.4 mm +0.3SD (range =12.0 to 12.6 mm; n
was found longer during monsoon with a mean =14). Again the mean BD of female goose during
length of 26.6 mm +0.2SD (range =26.2 to 26.9 monsoon was found to be 11.4 mm +0.2SD (range
mm, n=19) than the winter period with a mean of =11.4 to 11.9 mm, n =6) and during winter it
26.5 mm +0.2SD (range =26.2 to 26.9 mm, n =23). remains at 11.8 mm +0.2SD (range =11.5 to 12.0
No major differences were observed in the BL (BT mm, n =9).
to F) between the male and female during winter f. Head circumference (HC)
season (male: mean =26.5 mm +0.2SD, range =26.2 The mean HC of Cotton Pygmy-goose was found
to 26.9 mm, n =14; female: mean =26.5 mm to be 93.5 mm +0.1SD (range =92.7 to 94.2 mm, n
+0.2SD, range =26.2 to 26.8 mm, n =9). The mean =42, Table 5.1, Figure 5.1). The mean HC of male
BL (BT to FC) of the goose was found to be 22.8 goose was 93.9 mm +0.2SD (range =93.6 to 94.2
mm +0.2SD (range =22.3 to 23.2 mm, n =42). The mm, n =27) and of female goose was 92.9 mm
BL (BT to FC) in case of the male goose was found +0.2SD (range =92.7 to 93.2 mm, n =15)
greater with a mean 22.8 mm +0.3SD (range =22.4 irrespective of seasons of a year. The HC was found
to 23.2 mm; n =27), than the females where the greater during the monsoon season (93.5 mm
mean BL (BT to FC) was 22.6 mm +0.2SD (range +0.2SD, n =19) than during the winter season (93.4
=22.3 to 22.9 mm, n =15, Table 5.1). The Cotton mm +0.2SD, n =23) irrespective of the sex. During
Pygmy-goose, during the monsoon were found with the monsoon the male goose has a greater HC with
greater BL (BT to FC) than the goose observed a mean of 93.9 mm +0.2SD (range =93.6 to 94.2
during the winter (monsoon: mean 22.8 mm mm, n=13) than the female goose of the same
+0.9SD, range =22.4 to 23.2 mm, n =19; winter: season which has a mean HC of 93.0 mm +0.2SD
mean 22.7 mm +0.3SD, range =22.3 to 23.2 mm, n (range =92.7 to 93.2 mm, n =6).
=23). Again, the monsoon male were found to have g. Neck length (NL)
greater BL (BT to FC) than the monsoon females The mean NL of the Cotton Pygmy-goose was
(male: range 22.4 to 23.2 mm, mean 22.9 mm found to be 42.1 mm +0.6SD (range =40.0 to 43.5
+0.4SD; female: range 22.3 to 22.9 mm, mean 22.6 mm, n =42, Table 5.1, Figure 5.1) irrespective of
mm +0.2SD). sex and seasons of a year. The mean NL measured
d. Bill depth (BD) in male Cotton Pygmy-goose was 42.6 mm +0.4SD
In Cotton Pygmy-goose, the mean BD was (range= 42.0 to 43.5 mm, n =27), whereas in female
found to be 15.4 mm +0.1SD (range =15.1 to 15.6 goose the mean NL found was 41.4 mm +0.7SD
mm, n =42, Table 5.1, Figure 5.1). The male goose (range =40.0 to 42.1 mm, n =15. During the winter
has longer BD with a mean 15.4 mm+0.2SD (range season, the mean NL of the male was found to be
=15.1 to 15.6 mm, n =27), than the females who has 42.6 mm +0.4SD (range =42.0 to 43.2 mm, n =14),
a mean BD of 15.3 mm +0.1SD (range =15.1 to whereas the female has a mean NL of 41.3 mm
15.4 mm, n =15). During winter, the male goose has +0.7SD (range =40.0 to 42.1 mm, n =9). The
a mean BD of 15.4 mm +0.2SD (range =15.1 to female has mean NL of 41.8 mm +0.7SD (range
15.6 mm; n =14), whereas the female has a mean =40.5 to 42.1 mm, n =6), the male goose during
BD of 15.3 mm +0.1SD (range: 15.1 to 15.4 mm; n monsoon has a mean NL of 42.7 mm +0.5SD
=9). The BD found slightly greater in male than the (range = 42.0 to 43.5 mm, n =13).
194 Journal of Research in Biology (2011) 3: 191-201
Upadhyaya et al.,2011

h. Neck circumference (NC) (PWF) and 14-secondaries (SWF). The mean PWF
The mean NC of Cotton Pygmy-goose was found length was found to be 104.2 mm +0.06SD (range
to be 86.9 mm +0.2SD (range =86.5 to 87.2 mm, n =101.13 to 107.31 mm, n =42) irrespective of sexes
=42, Table 5.1, Figure 5.1) irrespective of sex and and seasons. The male goose has a mean PWF of
seasons of a year. The NC was slightly of greater 107.2 mm +0.07SD (range =107.07 to 107.31 mm,
value in male goose with a mean of 87.0 mm n =27), whereas the female PWF has a mean length
+0.2SD (range =86.7 to 87.2 mm, n =27) than the of 101.21 mm +0.06SD (range =101.13 to 101.31
female with a mean value of 86.8 mm +0.2SD mm, n =15). The PWF length was found
(range = 86.5 to 87.0 mm; n =15). During monsoon statistically significant at both 99% and 95%CL (p
season, the male goose has a greater NC with a <0.01, Student’s t-test) between the male and
mean of 87.0 mm +0.2SD (range =86.7 to 87.2 mm, female. During winter season, the mean PWF
n =13) than the female goose of the same season length measured in the male was 107.2 mm
with a mean NC of 86.8 mm +0.2SD (range =86.5 +0.07SD (range =107.07 to 107.31 mm, n =14),
to 87.0 mm, n =6). whereas in female goose the mean PWF length
i. Wing span (WS) remains at 101.23 mm +0.07SD (101.13 to 101.35
The mean WS of the Cotton Pygmy-goose was mm, n =9). During monsoon season, the mean PWF
found to be 425.3 mm +0.7SD (range 424.0 to was found to be 107.2 mm +0.07SD (range =107.07
426.0 mm, n =42, Table 5.1, Figure 5.1) during the to 107.31 mm, n =13) and 101.2 mm +0.05SD
study period. The male goose have a mean WS of (range =101.13 to 101.25 mm, n =6) respectively in
425.278 mm +0.7SD and female goose with 425.26 male and female gooses. The PWF length
mm +0.7SD (range =424.0 to 426.0 mm in both difference was found statistically significant in the
sexes, n =27 & 15). The mean WS of male during male and female at 99%CL (p <0.01, Students’
winter was found to be 425.428 mm +0.6SD (range Paired t-test) during winter and monsoon season.
=424.5 to 426.0 mm, n =14), whereas the female The length-wise arrangement of the PWF of male
has a mean WS of 425.388 mm +0.8SD (range was found as PWF1< PWF11< PWF10 < PWF9 <
=424.0 to 426.0 mm, n =9) in the same season. PWF8 < PWF7 < PWF2 < PWF6 < PWF5 < PWF3
Again, during monsoon the male goose has a mean < PWF4 (Table 5.2 & Figure 5.2). A more or less
WS of 425.11 mm +0.7SD (range =424.0 to 426.0 similar arrangement was found in PWFs of female
mm, n =13) and female has 425.08 mm +0.7SD goose with a difference in PWF2 and PWF6, where
(424.0 to 426.0 mm, n =6). PWF6 < PWF2.
j. Wing length (WL) The mean SWF length was found to be 83.1
The mean WL (flattened) in case of both the mm +0.06SD (range =80.5 to 85.7 mm, n = 42).
sexes of Cotton Pygmy-goose recorded was 169.2 The male goose have longer SWF length with a
mm +0.6SD (range =168.0 to 171.0 mm, n =42, mean 85.6 mm +0.06SD (range 80.6 to 85.6 mm, n
Table 5.1, Figure 5.1). The male goose has a mean =27) than the female goose with a mean 80.7 mm
WL of 169.3 mm +0.85SD (range =168.0 to 171.0 +0.05SD (range 80.6 to 80.8 mm, n =15)
mm, n =27), whereas the female has a mean WL of irrespective of seasons. During monsoon, the male
169.1 mm +0.92SD (range =168.0 to 171.0 mm, n has a mean SWF length of 85.6 mm +0.05SD
=15). The WL of the female goose during the (range =85.55 to 85.64 mm, n =13), whereas the
monsoon season was measured with a mean WL of female has a mean length of 80.6 mm +0.03SD
169.3 mm +1.1SD (range =169.0 to 171.0 mm, n (range 80.6 to 80.7 mm, n =6). During winter the
=6), whereas in male the same remains at 169.0
mm +0.7SD (range =168.0 to 170.0 mm, n =13).
During winter season, the mean WL of the male
goose was found to be 169.5 mm +0.9SD (range
=168.0 to 171.0 mm, n =14) and in female the
mean WL was found as 168.9 mm +0.8SD (range
=168.0 to 170.0 mm, n =9).
k. Wing feathers (WF) & wing feather length
(a) (b)
The number of WF in both male and female
Cotton Pygmy-goose was 25 with 11-primaries Plate 2 Secondary wing feathers of (a) male & (b)
female CPG (from inner to outer)

Journal of Research in Biology (2011) 3: 191-201 195
Upadhyaya et al.,2011

Table 2. Lengths of primary wing feathers of Cotton Pygmy-goose (n =42)
Feathers* Range (male) Mean +S.D. Range (female) Mean +S.D.
PWF-1 28.0- 28.2 28.1 +0.08 27.7 - 27.9 27.8 +0.08
PWF-2 121.0- 121.1 121.13 +0.05 118.3 - 118.5 118.4 +0.11
PWF-3 127.2 - 127.5 127.4 +0.11 122.0 - 122.5 122.3 +0.17
PWF-4 128.4 - 128.7 128.6 +0.12 123.3 - 123.5 123.4 +0.10
PWF-5 125.3 - 125.6 125.5 +0.10 119.2 - 119.6 119.4 +0.20
PWF-6 121.2 - 121.5 121.4 +0.12 115.4 - 115.8 115.6 +0.14
PWF-7 117.0 - 117.3 117.2 +0.1 109.4 - 109.7 109.5 +0.10
PWF-8 111.1 - 111.2 111.16 +0.05 103.4 - 103.8 103.7 +0.14
PWF-9 104.3 - 104.6 104.48 +0.12 97.3 - 97.5 97.4 +0.08
PWF-10 99.1 - 99.5 99.3 +0.16 91.0 - 91.2 91.1 +0.09
PWF-11 95.2 - 95.5 95.37 +0.10 84.9 - 84.1 85.0 +0.06
*PWF= Primary wing feather

male goose has a more or less similar mean SWF monsoon season the male goose have greater SFL
length with the monsoon season (mean: 85.6 mm with a mean SFL of 42.2 mm +0.3SD (range =41.9
+0.07SD, range =85.46 to 85.63 mm, n =14), to 42.5 mm, n =13) than the winter ones with a
whereas in the female goose the same remains at mean SFL of 42.0 mm +0.1SD (range =41.9 to 42.2
80.5 mm +0.07SD (range =80.52 to 80.75 mm, n mm, n =14). The scapulars found significantly
=9). The length-wise arrangement of the SWF in longer in males by 0.27 mm +0.13SD.
male was found to be SWF14 <SWF9 <SWF10 m. Tarsus length (TL)
=SWF13 < SWF8 < SWF7 < SWF6 < SWF5 < The mean TL in Cotton Pygmy-goose was
SWF4 <SWF1 <SWF3 <SWF11 <SWF2 <SWF12, found to be 29.1 mm +0.1SD (range =28.9 to 29.3
while in female the arrangement was found to be mm, n =42, Table 5.1, Figure 5.1). The mean TL in
SWF14 <SWF7 <SWF9 < SWF6 < SWF10 < both sexes of the goose was found to be 29.1 mm
SWF5 <SWF4 < SWF8 < SWF13 < SWF3 <SWF2 +0.1SD with a range between 29.0 to 29.2 mm
<SWF1 <SWF11<SWF12 (Table 5.3 & Figure (male: n =27; female: n =15).
5.2). The SWF length was found statistically n. Hind toe length (HTL)
significant at both 99% CL (p <0.01, Student’s t- In Cotton Pygmy-goose the mean HTL or
test) between the male and female. first toe length was found to be 8.1 mm +0.09SD
l. Scapular feathers (SF) and scapular feather (range =7.9 to 8.3 mm, n =42, Table 5.1, Figure
length (SFL) 5.1). The male goose have a slightly longer mean
There are 6 (six) SF in both male and female HTL with 8.2 mm +0.09SD (range 8.0 to 8.3 mm, n
Cotton Pygmy-goose with mean length of 42.0 mm =27) than the female goose which have a mean
+0.25SD (range =41.7 to 42.6 mm, n =42, Table HTL of 8.06 mm +0.09SD (range =7.9 to 8.2 mm, n
5.1). The male goose has a mean SFL of 42.2 mm =15) irrespective of the seasons of a year. During
+0.3SD (range =41.9 to 42.6 mm, n =27) whereas winter, the mean HTL of male goose was found to
the female goose has a mean SFL of 41.9 mm be 8.1 mm +0.1SD (range =8.0 to 8.3 mm, n =14),
+0.1SD (range =41.7 to 42.1 mm, n=15) whereas a mean length of 8.0 mm +0.08SD (range
irrespective of the seasons of a year. During =7.9 to 8.2 mm, n =9) was observed in female
goose during the same season. The male has a
greater HTL than the females during the monsoon
(male: mean 8.2 mm + 0.08SD, n =13; female:
mean 8.1 mm +0.1SD, n =6).
o. Inner toe length (ITL)
In Cotton Pygmy-goose the mean ITL or
second toe length was found to be 31.7 mm +0.1SD
(range =31.5 to 32.0 mm, n =42, Table 5.1, Figure
5.1). The male goose have a slightly longer mean
(a) (b) ITL with 31.8 mm +0.2SD (range 31.4 to 32.0 mm,
Plate 3 Rectrices of (a) male & (b) female CPG from n =27) than the female goose which have a mean
right to left) ITL of 31.6 mm +0.1SD (range =31.5 to 31.8 mm,
196 Journal of Research in Biology (2011) 3: 191-201
Upadhyaya et al.,2011

Table 3. Lengths of secondary wing feathers of Cotton Pygmy-goose (n =42)
Feathers* Range (male) Mean +S.D. Range (female) Mean +S.D.
SWF-1 88.9 - 89.7 98.3 +0.33 82.1 - 83.3 82.5 +0.43
SWF-2 91.0 - 91.5 91.3 +0.23 82.0 - 83.3 82.3 +0.51
SWF-3 89.2 - 89.5 89.4 +0.12 80.2 - 81.3 80.9 +0.39
SWF-4 87.1 - 87.5 87.3 +0.15 79.1 - 79.6 79.3 +0.17
SWF-5 86.0 - 86.7 86.3 +0.26 77.2 - 77.9 77.6 +0.27
SWF-6 84.3 - 84.6 84.5 +0.09 75.3 - 75.6 75.4 +0.12
SWF-7 83.4 - 83.8 83.6 +0.15 74.9 - 75.4 75.1 +0.18
SWF-8 82.3 - 82.5 82.5 +0.10 79.3 - 79.5 79.4 +0.08
SWF-9 80.4 - 80.8 80.6 +0.16 75.1 - 75.6 75.3 +0.19
SWF-10 82.1 - 82.5 82.4 +0.15 77.1 - 77.5 77.4 +0.16
SWF-11 89.3 - 89.7 89.5 +0.13 95.8 - 96.2 96.0 +0.13
SWF-12 94.2 - 94.5 94.3 +0.12 97.0 - 97.2 97.1 +0.08
SWF-13 82.2 - 82.5 82.4 +0.12 80.2 - 80.5 80.3 +0.12
SWF-14 74.8 - 75.2 75.0 +0.14 71.3 - 71.5 71.4 +0.11
*SWF= Secondary wing feather
n =15) irrespective of the seasons of a year. slightly longer MTL with a mean of 34.3 mm
During winter, the mean ITL of male goose was +0.2SD (range =34.0 to 34.5 mm, n =13) than the
found to be 31.7 mm +0.2SD (range =31.4 to 32.0 female goose with mean of 34.2 mm +0.2SD (range
mm, n =14), whereas a mean length of 31.6 mm =34.1 to 34.5 mm, n =6) during the monsoon
+0.1SD (range =31.5 to 31.8 mm, n =9) was season.
observed in female goose during the same season. q. Outer toe length (OTL)
The male has a greater ITL than the females during In Cotton Pygmy-goose the mean OTL or
the monsoon (male: mean 31.8 mm + 0.2SD, n =13; fourth toe length was found to be 33.3 mm +0.2SD
female: mean 31.6 mm +0.1SD, n =6). (range =33.0 to 33.5 mm, n =42, Table 5.1, Figure
p. Middle toe length (MTL) 5.1). The male and female goose have a more or
In Cotton Pygmy-goose the mean MTL or less similar measurement of OTL with a mean of
third toe length was found to be 34.3 mm +0.2SD 33.3 mm +0.2SD in both (range =34.0 to 34.5 mm,
(range =34.0 to 34.5 mm, n =42, Table 5.1, Figure n = male:27 & female:15) irrespective of the
5.1). The male and female goose have a more or seasons of a year. During winter and monsoon
less similar measurement of MTL with a mean of season, the mean OTL of male and female goose
34.3 mm +0.2SD in both (Male: range 34.0 to 34.5 was also found to be 33.2 mm +0.2SD (range =33.0
mm, n =27; Female: range 34.1 to 34.5 mm, N =15) to 33.5 mm, n =male: 14 & female: 9).
irrespective of the seasons of a year. During winter, r. Inner web distance (IWD)
the mean MTL of male goose was found to be 34.2 The mean IWD in Cotton Pygmy-goose was
mm +0.2SD (range =34.0 to 34.5 mm, n =14), found to be 20.3 mm +0.2SD (range =19.9 to 20.6
whereas a mean length of 34.3 mm +0.2SD (range mm; n =42, Table 5.1, Figure 5.1) irrespective of
=34.1 to 34.5 mm, n =9) was observed in female sexes and seasons. The male and female goose have
goose during the same season. The male has a a more or less similar measurement of IWD with a
mean of 20.3 mm +0.2SD in both (range =19.9 to
20.9 mm, n = male:27, female:15) irrespective of
the seasons of a year. During winter season, the
mean IWD of male and female goose was found to
be 20.2 mm +0.2SD (range: male =19.9 to 20.4
mm, n =14; female =19.9 to 20.6 mm, n =9).
Again during monsoon season, the mean IWD of
male and female goose was found to be 20.3 mm
+0.2SD (range: male =19.9 to 20.6 mm, n =13;
(a) (b) female =20.0 to 20.6 mm, n =6).
s. Outer web distance (OWD)
Plate 4 (a) Dorsal & (b) ventral view of a dead CPG The mean OWD in Cotton Pygmy-goose
(male) recovered from poachers’ hand near Borsola

Journal of Research in Biology (2011) 3: 191-201 197
Upadhyaya et al.,2011

was found to be 16.5 mm +0.2 (range 16.2 to 16.8 with mean TaL 64.4 mm +0.6SD (range =63.2 to
mm; n =42, Table 5.1) irrespective of sexes and 65.3 mm, n =9). The numbers of TF or rectrices in
seasons. The male and female goose have a more Cotton Pygmy-goose vary in both sexes. The male
or less similar measurement of OWD with a mean has 12 TF, whereas the female has 14 in numbers.
of 16.5 mm +0.2SD in both (range =16.2 to 16.8 The mean TFL was found to be 74.2 mm +0.08SD
mm, n= male-27 & female-15) irrespective of the (range =73.36 to 75.07 mm, n =42, Table 5.1)
seasons of a year. During winter season, the mean irrespective of sexes and seasons. The mean TFL of
OWD of male and female goose was found to be the male was 73.5 mm +0.09SD (range =73.4 to
16.5 mm +0.2SD (range =16.2 to 16.8 mm, n =male 73.6 mm, n =27) and in female it remains at 74.9
-14 & female-9). Again during monsoon season, the mm +0.08SD (range =74.8 to 75.1 mm, n =15).
mean OWD of male and female goose was found to During winter, in male goose the mean TFL found
be 16.5 mm +0.2SD and 16.4 mm +2SD (range: was 73.5 mm +0.09SD (range =73.4 to 73.7 mm, n
male =16.2 to 16.8 mm, n =13; female =16.2 to =14) and in female the mean value found to be 75.0
16.7 mm, n =6). mm +0.08SD (range =74.9 to 75.1 mm, n =9).
t. Tail length (TaL), tail feathers (TF) or Again, the male goose studied during monsoon
rectrices and tail feather length (TFL) shows a mean TFL of 73.5 mm +0.09SD (range
The mean TaL in Cotton Pygmy-goose was =73.4 to 73.6 mm, n =13), whereas the female
found to be 64.6 mm +0.8SD (range =63.2 to 65.9 goose has a mean TFL of 74.9 mm +0.09SD (range
mm, n =42, Table 5.1, Figure 5.1). The male has a =74.9 to 75.1 mm, n =6).
longer tail with a mean TaL of 64.6 mm +0.8SD Twenty pairs of body parameters were
(range =63.4 to 65.9 mm, n =27) than the female studied and relationship was correlated which
with a mean TaL 64.5 mm +0.5SD (range =63.2 to shows strong as well as negative relationship
65.3 mm, n =15). The goose studied during winter among them, and the value of ‘r’ ranges between -
season has greater TaL with mean 64.6 mm +0.6SD 0.9 to 0.9 (Table 5.4 & Figure 5.3). A strong
(range =63.2 to 65.9 mm, n =23) than the goose relationship was observed in case of female (r=0.8,
studied during monsoon season with a mean 64.5 Pearson Correlation) between BoW and BoL, while
mm +0.6SD (range =63.2 to 65.9 mm, n =19). The in the case of male a less strong correlation (r=0.2)
male goose captured during winter has longer tail was observed. It shows a higher tendency of
with mean TaL of 64.7 mm +0.7SD (range =63.4 to increase of body weight with an increase in BoL.
65.9 mm, n =14) than the female of the same season There exists a strong correlation between BoL and
Table 4. The co-efficient of correlation (r) among various body parameters of CPG
(male & female)
Parameters ‘r’ in male ‘r’ in female
1. Body wt. & Body length 0.2 0.8
2. Body length & Neck length 0.7 0.8
3. Body length & Bill length 0.9 0.1
4. Bill length & Bill depth 0.5 0.4
5. Body length & Middle toe length 0.7 -0.1
6. Tarsus & Middle toe length 0.1 0.5
7. Body length & Tail length 0.5 0.2
8. Wing length & Tail Length -0.7 -0.4
9. Wing feather length & Tail feather length -0.7 -0.1
10. Wing length & Body length -0.8 0.5
11. Bill length & Bill width -0.6 0.1
12. Wing Expansion & Body length -0.8 -0.7
13. Bill depth & Bill width -0.2 -0.1
14. Tarsus length & Hind toe length -0.2 -0.2
15. Tarsus length & Inner toe length 0.1 0.2
16. Hind toe length & Middle toe length -0.3 0.1
17. Tarsus length and Outer toe length -0.5 0.1
18. Outer toe length & Middle toe length 0.4
19. Hind toe length & Outer toe length 0.5 -0.7
20. Outer toe length & Inner toe length 0.5 0.9

198 Journal of Research in Biology (2011) 3: 191-201
Upadhyaya et al.,2011

Plate 5 An adult CPG (female) during measurement Plate 6 A pair of adult CPG recovered from
(in inset: an adult male) a poacher of Kadamani area.
NL in both male and female (r=0.7 & 0.8 irrespective of their sex except a male bird which
respectively) and a less strong correlation between may just arrived to the area and was trapped for the
the BoL and BL in female (r=0.1). The NL is study. The greater weight in the birds of the
highly related with the BoL and vice-versa, while vegetation cover area may be due to recent feeding
the tendency is lower in case of the male. A on the vegetation available in the wetland. The
moderately strong correlation was observed in Cotton Pygmy-goose studied from Borsola area
female (r=0.5) between TL and MTL. The co- were found greater weight than the goose captured
efficient of correlation between BoL and TaL was from other areas during the study period. The
found strong in male (r=0.5) and in female (r=0.2), reason might be the availability of food plants in the
whereas the WS and BoL in both male (r=-0.8) and wetlands of Borsola area.
female (r=-0.7) are oppositely related and increase The sex-wise differences of body length,
in WS decreases the BoL. An oppositely related plumage characteristics were found to be prominent
WFL and TFL were found in male (r = -0.7) and in the present context of the study. Deviations of
female (r= -0.1). Similarly the BD and BW in both the body lengths were more like those predicted by
male and female were oppositely related (r = -0.2 & the competition hypothesis in wetlands with low
-0.1 respectively). food abundance than in the wetlands with high food
abundance. The similar results were also discussed
DISCUSSION by Pöysä et al. (1994).
The present morphomatric study on forty T h e f u nct i on al s i gni f i c an ce of
two Cotton Pygmy-gooses, the average BoW of morphomatric study shows that due to differences
male and female is found to be 221.2 gm and 227.1 in neck and body length the Cotton Pygmy-goose
gm respectively. The study shows a low body use different feeding methods and depths for
weight of the Cotton Pygmy-goose in comparison feeding. The present findings were further
to other goose. A lower body weight of the supported by the works of Pöysä (1983a, 1983b,
Nettapus sp. > 500 gm has been confirmed. The 1986 & 1987). The wing span and the flattened
female Cotton Pygmy-goose was found heavier wing length are found quite correlative in response
during the monsoon season than the male. This to the flight adaptation. The relationship of wing
might be either due to excessive feeding for the span, wing length and tarsus length with the body
developing eggs during monsoon season or food weight is also supported by the works of Green et
reservation which may occur for future use during al. (2001). No relation was observed in between the
incubation of the eggs. The present investigation outer web distance and inner web distance. Fox et
also reveals that the female has slightly greater al. (1992) found significance differences between
body weight than the male bird during breeding male and female adult teal in tarsus and wing
season prior to egg laying. The weight of the birds length. The present findings are also more or less
congregated in vegetation covers area found to be supported by the findings of Moore and Battley
greater than the birds found in open area (2003) in Anas chlorotis.

Journal of Research in Biology (2011) 3: 191-201 199
Upadhyaya et al.,2011

The assumptions of the present study List of Threatened Species. CD ROM.
demonstrate a conclusion on the adaptiveness of the
morphological characteristics with relation to the Bock JW. 1959. Pre-adaptation and multiple
ecology of the habitat area of Cotton Pygmy-goose. evolutionary pathways. Evolution. 13: 123-131.
The morphology of birds has been greatly
influenced by the environmental condition of a Bock JW and Von Wahlert G. 1965. Adaptation
region. The new area eco-morphology of the broad and the form-function complex. Evolution 19:269-
subject ornithology provides the effects of various 299.
environmental as well as wetland factors on the
morphological features of the wetland birds. The Bock JW. 1980. The definition and recognition of
differences in their measurement from different biological adaptation. American Zoologist 20
wetland areas provide a clue for their adaptability of (1):217-227.
these birds with their natural habitat with niche
characteristics. The various measurements can be Fox AD, King R and J. Watkin. 1992. Seasonal
used for the formation of silhouettes. Based on the variation in weight, body measurements and
various parameters a bird baseline can be formed condition of free-living Teal. Bird Study 39: 53-62.
and differences between two sexes can be
visualized. Though the lengths of primaries, Green AJ, Figuerola J King R. 2001.
secondaries and tail feathers vary within limits Comparative inter-specific and intraspecific
across species, it is also true for population of an allometry in the Anatidae. J. Ornithology 142:321-
area. These measurements can be used in museum 334.
diagnosis for the identification of species and
geographic races in light of sexual variations. Grimmett R, Inskipp C and T. Inskipp, 1999.
Pocket guide to the Birds of Indian Sub- Continent.
ACKNOWLEDGEMENT Oxford Univ. Press 384.
Authors are very much thankful to Mr. C. K.
Bora, the then DFO, Sonitpur Forest Division (East) Moore SJ and Battley PF. 2003. The use of wing
for his active support during the study period. We remains to determine condition before death in
are also thankful to UGC (NERO) for providing brown teal (Anas chlorotis). Notornis 50:133-140.
financial assistance in the form of Minor Research
Project. Pöysä H. 1983a. Resource utilization pattern and
guild structure in a waterfowl community. Oikos,
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Original Research paper
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Oxidative stress as a significant contributor in the pathogenesis and white
cell changes in pre-eclampsia: A study in Owerri, Nigeria.
Journal of Research in Biology

Authors: ABSTRACT:
Alisi P. Nc,
Buseri FI and
Alisi CS.
Haeomatological and oxidative stress parameters were measured in Pre-
Institution: eclamptic [PE] women attending clinic in owerri South Eastern Nigeria. The effect of
Department of medical Pre-eclampsia on these parameters was assessed by juxtaposition with similar indices
Laboratory Science in normal pregnant and normal non-pregnant women. Result showed that pre-
Rivers State University of eclampia resulted in significant decrease (P< 0.05) in Lymphocyte count. There was a
Science and Technology significant increase in total white blood cell count, neutrophil count and mixed
Port Harcourt, Nigeria differential count. Pre-eclamptic resulted in significant increase (P<0.05) in
erythrocyte lipid peroxidation, met-haemoglobin concentration and percentage
Federal medical centre hydrogen peroxide-induced haemolysis. The result indicate that oxidative stress is a
PMB 1010 Owerri, Nigeria significant contributor in the pathogenesis and white cell changes seen in pre-
Department of
Biochemistry Federal
University of Technology
PMB 1526 Owerri.

Corresponding author: Keywords:
Alisi, Precious. Nc. Pre-eclampsia, oxidative stress,met-haemoglobin, lipid peroxidation.

Email: Article Citation: Alisi P. Nc, Buseri FI and Alisi CS.
Oxidative stress as a significant contributor in the pathogenesis and white cell changes
in pre-eclampsia: A study in Owerri, Nigeria.
Phone No:
Journal of research in Biology (2011) 3: 202-208

Web Address: Received: 21 May 2011 /Accepted: 26 May 2011 /Published: 18 Jul 2011
© Ficus Publishers.
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Pre-eclampsia is a pregnancy complication Study design
recognized by new-onset gestational hypertension It was a cross sectional case control study
and proteinuria. The disorder affects both mother conducted prospectively among antenatal women
and their infants. Once the disease is evident attending clinic at Holy Rosary, Federal Medical
clinically, it can be cured only by delivery. The Centre and General Hospitals Owerri. The study
indicated delivery of women to prevent the included fifty non-pregnant, fifty PE and fifty
progression of pre-eclampsia is responsible for 15% normotensive pregnant women of singleton
of all preterm births (Golderberg and Rouse, 1998). gestation in their third trimester.
The cause of pre-eclampsia remains largely Selection criteria
unknown, but poor placentation is an important The subjects were selected under defined
predisposing factor. A shallowly implanted placenta criteria. PE patients were at 28 to 42 wks of single-
which becomes hypoxic, leading to an immune diastolic pressure of 110mm hg or more, or two
reaction characterized by secretion of up-regulated measurements of 90mmhg or more on two
inflammatory mediators from the placenta, and consecutive occasions of 6 hours or more apart,
acting on the vascular endothelium. The shallow urinary protein 2+ or more. The exclusion criteria
implantation is thought to stem from the maternal include history of hypertension and proteinuria
immune system's response to the placenta. Not all before conception or before 20wks of gestation, a
women with reduced placental perfusion develop history of antioxidant vitamins therapy during the
pre-eclampsia. Other conditions recognized to last one year and smoking.
increase the risk of pre-eclampsia include obesity As cohort control, age and socio-
(Sibai et al., 1995), hypertension (Caritis et al., economically matched healthy normotensives at 28
1998), diabetes (Caritis et al., 1998), to 42 wks of singleton gestation with no urinary
hyperhomocysteinemia (Power et al., 2001), protein, were recruited by convenience. The non-
increased androgens (Laivuori et al., 1998), and pregnant controls were consisted of 50 healthy
black race (Eskenazi et al., 1993). Of course, these normotensive subjects. They were matched by
are all risk factors for cardiovascular disease in later group percent of age, education and income. Ethical
life. Appreciation that pre-eclampsia is a multi- clearances were obtained from the Heads of the
systemic syndrome characterized by vasoconstriction, three hospitals involved.
metabolic changes, endothelial dysfunction, activation Blood Collection
of the coagulation cascade, and increased 4mls of venous blood was collected from
inflammatory response, to mention only some of the each case and control subjects and added into a
organ systems involved, has redirected research bottle (8ml) containing 80μl of ethylene diamine
(Roberts et al., 2003). As a result, progress in tetra-acetic acid (EDTA). Samples were used for
understanding the disorder has accelerated greatly the various analyses. Analysis was done within
with attendant optimism for potentially effective 1hour of blood collection. The full blood count was
treatments (Roberts et al., 2005). Pre-eclampsia is done with the Erma automatic multi-parameter
associated with haematological complications. Altered
blood cell counter for in vitro diagnostic use in
erythrocyte aggregation has been observed in severe
clinical laboratories.
pre-eclampsia than in normal pregnancy (Sengupta,
1995). In some other stress associated diseases there Measurement of Hydrogen peroxide (H2O2)
has also been dramatic alteration in neutrophil and induced haemolysis
monocyte values but this has not been investigated in Equal volumes of blood were mixed with
PE (Oishi et al., 1999). H2O2 and the fraction of red cells lysed is
It has also been documented that PE is determined spectrophotometrically as reported by
accompanied by oxidative stress that contributes to Liu et al., 1990. Briefly, A 5% suspension of
vascular dysfunction (Shaarawy et al., 1998). In the washed, packed erythrocytes in buffer saline was
present paper we studied systemic oxidative stress vis- mixed with the same volume of 1% H2O2 solution
a-vis pre-eclampsia by assessing lipid peroxidation, and the final mixture was incubated at 37oC for 2
hydrogen peroxide induced haemolysis, hrs. At the end of incubation the extent of
methaemoglobin concentration and some haemolysis was determined by measuring released
haematological parameters in patients of pre- haemoglobin into the supernatant by absorbance
eclampsia. measurement in a spectrophotometer (Turner
Model390) at 540nm and was expressed on the
203 Journal of Research in Biology (2011) 3: 202-208
Alisi et al.,2011

basis of maximum absorbance (100%) in the increase in prenatal mortality and its socioeconomic
aliquots of erythrocyte completely haemolysed in impact on developing countries is huge, its
distilled water. pathophysiological changes include elevated
Measurement of Methaemoglobin (Hi) in blood systemic vascular resistance, generalized
Methaemoglobin has a maximum absorption vasoconstriction, and activation of the coagulation
at 630nm. When cyanide is added this absorption cascade, all of which may be explained by
band disappears and the resulting change in disruption of normal maternal endothelial function
absorbance is directly proportional to the (López-Jaramillo et al., 2001).
concentration of Hi. Total Haemoglobin in the Effect of pre-eclampsia on hydrogen
sample is then measured after complete conversion peroxide induced haemolysis in erythrocytes (figure
to HiCN by the addition of ferricyanide-cyanide 1), shows that erythrocytes from pre-eclamptic
reagent using the method of Evelyn and Malloy patients underwent more lysis than erythrocytes
(1938). from the normal pregnant control (PC) and the non-
Briefly, 0.2mls of blood was lysed in a pregnant control (NPC). It is known that the
solution containing 4ml of buffer and 6ml of increased lysis results from oxidative damage to the
nonidet P40. The lysate was divided into two equal erythrocyte membrane, causing a decrease in
volumes (A and B). The absorbance of A was membrane fluidity and reducing its ability to
measured in a spectrophotometer at 630nm (D1). 1 withstand osmotic changes. Hydrogen peroxide
drop of KCN solution was added and absorbance participates in the Haber–Weiss reaction: O2•ˉ +
was measured after mixing (D2). 1 drop of K3Fe H2O2 → O2 + •OH + OHˉ. Increase in the H2O2
(CN)6 was added to solution B and after 5mins induced haemolysis in pre-eclampsia could indicate
absorbance was measured at the same wavelength a lesser ability to neutralize hydrogen peroxide
(D3). Then 1 drop of KCN was added to solution to (H2O2 + 2H+ + 2eˉ → 2H2O). Our observation
B and absorbance was measured after mixing (D4). could mean that there is a decreased antioxidant
Measurements were made against a blank capacity in pre-eclampsia. Increase in osmotic lysis
containing buffer and detergent in the same have been reported in pre-eclampsia in the united
proportion as present in the sample. % kingdom and the Netherland (Courinne et al., 1998;
methaemoglobin concentration was calculated thus: Armutcu, 2005).
Excessive formation of methaemoglobin was
% Methaemoglobin = D1-D2 C 100 seen in the erythrocytes of the preclamptic patient
D3-D4 (figure 2) in comparison with the control groups.
Measurement of Lipid Peroxidation This could be as a result of the oxidation of
Lipid peroxidation in the erythrocyte was erythrocyte protein (haemoglobin) which leads to
determined spectrophotometerically by assessing accumulation of methaemoglobin. The above is
the level of thiobarbituric acid reactive substance similar to accumulation of methaemoglobin seen in
(TBARS) as described by Liu et al. (1990).
Absorbance was measured at 532 nm using a 100
% Hydrogen Peroxide induced hemolysis

spectrophotometer to know Malonyldialdehyde
(MDA) an end product of lipid peroxidation, which
reacts with thiobarbituric acid to form pink 75
chromogen–thiobarbituric acid reactive substance.
Calculations were made using a molar extinction
coefficient of 1.56×105M−1 cm−1and expressed as 50
micromoles /l.
Statistical Analysis
Data obtained from the study were analyzed by the 25
use of two-way analysis of variance (ANOVA) and
values for P < 0.05 were considered statistically
significant. 0
Pre-eclampsia is associated with a five-fold Figure 1: Effect of pre-eclampsia on hydrogen
peroxide-induced haemolysis

Journal of Research in Biology (2011) 3: 202-208 204
Alisi et al.,2011

oxidative damage by drugs and chemical agents. The total white cell count (table 1) in pre-
Increases observed in methaemoglobin eclamptic women was significantly elevated than
concentration in pregnancy and pre-eclampsia the NPC. A significant increase in total white cell
implied a shift in pro-oxidant/antioxidant count was also found in PE when compared to PC
equilibrium in favour of the pro-oxidant, resulting women. This observation implied that pre-
in stress. Increased concentration has been eclampsia further exacerbated leukocytosis as
postulated in diseases associated with oxidative pregnancy is universally known to induce
stress (Dacie and Lewis 2006). leukocytosis. This is in agreement with Balloch
In this study, erythrocyte lipid peroxidation and Cauchi (1993). Schuiling et al., (2000) found
(LPO) as seen from the malondialdehyde an increased activated white blood cell in
concentration were significantly higher in the blood preeclampsia than in normal pregnancy, while
of pre-eclamptic women (figure 3) than the Sargent et al., (1998) found an increase in activated
controls. However, the RBC LPO in the pregnant white blood cells both in normal pregnancy and
control was also higher than the non-pregnant preeclampsia which was more in preclampisa
control. This shows that during uncomplicated resembling sepsis condition. On the other hand
pregnancy, there is an increased production of pro- Ceyhan et al., (2006) and Heilman et al., (2004)
oxidants that is balanced by the synthesis of recorded in their study an increase in total white
antioxidants (Scholl et al., 2005). Sëimsë et al., cell count in preeclampsia that was not statistically
(1998) also reported that serum lipid peroxide significant. Correlation has been found to exist
concentrations in pregnant subjects were between stress conditions and higher total white cell
significantly higher than those in non-pregnant count (Allsop et al 1992). This could lead one to
subjects. The significant variation seen in the pre- say that white blood cells are activated in pre-
eclamptic group and pregnant control is suggestive eclampsia and that pre-eclampsia may be partly an
that pre-eclampsia resulted from an imbalance inflammatory disorder since the activation of white
between pro-oxidant production and probably the cells were similar to that seen in inflammatory
antioxidant defenses. This imbalance could disorder (Sargent et al.,1998).
contribute to vascular dysfunction (Shaarawy et al., The percentage lymphocyte in pre-eclamptic
1998). The nature and extent of oxidative injury women was significantly decreased when compared
contributing to vascular dysfunction would depend to the normal pregnant controls. The result of this
on the concentration of free radicals/ reactive study agreed with reports from Israel (Lurie et al.,
oxygen species. Increases in lipid peroxidation 1998) but reports from Louisiana (Canzoneri et al.,
products like malondialdehyde correlates directly 2009) was different, no statistical significant
with increased imbalance in pro-oxidant/antioxidant difference was seen in lymphocyte between the PE
concentrations. and normal PC. The variation in this parameter
could come from the increase in the percentage


Percentage (%) Methaemoglobin

MDA (μmol/l)



Ser i es1

Figure2: Effect of Pre-eclampsia on Figure3: Effect of Pre-eclampsia on Lipid
Methaemoglobin concentration in Humans peroxidation

205 Journal of Research in Biology (2011) 3: 202-208
Alisi et al.,2011

Table 1: Effect of pre-eclampsia on white blood cell parameters
Non pregnant Pregnant
Pre-eclampsia P-value
controls controls
Total White Blood Cell (x10 /L) 7.57 ± 3.06 15.09± 2.40 17.56 ± 2.18 3.48E-43***
Neutrophils (%) 46.08 ± 7.82 63.44 ± 7.01 71.46 ± 7.53 3.12E-27***
Lymphocytes (%) 42.78 ± 8.25 26.71±13.72 14.25 ± 8.49 7.18E-27***
Mixed Differential count (%) 11.14 ± 5.10 9.85± 6.68 14.29 ± 3.50 0.00406**
Absolute lymphocyte count 3.24 ± 1.57 4.03 ± 2.13 2.52 ± 1.56 5.64E-7***
Absolute Neutrophil count 3.73 ± 2.09 9.61 ± 2.05 12.52 ± 1.92 2.72E-30***
Absolute Mixed Differential count 0.86 ± 0.53 1.49 ± 1.04 2.57 ± 0.79 4.93E-10***
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Original Research paper
Publication group

Antimicrobial action of methanol extract of Chromolaena odorata-Linn is
logistic and exerted by Inhibition of Dehydrogenase Enzymes
Journal of Research in Biology

Authors: ABSTRACT:
Alisi CS, Nwaogu LA,
Ibegbulem CO and
Inhibition of total dehydrogenase enzyme activity in pathogenic gram
Ujowundu CU.
positive and gram negative micro organisms exposed to methanol extract of
Chromolaena odorata was used as an index for assessment of its antimicrobial
Institution: activity. Assay of total dehydrogenase enzyme activity was done in the test organisms
Department of Biochemistry, (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli) using 2,3,5-
Federal University of triphenyltetrazolium chloride (TTC) as an artificial electron acceptor which was
Technology, reduced to the red-coloured triphenyl-formazan. Response of the bacterial isolates
PMB 1526 Owerri, Imo varied with extract concentration. Dehydrogenase activity was progressively inhibited
State, Nigeria. in a logistic dose-response fashion. The gram negative Escherichia coli responded
more markedly than Pseudomonas aureginosa and gram positive Staphylococcus
aureus. Inhibitory concentrations (IC50) of the methanol extract against Escherichia
Corresponding author: coli, Staphylococcus aureus, and Pseudomonas aeruginosa were 208.49 µg/ml,
Alisi CS 1361.01 µg/ml, and 903.08 µg/ml respectively. Preliminary phytochemical screening
of the extract gave positive reactions for alkaloids, flavonoids, tannins, 4-
hydroxybenzoic acid, and glycosides. These phytochemicals may be responsible for
Email: the observed inhibition of total dehydrogenase enzyme activity that translates to antimicrobial action in these pathogenic organisms.

Oxidoreductases, toxicity, enzyme inhibition, wound isolates, phytochemicals,
and bacterial response.

Article Citation:
Web Address:
Alisi CS, Nwaogu LA, Ibegbulem CO and Ujowundu CU.
Documents/RA0016.pdf. Antimicrobial Action of Methanol Extract of Chromolaena Odorata-Linn is
Logistic and Exerted by Inhibition of Dehydrogenase Enzymes.
Journal of research in Biology (2011) 3: 209-216

Received: 05 May 2011 /Accepted: 09 May 2011 /Published: 18 Jul 2011

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

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INTODUCTION culture method for enumeration of microorganisms
Selection of scientific and systematic which can underestimate number of viable cells due
approach for the biological evaluation of plant to lack of homogeneity in distribution or difficulty
products based on their use in the traditional in being readily desorbed from the substrate matrix
systems of medicine has continued to form the basis (Oberbremer and Muller- Hurtig, 1989;
for an ideal approach in the development of new Torstensson, 1997). Dehydrogenase assay is also an
drugs from plants. Chromolaena odorata (L.) R. effective primary test for assessing the potential
KING & H. ROBINSON (formerly Eupatorium toxicity of metals to planktonic (Nweke, et al.,
Journal of Research in Biology

odoratum L.), a perennial belonging to the plant 2006), and heterotrophic (Nweke, et al., 2007)
family Asteraceae (=Compositae), is a diffuse, bacteria. We had earlier also assessed toxicity of
scrambling shrub that is mainly a weed of antimicrobial agent to pathogenic bacteria using the
plantation crops and pastures of Southern Asia and dehydrogenase assay (Alisi, et al., 2008; Nwaogu,
Western Africa. It is a weed of 13 crops in 23 et al., 2008; Nwaogu, et al., 2007).
countries and has been described as the world’s Results of an earlier study showed that the
worst weed (Holme et al., 1977). This common extract of the leaves of C. odorata inhibited the
plant called Siam weed is known among the Igbos growth of some bacteria (Alisi and Onyeze, 2009).
of the South-Eastern Nigeria as: ‘Elizabeth’, Inhibition of dehydrogenase enzymes in pathogenic
‘Independence leaf’, ‘Enugu plantation weed’, or pure microbial cultures by methanol extracts of C.
‘Awolowo weed’. Chromolaena odorata is used as odorata has not been demonstrated. This work is
a gargle for sore throat and cold. Traditionally, therefore aimed at studying the inhibition of total
fresh leaves or a decoction of C. odorata have been dehydrogenase enzymes in pathogenic pure
used throughout Vietnam for many years as well as microbial cultures exposed to methanol extract of
in other tropical countries for the treatment of leech Chromolaena odorata.
bite, soft tissue wounds, burn wounds, skin
infection and dento-alveolitis (Le, 1995). MATERIALS AND METHODS
Decoctions of leaves have been used by traditional Plants
healers in the South-Eastern Nigeria for the Fresh aerial parts of C.odorata were
treatment of liver diseases. collected from Egbu and Ihiagwa in Owerri North
Several subclasses of flavonoids have been and Owerri West local Government areas of Imo
isolated from C. odorata extracts. The phenolic State respectively. Plant was authenticated by
acids present in ethanol extract of C.odorata are Professor S.E.Okeke, a plant taxonomist, of the
protocatechuic, p-hydroxybenzoic, p-coumaric, Department of Plant Science and Biotechnology,
ferulic and vanillic acids. The complex mixtures of Imo State University Owerri, Imo State. Voucher
lipophilic flavonoid aglycones present in C.odorata specimen is deposited in the author’s laboratory.
included flavanones, flavonols, flavones and Extract Preparation
chalcones. C. odorata also contains high The aerial part of C. odorata was shed dried
concentrations of amino acids (Phang, et al., 2001). at room temperature and reduced to a coarse
The use of the total dehydrogenase assay has been powder in a mill (Kenwood BL357). The powder
used as a tool in probing response of was extracted with methanol. The extract was
microorganisms to antibacterial agents (Alisi et al., recovered by distillation under reduced pressure at
2008) and is recognized as a useful indicator of the 49oC in a rotary evaporator-Buchi rotavapour
overall measure of the intensity of microbial, (Switzerland). The extracts were then dried to solid
metabolism (Tabatabi 1982; von Marsi and forms in vacuum desiccators, and stored in a freezer
Schinner, 1991). This method is preferred over (<-4.0 0C).
Table 1: Phytochemical constituents of Chromolaena.odorata methanol extracts
Cardiac Steroidal benzoic
Alkaloids Flavonoids Tannins Saponins Glycosides glycosides aglycone Protein acid)
plant + + + + + + + + +
MECO + + + - + + + + +

Key: + = presence, - = absence,
210 Journal of Research in Biology (2011) 3: 209-216
Alisi et al.,2011
Journal of Research in Biology

Isolation of bacterial strains and culture exponential phase in nutrient broth (Lab M) on a
conditions Marrienfeld rotary incubator (150 rpm) at room
Pathogenic bacteria (Pseudomonas sp., temperature (28 ± 2oC). The cells were harvested by
Staphylococcus sp., and Escherichia sp. were centrifugation at 4000 rpm for 10 min. Harvested
obtained from degenerated wound. Isolates were cells were washed twice in deionised distilled water
purified on nutrient agar (Fluka) plates and and re-suspended in water. The re-suspended cells
characterizations were done using standard were standardized in a spectrophotometer to an
microbiological methods. Identifications to the optical density of 0.70 at 420 nm. The dry weights
generic level followed the schemes of Holt et al. of the standardized cells were determined by drying
(1994). The bacterial strains were grown to mid volumes of cell suspension to constant weight in an
Table 2: Showing the threshold inhibitory concentrations of methanol extracts of C.odorata against the total
dehydrogenase activity (DHA) of some wound isolates (Escherishia.coli, Staphylococcus.aureus, and
Inhibitory Concentrations Against Wound Isolates
IC20 IC50 IC70 IC80 IC100
Escherishia coli 19.15 208.49 * 4736.25 9755.00
Staphylococcus aureus 111.11 1361.01 * ND ND
Pseudomonas aureginosa 206.27 903.08 1843.57 2605.12 5473.75

Journal of Research in Biology (2011) 3: 209-216 211
Alisi et al.,2011
R2 =

1.5 2

Log % Inhibition

1.25 1.5

Journal of Research in Biology

1 1


0.75 0.5

10 100 1000 10000


0 500 1000 1500 2000
MECO (ug/ml)
Figure 1b: Plot of Log percentage inhibition of DHA
against graded concentration of methanol extract of
chromolaena odorata

Determination of Antimicrobial Potentials of C.
odorata extracts by total dehydrogenase activity
(DHA) assay
Total dehydrogenase assay method as
described by Alisi et al. (2008) was employed
Briefly, total dehydrogenase activity was
determined using 2,3,5-triphenyltetrazolium
chloride (TTC) (BDH England) as the artificial
electron acceptor, which was reduced to the red-
colored triphenyl-formazan (TPF). The assay was
done in 4 ml volumes of nutrient broth-glucose-
TTC medium supplemented with varying
concentrations (0 – 2000 μg/ml) of extract in
Figure 1a: Inhibition of dehydrogenase activity (mg separate 20 ml screw-capped test tubes. Portions
Formazan/mg cell dry weight/hr) in pathogenic (0.3 ml) of the bacterial suspensions were
(wound isolates) bacteria (Escherichia.sp) by ethanol inoculated into triplicate glass tubes containing 2.5
extract of Chromolaena odorata- showing ml of phosphate-buffered (pH 6.8) nutrient broth-
dehydrogenase activity, percentage inhibition and glucose medium amended with Chromolaena
log %inhibition with graded concentrations of odorata extract and pre-incubated on a rotary
methanol extract of C.odorata. incubator (150 rpm) at room temperature (28 ± 2
C) for 30 min. Thereafter, 0.1 ml of 1 % (w/v)
oven at 110oC. These standardized cell suspensions TTC in deionised distilled water was added to each
were used as inoculums in the dehydrogenase tube to obtain final extract concentrations of 0-
activity assay. 2000 μg/ml in different test tubes. The final
Screen Test for TTC reduction (Dehydrogenase concentrations of nutrient broth, glucose and TTC
activity) in the medium were 2, 2 and 0.25 mg/ml,
On a colony of each bacterial isolate respectively. The controls consisted of the isolates
growing on nutrient agar, one drop of 1:1 mixture and the media without Chromolaena odorata
of aqueous solution of TTC (0.4 %w/v) and glucose extract. The reaction mixtures were further
(2 & w/v) was placed. The plates were incubated at incubated statically at room temperature (28 ± 2 oC)
room temperature for 10 minutes. Production of red for 8.0 h. The TPF produced were extracted in 4 ml
coloured formazan was suggestive of TTC of amyl alcohol and determined
reduction. spectrophotometrically at 500 nm (λmax). The
212 Journal of Research in Biology (2011) 3: 209-216
Alisi et al.,2011

Staphylococcus sp.
1.1 1.1

DHA (mgFormazan/mg cell dry wt/hr)
1 0.9
0.9 0.7
Journal of Research in Biology

0.7 0.01 0.1 1 10 100 1000 10000



0 500 1000 1500 2000
B ETECO(ug/ml)


40 60


10 10

0 -10
0.01 0.1 1 10 100 1000 10000

0 500 1000 1500 2000
C ETECO(ug/ml) D
1.5 1.75



0.5 1


-0.5 0.25
0 500 1000 1500 2000 0 500 1000 1500 2000
ETECO(ug/ml) ETECO(ug/ml)
MECO (µg/ml)

Fig.2 : Inhibition of dehydrogenase activity (mg Formazan/mg cell dry weight/hr) in Pathogenic (wound
isolates) bacteria(Staphylococcus. sp.) by methanol extract of Chromolaena odorata. Plots (A,B,C,and D) show
dehydrogenase activity, percentage inhibition of DHA, gamma parameters and log %inhibition of DHA on y-
axis respectively against graded concentrations of extract on x-axis.

amount of formazan produced was determined from inhibition data generated are fitted into the model
a standard dose-response curve [0 - 20 μg/ml TPF equation (2) which is a logistic dose response
(Sigma) in amyl alcohol; y = 0.0487x; R2 = equation. The parameters were estimated by
0.9977]. Dehydrogenase activity (DHA) was iterative minimization of least squares using
expressed as milligrams of TPF formed per mg dry Levenberg-marquardt algorithm (Table curve 2D
weight of cell biomass per hour. systat USA) Marquardt (1964). The data of %
Data Analysis inhibition fitted into equation (2) were used to
Percentage Inhibition of dehydrogenase evaluate the toxicity thresholds IC5, IC20, IC50, IC70,
activity by MECO was calculated relative to the IC80, IC100 which are the concentrations of the
control as shown in equation (1) (table 1). The extracts that inhibited 5%, 20%, 50% 70%, 80%

Journal of Research in Biology (2011) 3: 209-216 213
Alisi et al.,2011

Pseudomonas sp
and 100% of controls respectively. Where IC100 was
A 0.9 not determinable (ND) by fitting Eqn (1) into the
model (Eqn 2), Equation (1) was transformed to
DHA (mg Formazan/mg cell dry wt/hr)


0.8 0.7 their natural logarithms. Log y was plotted against
x. x values at y = 2 were taken as IC100. Data that
did not fit into logistic dose response model
Equation 2 were fitted into the WEIBULLCUM
0.6 0.4
model (Eqn 3). IC50 were obtained from Ѓ-
Journal of Research in Biology

parameter plot where data gave high R2-value with
0.5 0.2
0.01 0.1 1 10 100 1000 10000 Eqn 4. Data whose IC100 was non-determinable
using Equation 2 and 3, were fitted into an inverse
Log(x) polynomial equation (Eqn 5). By solving for
x in Eqn 5, IC100 (the concentration at which MECO
will exert total inhibition against the tested
0.2 organism) was calculated.
0 500 1000 1500 2000
MECO (ug/ml)
The plant C.odorata was found to contain
Flavonoids, Tannins, Saponins, Glycosides,
%Inhibition of DHA in Pseudomonas

Steroidal aglycones, Alkaloids and 4 -
60 hydroxybenzoic acid. The methanol extract
however did not show a positive reaction for
saponins (Table 1). These phytochemicals have
40 60
been found to have medicinal properties and health
promoting effects (Raza and John, 2007; Salah et
40 al., 1995; Del-Rio et al., 1997; Okwu, 2004; Liu,
30 2004). The use of C.odorata in ethno-medical
practice may be due to the medicinal effects of
10 10
these phytochemicals.
0.01 0.1 1 10 100 1000 10000
Methanol extract of C.odorata inhibited
0 500 1000 1500 2000 dehydrogenase activity in the organisms in a
MECO (ug/ml) logistic dose dependent manner. Inhibition of
dehydrogenase activity in Staphylococcus aureus,
2.5 and Pseudomonas aerugenosa followed a logistic
dose response abcd model (Eqn 1) while E.coli,
followed weilbullcum abcde model Eqn (3) and

(Eqn 5).
1.5 Threshold inhibitory concentrations of the
extracts (Table 2) showed that Pseudomonas
auregenosa responded gradually but steadily. At
lower concentrations, the extracts exerted stronger
inhibitory effect on the dehydrogenase activity of
0 Escherishia coli and Staphylococcus aureus than
0 500 1000 1500 2000
MECO (ug/ml) Pseudomonas auregenosa. The rate of inhibition of
total dehydrogenase enzyme activity in Escherishia
Fig.3: Inhibition of dehydrogenase activity (mg coli and Staphylococcus aureus was not sustained
Formazan/mg cell dry weight/hr) in Pathogenic as Pseudomonas auregenosa responded more at
(wound isolates) bacteria(Pseudomonas sp..) by higher concentrations of C.odorata extracts, making
methanol extract of Chromolaena odorata. Plots IC100 in Escherishia coli and Staphylococcus aureus
(A,B,and C) show dehydrogenase activity,
percentage inhibition of DHA and gamma
parameters on y-axis respectively against graded The gamma parameter model (Eqn 4) gave a
concentrations of extract on x-axis. strong linearization of percentage inhibition of
DHA by methanol extract of C. odorata against
214 Journal of Research in Biology (2011) 3: 209-216
Alisi et al.,2011
Table 3: Showing models and equations for dehydrogenase inhibition
with pearson correlation coefficient in the pathogenic organisms.
Organisms Model Equation R2-value
WEIBULLCU Eqn (3) 0.9969
Escherishia coli M (abcde)
inverse Log(x) Eqn (5) 0.9989
Journal of Research in Biology

Staphylococcus logistic dose Eqn (2) 0.9938
aureus response (abcd)
Pseudomonas logistic dose Eqn (2) 0.9981
aureginosa response (abcd)

Pseudomonas aerugenosa. This makes this the Ibegbulem CO. 2008. Inhibition of dehydrogenase
model of choice in the determination of IC50 in activity in pathogenic bacteria isolates by aqueous
Pseudomonas aerugenosa. All equations used in the extracts of Musa paradisiaca (Var Sapientum). Afr.
modelling of the results gave high correlation Journ. Biotech. 7(12):1821-1825.
coefficient (R2 ≥ 0.99), with very low Fit standard
errors. Inhibition of dehydrogenase activity in the Aziz NH, Frag SE, Mousa LA and Abo-Zald
wound isolates showed antimicrobial activity. The MA. 1998. Comparative antimicrobial and
extract was toxic to the organism at all antifungal effects of some phenolic compounds.
concentrations and the nature of inhibition was Microbios 93(374):43-54.
logistic rather than hormetic (Table 3).
Extracts of the plant C. odorata have earlier DelRío A, Obdulio BG, Castillo J, Marin RR,
been shown to contain phenolic compounds (Phan, and Ortuno A. 1997. Uses and properties of citrus
et al., 2001). The presence of phenolic compounds flavonoids. J. Agric. Food Chem. 45:4505-4515.
in the plant C.odorata has been demonstrated by
earlier worker (Phan et al., 2001). Derivatives of 4- Evans WC. (ed) 2002. Trease and Evans
hydroxybenzoic acid are used as anagelsics and pharmacognosy: Edinburg:W.B.Saunders.
antimicrobial substances. Also, Tannins,
flavonoids, and saponins are known to have Holme GLR, Plucknet DL, Pancho JV and
antimicrobial activities (Aziz et al., 1998; Evans, Herberger JP. 1977. Chromolaena odorata, The
2002). Hydroxybenzoic acid found in the extract is World’s Worst Weeds: Distribution and Biology:
known to possess antimicrobial activity. The The University Press of Hawaii.
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extract may be acting synergistically to bring about Le TT. 1995. The 5th European Tissue Repair
the observed inhibition of dehydrogenase activity. Society Annual Meeting, Padova, Italy, (Abst. 30).
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Alisi CS and Onyeze GOC. 2008. Nitric oxide Nweke CO, Okolo JC, Nwanyanwu CE and Alisi
scavenging ability of ethyl acetate fraction of CS. 2006. Response of planktonic bacteria of new
methanolic leaf extracts of Chromolaena odorata Calabar river to zinc stress. Afr. Journ. Biotechnol 5
(Linn.). Afr. Journ. Bioch. Res. 2 (7):145-150. (8):653-658.

Alisi CS and Onyeze GOC. 2009. Biochemical Nweke CO, Alisi CS, Okolo, JC and Nwanyanwu
mechanisms of wound healing using extracts of CE. 2007. Toxicity of Zinc to heterotrophic
chromolaena odorata-linn* Nig. Journ. Bioch and Bacteria from a tropical river sediment. Applied
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216 Journal of Research in Biology (2011) 3: 209-216
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Development trend and status of Pressurized Irrigation in Golestan
Province, Iran
Journal of Research in Biology

Authors: ABSTRACT:
Kami Kaboosi In recent decades, specific big share of executive capabilities, capital and
activities of the Iranian government have been allocating to the development of
pressurized irrigation systems. Concluding these programs and specifying the
development status of these systems in different locations of country can be regarded
as a guideline to assign future strategies. Reliable information on irrigation methods is
important for determining agricultural water demand trends. Therefore, this research,
in addition to surveying of pressurized irrigation projects performed from 1990 to
Institution: 2007 in Golestan province; consider development trend and status of pressurized
Islamic Azad University, irrigation systems in this province. Based on the result, totally 1020 pressurized
Gorgan branch, irrigation projects with 22990.9 hectares area have been performed in Golestan
Gorgan, Iran. province, which have covered only 6.64 percent of irrigated lands area or 3.2 percent
of total agricultural lands area and shows the lack of pressurized irrigation
development. Comparing the pressurized irrigation development trend in Golestan
province with the country and their coordination shows that the most important
factor in the lack of pressurized irrigation development in Golestan province is general
policies on the country scale. The most important reason of very fluctuation in
developing of these systems is continuous changes in policies and instability of
Corresponding author: adopted policies while, technical, social, cultural and executive factors have the least
Kami Kaboosi effects. Results show more than 90 percentage of sprinkler irrigation systems area
allocated to cotton, soybean, wheat and barley. Also, because of petty landowner of
agricultural lands in Golestan province, area average of personal farms that equipped
with pressurized irrigation systems is very lesser than cooperative farms. This paper
present comprehensive conclusion of sprinkler and micro irrigation projects from
various aspects in Golestan province in Iran.

Email: Keywords: Development, Pressurized Irrigation, Iran.

Web Address: Article Citation: Kami Kaboosi.
Documents/RA0014.pdf. Development Trend and Status of Pressurized Irrigation in Golestan Province, Iran.
Journal of research in Biology (2011) 3: 223-229

Received: 03 May 2011 /Accepted: 13 May 2011 /Published: 20 Jul 2011

© Ficus Press.
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INTRODUCTION Iran and (2) to consider development trend and
Reliable information on irrigation methods status of pressurized irrigation systems in this
is important for determining agricultural water province.
demand trends. In addition, trend of future water Golestan province is located in the north part
demand by agriculture is extremely important for of Iran. It consists of 11 townships. Province area is
water resource planning. The absence of reliable 20437.7 Km2 that consist 1.25 percentage of Iran
information can severely limit usefulness of long- (MPOG, 2005; MPOG, 2006; Abedi and Hatami,
term water planning. Water resource projects 2006). Because of fertile soil and good weather
Journal of Research in Biology

planning require reliable estimates of crop and condition, this province has important role in the
irrigation system combinations, which are important agricultural production of country. Based on
components of water budget. To update records on published statistical information by agriculture
irrigation methods used within each region, survey ministry’s head-office of Statistics and Information
should been conducted. Then, survey data are (2005), total agricultural land area of Golestan
analyzed and compared with earlier surveys to province is 717594.6 hectares that is equal to 5.32
study how irrigation methods are changing and to percentage of total agricultural land area of country.
make projections of future changes for long-term
Iranian government has particularly To update records on pressurized irrigation
attentions to the development of pressurized methods, a survey was conducted. In Iran, farmers
irrigation systems in order to improve water use equip agricultural lands to pressurized irrigation
efficiency and water application efficiency. So, in systems by governmental loans. Therefore, data of
recent decades, specific big share of executive farms that equipped with pressurized irrigation
capabilities, capital and activities of the Iranian systems are available. Those are with Iranian
government have been allocating to the general office on pressurized irrigation. Pressurized
development of pressurized irrigation systems. irrigation office in Golestan province is a branch of
Concluding these programs and specifying the Iranian general office on pressurized irrigation. In
development status of these systems in different the first step, total information about pressurized
locations of country can be regarded as a guideline irrigation projects that performed from 1990 to
to assign future strategies. 2007 in Golestan province gathered from
To update California’s records on crops and pressurized irrigation office in Golestan province.
irrigation methods, the California department of Then, these data (projects) from the viewpoints of
water resources conducts survey every 10 years number and area of projects, variety of user, variety
during recent decades (Orang et al, 2008). of pressurized irrigation systems, crops, etc were
Therefore, Orang et al. (2005 and 2008) conducted analyzed. Finally, on the basis of results, it was
a survey of irrigation methods in California in 2001. analyzed development trend and status of
Kahlown and Kemper (2007) evaluated the pressurized irrigation in Golestan province.
performance of trickle systems installed in
Balochistan, Pakistan during 1982–2002 by field RESULTS
surveys, physical verifications and interviews with 1- Number and area of pressurized irrigation
farmers. projects
Akhavan Giglo (2006) and Akhavan Giglo Number and area of pressurized irrigation
and Kanooni (2008) presented the situation of projects that performed from 1990 to 2007 in
pressurized irrigation systems in private and Golestan province is present in table 1. As shown,
governmental agricultural lands of Ardebil totally 1020 pressurized irrigation projects with
province, Iran. 22990.9 hectare land usage were performed that are
The purposes of this research is twofold; (1) mostly sprinkler systems and only 6 percentage of
to survey of pressurized irrigation projects that the number of pressurized irrigation projects (equal
performed from 1990 to 2007 in Golestan province, to 12.45 percentage of area) are allocated to micro
Table 1- Number and area (hectare) of pressurized irrigation projects
Sprinkler systems Micro systems Pressurized systems
number % area % number % area % number area
956 93.73 20129.3 87.55 64 6.27 2861.6 12.45 1020 22990.9
224 Journal of Research in Biology (2011) 3: 223-229

irrigation systems. Main reason of non-development which can cause irrigated land area increases. As
of micro irrigation in Golestan province is the little mention previously and shown in table 2, in
area of orchards in this province. Golestan province, area of orchards is much lesser
2- Percentage of agricultural lands equipped than farmland area. Therefore, in spite of little area
with pressurized irrigation systems of micro irrigation projects, its percentage is more
Area and percentage of agricultural lands than the percentage of sprinkler irrigation projects.
equipped with pressurized irrigation systems in 3- Comparing development of pressurized
Journal of Research in Biology

Golestan province is present in the table 2. irrigation in Golestan province with the country
According to this table, only 6.64 percentages of Area and number (N.) of pressurized
total irrigated agricultural lands is equipped with irrigation projects that performed in Golestan
pressurized irrigation systems that it is equal to 3.2 province within a period of 18 years are presented
percentages of total agricultural lands. It was clearly in table 3 and figure 1. In addition, area of
shown that in a period of 18 years, pressurized pressurized irrigation projects that performed in
irrigation systems have not developed in this country (Iran) after Zareii and Sadre Ghaen (2005)
province and at the present about 95% of total are shown in this table and figure. As seen,
irrigated agricultural land area is irrigating with development trend of pressurized irrigation systems
traditional surface method. Whereas more than 50 in Golestan province is completely similar to that of
percentages of total agricultural lands in Golestan the country. The most number and area of
province is rainfed, obviously water saving by the pressurized irrigation systems in Golestan province
development of pressurized irrigation systems and country are performed in 1996.
Table 2- agricultural land area (hectare) equipped with pressurized irrigation
area of
Type cultivation area of land % of total %
irrigated 327248 45.6 6.15
farmland 20129.3
total 684766 95.43 2.94
irrigated 19243.4 2.68 14.87
orchards 2861.6
total 32828.6 4.57 8.72
irrigated 346491.4 48.29 6.64
total 22990.86
total 717594.6 100 3.2

Table 3- comparing development trend of pressurized irrigation in Golestan with the country
Sprinkler systems Micro systems Pressurized systems
year Golestan Iran Golestan Iran Golestan Iran
N. area area N. area area N. area area
1990 0 0 2450 0 0 350 0 0 2800
1991 2 21 8594 0 0 2406 2 21 11000
1992 4 65.5 10629 0 0 2098 4 65.6 12727
1993 35 577 10778 1 3 2695 36 580 13473
1994 74 1459.6 23476 2 1 3024 76 1460.3 26500
1995 38 684.6 9875 1 1 1725 39 685.6 11600
1996 262 4454.7 66112 1 1.6 9288 263 4456.3 75400
1997 195 3304 37058 4 43 8442 199 3346.9 45500
1998 87 2031.5 27939 1 5 11061 88 2036.5 39000
1999 57 1735.1 20308 1 48.6 11692 58 1783.7 32000
2000 69 1501.8 19293 2 151 13607 71 1652.8 32900
2001 20 287.1 14467 2 126.2 9710 22 413.3 24177
2002 9 83.2 12499 3 101 15294 12 184.2 27748
2003 23 895.3 28061 4 844.4 27138 27 1739.7 55199
2004 29 526 * 5 598.8 * 34 1123.8 *
2005 16 902.4 * 8 233.9 * 24 1136.3 *
2006 25 1391.5 * 17 559.6 * 42 1951.1 *
2007 11 208.9 * 12 144.5 * 23 353.4 *
total 956 20129.3 * 64 2861.6 * 1020 22990.9 *
* Non available data
Journal of Research in Biology (2011) 3: 223-229 225
Journal of Research in Biology

Figure 1- comparing development trend of pressurized irrigation in Golestan with the country

As mentioned previously, In Iran, farmers Comparing development trend of
equip agricultural lands to pressurized irrigation pressurized irrigation in Golestan province with the
systems by governmental loans. Within a period of country and their coordination shows that the most
1990-96, governmental loans for developing of important factor in the lack of pressurized irrigation
pressurized irrigation system increases but it development in Golestan province is general
decreases within a period of 1996-2002. policies on the country scale. The most important
Consequently, development of pressurized reason for very fluctuation in developing of these
irrigation systems increases in 1990 to 1996 but systems is continuous changes in supporting
decreases in 1966 to 2002. Governmental support policies and instability of adopted policies while
henceforth increases again and it causes developing technical, social, cultural and executive factors have
pressurized systems. the least effects.
Table 4- Variety of sprinkler and micro irrigation systems in Golestan province
Number Area (ha)
system average
Number % Area (ha) %
Solid Set 7 0.73 474.63 2.36 67.8
Hand Move 1 160 16.74 5127.21 25.47 32.1
Hand Move 2 427 44.67 4921.7 24.45 11.5
Unknown Hand Move 147 15.38 2652.54 13.18 18
Side Roll 17 1.78 877.7 4.36 51.6
Sprinkler Travelling Gun 168 17.57 3254.69 16.17 19.4
Center Pivot 6 0.63 1281.1 6.36 213.5
Linear Move 3 0.31 352 1.75 117.3
Complex of two systems 13 1.36 1067.11 5.30 82.1
Unknown Sprinkler 8 0.84 120.58 0.60 15.1
Total of Sprinkler 956 100 20129.26 100 21.1
Line Source Emitter 7 10.94 809.06 28.27 115.6
Micro Point Source Emitter 57 89.06 2052.54 71.73 36
Total of Micro 64 100 2861.6 100 44.7
Total of Pressurized 1020 - 22990.86 - 22.54
Hand Move 1 refers to lateral hand move but Hand Move 2 refers to a system that lateral,
manifold and main pipes and pump move by hand.
226 Journal of Research in Biology (2011) 3: 223-229

4- Variety of sprinkler and micro irrigation 5- Irrigated crops by pressurized irrigation
systems in Golestan province systems in Golestan province
Table 4 shows the variety of sprinkler and Results showed that sprinkler irrigation
micro irrigation systems in Golestan province that systems in Golestan province are mostly used for
performed from 1990 to 2007. As shown, hand farmland crops. Based on the results, more than 90
move 1 and 2 and travelling gun systems allocated percentage of sprinkler irrigation system area (more
25.5, 24.5 and 16.17 percentage of total sprinkler than 18100 hectare) are allocated to cotton,
Journal of Research in Biology

systems area, respectively. However, sum of the soybean, wheat and barley. Others sprinkler
number and area of center pivot, linear move and projects are performed for corn and canola. Based
side roll systems in Golestan province is in 26 on published statistical information by agriculture
projects and 2510.8 hectare, respectively. This is ministry’s head-office of Statistics and Information
equal to 2.72 and 12.47 percentage of total number (2005), 76 percentage of total farmlands area in
and area of sprinkler irrigation systems. The most Golestan province (more than 520000 hectare) are
important reasons of little development in center allocated to these four crops (cotton, soybean,
pivot, linear move and side roll systems in Golestan wheat and barley), in that 43 percentage of it is
province are the existence of cheap workers in the irrigated farmland. Therefore, at present only 8
region and petty landowners. Since too much percentage of irrigated land area of these four crops
percentage of agricultural lands in Golestan is equipped with sprinkler irrigation systems.
province have small area; developing of these Staple percentage of micro irrigation systems in
systems is difficult. Golestan province is point source emitter system
Hand move 2 system has the least amount of that allocated to orchards as plum, peach,
area average. Its area average is 11.5 hectare. eucalyptus, kiwi, olive and citrus trees while line
However, center pivot and linear move systems source emitter systems mostly allocated to some of
have the most area average. Their area averages are the farmland crop as cotton, tomato and soybean.
213.5 and 117.3 hectare, respectively. 6- Variety of users of pressurized irrigation
As seen in table 4, staple percentage of systems in Golestan province
micro irrigation systems in Golestan province is In this research, users of pressurized
point source emitter system that allocated to irrigation systems in Golestan province is divided to
orchards while 28.27 percentage of micro irrigation 3 groups: 1- personal or individual farms 2-
systems area is line source emitter system that Governmental farms and 3- cooperative farms.
allocated to some of the farmland crop. Table 5 shows the variety of users of pressurized
irrigation systems in Golestan province. As seen,
staple percentage of pressurized irrigation projects

Table 5- Variety of users of pressurized irrigation systems in Golestan province
system index index sprinkler micro total
number 920 47 967
% 96.23 73.44 94.80
Area area 15872.17 1314.01 17186.18
(ha) % 78.85 45.92 74.75
average 17.25 27.96 17.77
number 15 3 18
% 1.57 4.69 1.76
Area area 575.29 2.60 577.89
(ha) % 2.86 0.09 2.51
average 38.35 0.87 32.11
number 21 14 35
% 2.20 21.88 3.43
Area area 3681.80 1544.99 5226.79
(ha) % 18.29 53.99 22.73
average 175.32 110.36 149.34
number 956 64 1020
total Area (ha) 20129.26 2861.6 22990.86
average 21.06 44.71 22.54
Journal of Research in Biology (2011) 3: 223-229 227
Table 6- frequency distribution and average of pressurized irrigation projects
sys. Area (ha) <2 2-5 5-10 10-20 20-30 30-40 >40 total average
number 5 91 293 309 153 37 68 956 21.06
sprinkler % 0.52 9.52 30.65 32.32 16 3.87 7.11 100 -
cumulative 0.52 10.04 40.69 73.01 89.02 92.89 100 - -
number 9 10 17 8 4 3 13 64 44.71
micro % 14.06 15.63 26.56 12.5 6.25 4.69 20.31 100 -
cumulative 14.06 29.69 56.25 68.75 75 79.69 100 - -
Journal of Research in Biology

number 14 101 310 317 157 40 81 1020 22.54
total % 1.37 9.9 30.39 31.08 15.39 3.92 7.94 100 -
cumulative 1.37 11.27 41.66 72.75 88.14 92.06 100 - -
are performed in personal farms so that 967 projects 93.73 percentage of pressurized irrigation project
(about 95 percentages of total projects) with area are allocated to sprinkler irrigation projects.
17186.18 hectare (75 percentage of total area) were However, it is relatively different when frequency
performed in personal farms. distribution percentage of area of pressurized
Because of petty landowners of agricultural irrigation projects on comparing with micro
lands in Golestan province, area average of personal irrigation projects.
farms that equipped with pressurized irrigation 8- Type of used water resource for pressurized
systems is much lesser than cooperative farms. irrigation projects in Golestan province
Ratio of area average of pressurized irrigation Based on the results, water for more than 90
systems in cooperative farms to personal farms is percentage of pressurized irrigation projects are
8.4 (17.77 against 149.34 hectare). This ratio for supplied from ground water (shallow and semi deep
sprinkler projects is more than 10 (175.32 against wells). This awareness can assist in better water
17.25 hectare) and for micro projects is about four resource planning.
(110.36 against 27.96 hectare).
When data of table 5 reviews again, this CONCLUSION
found that the area average of pressurized irrigation Reliable information on irrigation methods
projects (22.54 hectare) is approximately equal to is important for determining agricultural water
the area average of personal farms pressurized demand trends. The purposes of this research is to
irrigation projects (17.77 hectare). Its reason is that survey the pressurized irrigation projects that
more percentage of projects is performed in performed from 1990 to 2007 in Golestan province,
personal farms. Iran and to consider the development trend and
7- Analysis of frequency distribution and area status of pressurized irrigation systems in this
average of pressurized irrigation projects in province. Based on the results, totally 1020
Golestan province pressurized irrigation projects with 22990.9
In order to make easier, analyzing of hectares area have been performed in Golestan
projects from the point of view of area, projects are province, which have covered only 6.64 percent of
divided to 7 classes (less than 2 ha, 2-5 ha, 5-10 ha, irrigated land area or 3.2 percent of total
10-20 ha, 20-30 ha, 30-40 ha and greater than 40 agricultural land area and shows the lack of
ha). Number of pressurized irrigation projects for pressurized irrigation development. Coordination of
each class in Golestan province is present in table 6. pressurized irrigation development trend in
As shown, the most percentage of pressurized Golestan province and the country showed that the
irrigation projects performed in farms with 10-20 ha most important factor in the lack of pressurized
area and 50-10 ha area, respectively so that these irrigation development in Golestan province is
two classes respectively allocated 31.08 and 30.39 general policies on the country scale. Results
percentage of total projects. Although area average showed that more than 90 percentage of sprinkler
of pressurized irrigation projects is 22.54 hectare irrigation systems area are allocated to cotton,
and it is seemingly good, however, table 6 shows soybean, wheat and barley. In addition, because of
that the area of more than 72 percentages of petty landowner of agricultural lands in Golestan
projects is less than 20 hectare. province, area average of personal farms that
In addition, frequency distribution equipped with pressurized irrigation systems is very
percentage of the area of sprinkler and pressurized lesser than cooperative farms. Because of cheap
irrigation projects is approximately equal because workers in the region and petty landowners,
228 Journal of Research in Biology (2011) 3: 223-229
development of center pivot, linear move and side Kahlown MA and Kemper WD. 2007. Factors
roll systems in Golestan province is difficult. Affecting Success and Failure of Trickle Irrigation
Systems in Baluchistan, Pakistan. Irrig. Sci., 26:71-
Abedi G and Hatami A. 2006. Economical, Social
and Cultural Indices of Golestan province and Management and Planning Organization of
comparing it with country,Golestan, Iran (in Golestan province (MPOG). 2006. Economical,
Journal of Research in Biology

Persian). MPOG 240. Social and Cultural Indices of Golestan province,
Golestan, Iran (in Persian). 231.
Akhavan Giglo K. 2006. Consideration of
Pressurized Irrigation Situation. seasonal magazine Management and Planning Organization of
of Agricultural and Natural Resources Engineering Golestan province (MPOG). 2005. Summery of
Regulation (ANRER) 12:30-35. basic Indices of Golestan province, Golestan, Iran
(in Persian). 230
Akhavan Giglo K and Kanooni A. 2008.
Operation Evaluation of Pressurized Irrigation Orang MN, Matyac JS and Snyder R. 2008.
Systems in Private and Governmental Land of Survey of Irrigation Methods in California in 2001.
Ardebil Province. Proceeding of the Scientific J. Irrig. and Drain. Eng., ASCE 134:96-100.
Seminar of National Project of Pressurized
Irrigation and Sustainable Development, Iranian Orang MN, Snyder R and Matyac JS. 2005.
National Committee on Irrigation and Drainage Survey of Irrigation Methods in California in 2001.
(IRNCID), Tehran, Iran. California water plan update.

Agriculture ministry’s head-office of Statistics Zareii G and Sadre Ghaen SH. 2005. Perspective
and Information. 2005. Iranian Agriculture of Sprinkler Irrigation Development in Iran on 1400
Ministry, Horizon. Proceeding of the Technical Workshop of
Sprinkler Irrigation, Iranian National Committee on
Irrigation and Drainage, Tehran, Iran.

Journal of Research in Biology (2011) 23: 223-229 229
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Patterns of butterfly biodiversity in three tropical habitats of the eastern part
of Western Ghats
Journal of Research in Biology

Authors: ABSTRACT:
Murugesan S and
Muthusamy M. The Western Ghats is one among the hot spots in 25 biodiversity countries
identified in the world. The Coimbatore city is situated at the foot hills of Western
Ghats (a part of Nilgiri Biosphere Reserve) are recognized as a paradise of butterflies
due to its salubrious climate. Butterflies in the Western Ghats belong to 5 families,
Post graduate and Research 166 genera and 330 species, of which 37 species are endemic and they depend on
Department of Zoology, more than 1000 plant species for feeding and breeding. The present study surveyed
Government Arts College 103 individual butterfly species belonging to 5 families namely Nymphalidae (32),
(Autonomous) Pieridae (23), Lycaenidae (19), Hesperiidae (15) and Papilionidae (14), which revealed
Coimbatore 641 018. that Nymphalidae and Pieridae are the rich dominant families, while Hesperiidae and
Tamil Nadu, India. Papilionidae are less dominant. High incidences of butterfly population with wide
distribution were observed during the months of March-April and the monsoon
seasons (September - November) which diminish during December-January. It was
observed that the occurrence and distribution of butterflies are closely associated
Corresponding author: with the availability of its larval and adult host plants. The butterfly population of a
Murugesan S species is gradually decreasing in number due to human interference in the habitat
and the destruction of host plants.

Email: Keywords:
Biodiversity, Bioregion, Butterfly population, Host plant, Western Ghats.

Web Address: Article Citation:
Murugesan S and Muthusamy M.
Patterns of butterfly biodiversity in three tropical habitats of the eastern part of
Western Ghats.
Journal of research in Biology (2011) 3: 217-222

Received: 06 Jul 2011 /Accepted: 08 Jul 2011 /Published: 20 Jul 2011

© Ficus Publishers.
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217-222 | JRB | 2011 | Vol 1 | No 3
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INTRODUCTION Chromolaena odorata, and Parthenium
Preserving our planets irreplaceable flora hysterosporus are the prominent weeds in the open
and fauna in the race against extinction has become areas of the landscape.
most significant of all environmental issues. In the
recent past, the Western Ghats of India is one of the MATERIALS AND METHODS
34 biodiversity hot spots of the world. A great The butterflies were observed and collected
variety of vegetations are found all along the on either side of the transect path in each site.
Western Ghats. Butterflies and Moths (Order: Transects were surveyed twice in a month.
Lepidoptera) offer good opportunities for studies on According to Wynter – Blyth (1957) the study
population and community ecology (Pollard, 1991). period was chosen from September 2005 to May
Larsen (1987) made a detailed survey of butterflies 06. The butterflies were initially identified in the
on Nilgiri Mountains and recorded nearly 330 field condition and unidentified butterflies were
species including endemics. Previous workers collected by using nylon net and brought to the
(Ugarte & Rodricks, 1960; Larsen, 1987; laboratory for better identification. The collected
Asaithambi, 1994; Kunte, 1997; Arun, 2000; butterflies were placed in the killing jars (wide
Eswaran & Pramod, 2005) have studied the mouthed bottle containing a piece of cotton soaked
diversity and seasonal pattern of butterflies in the in ethyl acetate) for 1 h. The killed butterflies were
Western Ghats. Butterflies are an essential part of preserved in a paper envelope and fixed on the
any natural ecosystem as their adults perform spreading board. The colleted butterflies were
pollination and larvae act as primary herbivores identified by using the keys of Ackery (1988);
thereby transferring radiant energy trapped by Evans (1932) and Gonakar (1996).
plants to the next tropic level; rendering dual roles
as pollinators and energy transferors. Butterflies are RESULTS
being good indicators of climatic conditions as well Butterfly belongs to the order Lepidoptera,
as seasonal and ecological changes; they can serve and approximately 15,000 species were so far
in formulating strategies for conservation. It is recorded in the literature (Shieldes, 1989). The
hence encouraging that butterflies are now being Coimbatore bioregion comprises a wide variety of
included in biodiversity studies and biodiversity butterflies with abundant distribution in the foot
conservation prioritization programme. The present hills of Western Ghats. In present study, there are
paper deals with the occurrence and diversity of about 103 species of butterflies belonging to five
butterflies in the different sites of Western Ghats. families were identified and recorded. The family
Hesperiidae, popularly known as Skippers was
STUDY AERA characterized by short use antennae which bear
The Siruvani hills (10o56’ – 10o57’ N & hooks at the tip. In Hesperiidae around 3500 species
076 43’ - 076o44’ E); Maruthamalai hills (11º02’ –
were recorded in the world of which about 145
11º03’ N & 076º52’ – 076º54’ E) and Anaikatti species are found in the Indian Sub Continent
hills (11o06’ – 11o08’ N & 076o44’ – 076o 45’ E) of (Meena Haribal, 1992). In the present survey, 15
Coimbatore bioregions were selected for the study. individual butterfly species were identified in the
The places are well known for its good climate and family Hesperiidae (Table 1).
consist of rich variety of butterfly host plants. The The family Papilionidae includes the
area receives an average rainfall of 668 mm per Swallowtails; they are large in size more attractive
year recorded over the last 10 years. Maximum and the most endangered groups. In the present
temperature varies from 27-35 degrees. Trees such study there are about 14 species of butterflies were
as Albizia amara, Albizzia lebberck, Acacia identified and recorded (Table 2). The family
leucophloea, Acacia polyacantha, Ziziphus Pieridae includes 1000 species in the world. In the
mauritiana, Chloroxylon swietenia, Tamarindus present study, there are 23 species of butterflies
indica, Tectona grandis and Eucalyptus are the belonging to the family Pieridae were identified in
dominant trees in these areas. Dominant shrubs are Coimbatore region (Table 3). The butterflies of
Cassia auriculata, Cassia fistula, Capparis grandis, family Pieridae was identified by using the clues of
C. roxburghii, C. grandiflora, C. sepiaria, hifid pretarsal claws, using scales containing ptarine
Flacourtia indica, Elaeodendron glaucam, type of pigments and imperfect forelegs. The
Clausina heptaphylla, Randia dumetorum, Premna family Nymphalidae is known as the “Brash foot
tomentosa and Pavetta indica. Lantana camara, butterflies” which is a diverse family, consists of
218 Journal of Research in Biology (2011) 3: 217-222
Murugesan et al.,2011

Month of Site of
Family name Common name Species Name
collection Collection
The dart Potanthus pava pava March-06 SVH
Dusky yellow brash flat Daimio phisara phisara February-06 MAH
Chestnut angle Odentoptilun angulata angulata February-06 MAH
Common Red eye Matapa aria February-06 MAH
Giant Red eye Gangara thyrsis thyrsis February-06 MAH
Small grass owlet Bibasis amara January -06 SVH
Chestnut bob Lambrix salsala salsala March - 06 SVH
Brown awl Badamia exclamationis January- 06 SVH
Rice Swift Borbo cinnara March -06 SVH
Common Dart Potanthus pallida March- 06 SVH
Indian Skipper Spialia galba November-05 AKH
Common Spottedflat Celaenorrhinus leucocera November-05 AKH
Spotted Demon Notocrypta fiesthamelii alysos November -05 AKH
Common Awl Hasora badra badra October-05 AKH
Small Common Flat Sarangesa dasahara dasahara October-05 AKH

Month of Site of
Family name Common name Species Name
collection Collection
Blue mormon Princeps polymnestor October-05 SVH
Common mormon Princeps polytes romulus December-05 MAH
Common rose Pachliopta aristoloachiae aristoloachiae December - 05 MAH
Common bluebuttle Graphium sarpedon sarpedon December - 05 MAH
Crimson rose Pachliopta hector December - 05 MAH
Tailed Jay Graphium agammemnon agammemnon January-06 MAH
Papilionidae Common Jay Graphium doson axion December- 05 SVH
Lime Butterfly Princeps demoleus January- 06 MAH
Great zebra Pathysa xenocles phrontis November- 05 AKH
Glassy bluebottle Graphium cloanthus November- 05 AKH
Great jay Graphium eurypylus cheronus November- 05 AKH
Common raven Princeps castor polas September-05 AKH
Common Mime Chilasa clytia clytia September-05 AKH
Veined jay Graphium bathycles Chiron November-05 AKH

14 sub families out of which 10 sub families were related to the available nutritional values of the host
recorded in Peninsular India. In the present work plant (Slanksy, 1992; Scribes, 1995 and Thomas,
there are 32 species of Nymphalidae butterflies 1995). The growth rate of Lepidopteran depends on
were identified (Table 4). The family Lycaenidae, the host plants consisting of nutritional composition
otherwise known as “Blues” is the world’s largest and plant secondary compositions (Salnsky, 1992).
family of butterflies, which about 6,000 species. In the Southern Nilgiris and on the lower plateau,
India has approximately 450 species, which is 21% March and September - October is the best seasons
of its total number of butterfly species. The present
for butterflies; May is comparatively poor month
studies viewed around 19 species of lycaenidaes (Wyhter & Blyth, 1957) for butterflies. The
were identified in the foothills of Western Ghats maximum number of species was observed in the
(Table 5). Family Nymphalidae throught out the entire study
area. Many members of this family were
DISCUSSION polyphagous which would help them to live in all
Climatic changes are thought to alter the habitats and in different elevation gradients
distribution and availability of animals and plants (Sreekumar & Balakrishnan, 2001). They also
through out the world (Warren et al., 2001). reported that the May month was comparatively
Feeding and reproduction of butterflies are closely poor month for butterfly population.

Journal of Research in Biology (2011) 3: 217-222 219
Murugesan et al.,2011

Family Month of Site of
Common name Species Name
name collection Collection
Common albatross Appias albina darada January-06 MAH
Three spot grass yellow Eurema blanda silhetana December-05 MAH
Common grass yellow Eurema hecabe contubernalis December-05 MAH
Common Emigrant Catopsilia Pomona January-06 MAH
Common wanderer Pareronia valeria hippia March-06 SVH
Great orange tip Hebomoia glaucippe glaucippe March-06 SVH
Mottled emigrant Catopsilia pyranthe January-06 MAH
Tree yellow Gandaca harina assamica December-05 MAH
White orange tip Irias Marianne cramer December-05 SVH
Pioneer Anapheis aurota aurota January-06 MAH
Common jezebel Delias eucharis March- 06 SVH
Pieridae Common gull Cepora nerissa nerissa December- 05 MAH
Chocolate albatross Appias lyncida elenora March-06 SVH
Yellow orangetip Ixias pyrene familiaris January-06 MAH
Small orange tip Coloti etrida February -06 MAH
Dark clouded yellow Colias fieldii January-06 MAH
Psyche Leptosia nina nina February-06 MAH
Indian cabbagewhite Pieris canidia indica December-05 MAH
Plain puffin Appias indra indra November-05 AKH
Yellow jezebel Delias agostina agostina November-05 AKH
Spot puffin Appais lalage durvasa November-05 AKH
Hill jezebel Delias belladona ithiela October -05 AKH
Small grass yellow Eurema brigitta rubella October -05 AKH

Month of Site of
Family name Common name Species Name
collection Collection
Common sergeant Parathyma perius March- 06 SVH
Common sailer Neptis hylas varmona March-06 SVH
Lemon pansy Precis lemonias lemonias December-05 MAH
Common fourring Ypthima hubenri hubenri February-06 MAH
Tawny coster Acraea violae December-05 MAH
Common five ring Ypthima baldus baldus March - 06 SVH
Angled caster Ariadne ariadne pallidior December -05 MAH
Danaid eggfly Hypolimnas misippus February -06 MAH
Rustic Cupha erymanthis lotis March -06 SVH
Darkbrand bushbrown Mycalesis mineus mineus December-05 MAH
Great eggfly Hypolimnas bolina February -06 BUC
Nymphalidae Peacock pansy Precis almana almana December -05 MAH
Yellow pansy Precis hierta magna December-05 MAH
Dark evening brown Melanitis phedima bela December-05 MAH
Common bushbrown Mycalesis perseus blasius December -05 MAH
Gray pansy Precis atlites atlites March - 06 SVH
Tabby Psuedergolis wedah February - 06 MAH
Baronet Symphaedra nais March - 06 SVH
Common baron Euthalia aconthea suddhodana December -05 MAH
Common tiger Danus genutia December -05 MAH
Blue king crow Euploea klugii klugii December -05 MAH
Common crow Euploea core core December -05 MAH
Plain tiger Danus chrysippus December -05 MAH

220 Journal of Research in Biology (2011) 3: 217-222
Murugesan et al.,2011
Month of Site of
Family name Common name Species Name
collection Collection
Blue tiger Tirumala limniace leopardus March- 06 SVH
Dark blue tiger Tirumala septentrionis March-06 SVH
Glassy tiger Parantica aglea melanoides March- 06 SVH
Common evening brown Melanitis leda ismene November-05 AKH
Nymphalidae Common duffer Discophora sondiaca zal November-05 AKH
Bright eye bush brown Mycalesis nicotia November- 06 AKH
Common brown Euthalia aconthea suddhodana November- 06 AKH
Leopard lacewing Cethosia cyane September -05 AKH
Indian Fritillary Argyreus hyperbius hyperbius October -05 AKH

Month of Site of
Family name Common name Species Name
collection Collection
Indian red flash Rapala manea March- 06 SVH
Common pierrot Castalius rosimon rosimon March- 06 SVH
Zebra blue Syntarucus plinius March- 06 SVH
Common tip Hypolycaena erylus himavantus February-06 MAH
Hybrid sapphire Heliophorus hybrida February -06 MAH
Common cerulean Jemides celeno celeno February-06 MAH
Pale grass blue Pseudozizeeria maha March-06 SVH
Metallic cerulean Jemides alecto eurysaces March-06 SVH
Red Pierrot Talicada nyseus guerin meneville March -06 SVH
Lycaenidae Grass jewel Zizeeria trochilus trochilus February-06 MAH
Striped pierrot Tarucus nara March -06 SVH
Tiny grass blue Zizula hylax February -06 MAH
Lime blue Chilades laius March -06 SVH
Margined hedge blue Lycaenopsis marginata November-05 AKH
Silver royal Ancema blanka argentea November -05 AKH
Common acacia blue Surendra quercetorum quercetorum September-05 AKH
Common leaf blue Amblypodia anita anita September- 05 SVH
Common Cerulean Jamides celeno celeno November- 05 AKH
Dark grass blue Zizeeria knyasna November -05 AKH
SVH : Siruvani Hills
MAH : Maruthamalai Hills
AKH : Anaikatti Hills
The Princeps demolens, Papilionidae plants may be the factors for the emergence of
hector, P. aristolochiae are more in number during butterflies during the same seasons. The butterfly
the months of October and December. The present community did not show much variation between
investigation showed that the butterfly population is the sampling locations of the study areas, hence the
greatly increased during the period of October – whole area can be considered as one unit while
November due to the availability of more larval planning conservation measures.
host plants and high flower density. Therefore, this
period might be the best season for butterflies of
these areas. Though the Danaid butterflies are found
throughout the year, they found abundantly during
the months of October - December and also it was
found more during March and April. The cool
climate, increased moisture content, emergence of
fresh larval host plants and flowering of adult host

Journal of Research in Biology (2011) 3: 217-222 221
Murugesan et al.,2011

REFERENCES Shields O. 1989. World number of butterflies
Ackery PR. 1988. Host Plants and classification: A J.Lep.soc. 5431(3):178-183(G).
review of Nymphalidae butterflies Biol.J.Linn.soc.,
33:95-203(CE). Slanksy JJ. 1992. National ecology the
fundamental guest for nutrients. In Ecological and
Arun PR. 2000. Seasonality and abundance of evolutionary (constraints on fording by caterpillars.
insects with special reference to butterflies (Eds: Stamps NE. and Case TM.) (Chapman and
(Lepidoptera: Rhapalocera) in a moist deciduous Hall, Newyork). 135-174.
forest of Siruvani, Nilgiri Bioshere reserve, South
India. Ph.D Thesis submitted to Bharathiar Sreekumar PG and Balakrishnan M. 2001.
University, Coimbatore, India. Habitat and altitude preferences of butterflies in
Aralam Wildlife Sanctuary, Kerala. Tropical
Asaithambi P. 1994. Butterflies of Mudumalai Ecology 42(2):277-281.
Wildlife sanctuary, Tamil Nadu. Zoos’ Print. 12
(11):1. Thomas JA. 1995. The ecology and conservation
of Maculinea arion and other European species of
Eswaran R and Pramod P. 2005. Structure of large blue butterfly. In: A.S. Pullin (ed.) Ecology
butterfly community of Anaikatty hills, Western and conservation of butterflies. Chapman and Hall,
Ghats. Zoo’s print. 20(8):1939-1942. London. 180-210.

Evans WH. 1932. Identification of Indian Ugarte E and Rodricks. 1960. Butterflies of Palani
butterflies Bombay natural History, Bombay (TA). hills: A complementary list. Journal of the Bombay
Natural history society. 57(2):270-277.
Goankar H. 1996. Butterflies of Western Ghats,
India (including Srilanka): A biodiversity Warren M, Shill J, Kthomes JA, Asher J, Jox R,
assessment of a threatened mountain system. Report Huntlay B, Roy DB, Felter MG, Jeffcate S,
submitted to the center for Ecological Sciences, Harding P, Teffcoate G, Willis SG, Greateo Rex,
Bangalore (unpublished). Davies JN, Moss D and Thomas CD. 2001.
Nature 414:65-69.
Kunte K. 1997. Seasonal patterns in butterfly
abundance and species diversity in four tropical Wynter-Blyth. 1957. Butterflies of the Indian
habitats in northern Western Ghats. Journal of region. Bombay Natural History Society. Bombay.
biosciences 22(5):593-603. 523.

Larsen TB. 1987. The butterflies of the Niligiri
mountains of South India (Lepidoptera:
Rhopalocera) journal of the Bombay Natural
History Society 84:26-43.

Meena Haribal. 1992. The butterflies of Sikkim
Himalaya and their Natural history. Publish. SNCF,
Sikkim. 53-6.

Pollard E. 1991. Monitoring butterfly numbers; in
Monitoring for conservation and ecology (ed.) F.B.
Goldsmith (London: Chapman and Hall). 87.

Scribes JM. 1995. Overview of swallowtail
butterflies taxonomic and distributional latitude of
swallowtail butterflies; their ecology and
evolutionary biology (Ed: by Scribes JM, Tsubaki
Y and Leder house RC.), Scientific Publishers,
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222 Journal of Research in Biology (2011) 3: 217-222
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An International Online Open Access
Original Research paper
Publication group

Habitat diversity, Morphological and systematic analysis of multipotential species of Aloe
barbadensis Mill. (Liliaceae) from the Southern Western Ghats of Tamil Nadu, India
Journal of Research in Biology

Authors: ABSTRACT:
Parthipan M, Binu
Thomas and Rajendran A.

The present paper highlights the habitat variability, morphological features,
Department of Botany,
School of life Sciences systematic analysis and multi potentiality of Aloe barbadensis Mill. were collected
Bharathiar University, from Nilgiri district of Southern Western Ghats of Tamil Nadu, India. The epiphytic
Coimbatore – 641046 nature of Aloe barbadensis Mill. is quite interesting than other habitats.
Tamil Nadu, India

Corresponding author:
Binu Thomas

Email: Aloe barbadensis, Habitat diversity, Southern Western Ghats, Nilgiri, Tamil Nadu.

Web Address: Article Citation: Parthipan M, Binu Thomas and Rajendran A.
Documents/RA0068.pdf. Habitat diversity, Morphological and systematic analysis of multipotential species of
Aloe barbadensis Mill. (Liliaceae) from the Southern Western Ghats of Tamil Nadu,
Journal of research in Biology (2011) 3: 237-241

Received: 24 Jul 2011 /Accepted: 25 Jul 2011 /Published: 30 Jul 2011

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

237-241 | JRB | 2011 | Vol 1 | No 3
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INTRODUCTION they are not found in cold habitat regions and they
Most of the Angiosperm species shows are mostly found in tropical and subtropical regions
varied habitat specificity such as Lithophytes, of the country (Frame, 2003).
Chasmophytes, Psammophytes, Hydrophytes,
Xerophytes, Parasites and Epiphytes (Annaselvam RESULTS:-
and Parthasarthy, 2001; Das et al. 2006). Habitat diversity of Aloe barbadensis Mill
Angiosperms have been so successful in terrestrial The Aloe barbadensis Mill. is a perennial
and aquatic ecosystems that they represent majority plant with fleshy leaves. A native of North Africa.
of the herbs and shrubs and many of the trees as It is planted as hedge in house premises and also
well. Depend up on their mode of nutrion and run wild in the desert conditions and poorest soils
attachment pattern they are of different types and and also extensively cultivated throughout India for
shows variety of growth forms and also adapted to its medicinal value. The habitat variability of the
various habitats for their survival (Rao, 1994). Aloe barbadensis Mill, reveals that it is not only in
The species Aloe barbadensis Mill, comes terrestrial habitat but also in rocky habitats as
under the family liliaceae. Most of the members of chasmophytes in the Southern Western Ghats of
the family liliaceae are herbs, sometimes climbers Coimbatore district of Tamilnadu by Binu Thomas
and rarely as shrubs. The roots are fibrous, tuberous et al. (2009). The present study observed that the
or a creeping rhizome. Leaves various, cauline or species Aloe barbadensis Mill. shows an intresting
radical and sometimes functionally replaced by epiphytic habitat of Aloe barbadensis Mill. in the
cladodes, fleshy, usually parallel veined. Flowers blue mountains (2,240 meters above sea level) of
usually regular and 2-sexual, axillary or terminal Nilgiri District of Southern Western Ghats of Tamil
solitary. Fruit become berry, seeds globose or Nadu shows an intresting epiphytic habitat of Aloe
flattened. The main characteristic feature of the barbadensis Mill. It is well established in tree
species Aloe barbadensis Mill. having thick and trunks of Grevillea robusta Cunn. (Proteaceae).
fleshy leaves. The margins of the leaves become The nature of unusual/uncommon habit of Aloe
spinous and forming rosettes. It is introduced from barbadensis Mill in epiphytic habitat is luxuriant
tropical Africa and naturalized in India growing when comparing with the same species grown as
and run wild, especially in hedge- rows in the drier terrestrial habitat of neighbouring area of Nilgiri
localities up to 2,500 feet (Gamble and Fischer, district. The interactions between climate and
1915-1936). vegetation results in the marvelous vegetation in
Aloe barbadensis Mill. is a semi-tropical Nilgiri district is very important in the contribution
plant which looks more like a cactaceous member of biodiversity of the Western Ghats (Plate-1)
of lily family which usually grows in the African Systematic and Morphological analysis of Aloe
continent. There is an evidence to suggest that Aloe vera (Liliaceae).
barbadensis originated from the warm, dry climates Aloe barbadensis Mill. f.,Fl. Ind. 83.1768;
of Africa (Moon, 1824). Now the plant is much Aloe perfoliata L., Sp. Pl. 320. 1753; Gamble’s Fl.
more widespread and can be found growing Pres. Madras 1520. 1928; Henry et al., Fl. Tamil
throughout Europe and North America as well as Nadu 37.1989; (“Kathalai”).
South America, the Middle East, China, India, The dwarf plant with radical rosettes leaves,
Pakistan and Australia (Eldridge, 1978). The habitat ensiform, 40-60 × 2-8 cm, succulent, spiny. Scapes
of Aloe vera means Woodlands, Mediterranean 1-3; racemes to 40 cm. Flowers bisexual, yellow,
forests and Scrub according to the World Wildlife 2.5-3.5 cm long. Perianth-tube terete, 1-1.5 cm,
Federation ( Balakrishnan,1974). somewhat curved; lobes 6, orange, oblong, 1× 0.5
The Aloe barbadensis Mill. habitat needs cm, 3- nerved. Stamens 3 + 3. Ovary 3-celled;
direct rays of the sun and a well drained soil. When ovules α, axile; style elongate to 2.5 cm; stigma
these plants are grown outdoors then it needs obscurely lobed. Capsule ellipsoid-oblong to 1.5 ×
warmth sun rays and protection from the winters. 1 cm.
Sometimes the habitat is destroyed due to various The Aloe barbadensis Mill. plant has thick
factors. The reasons are settlement of area by juicy leaves with sharp points, which grow to a
humans and deforestation (Wilfred and Claudia, height of twelve to sixteen inches. The leaves of
2007). It disturbs the natural balance of the area and aloe vera have no stem and are greenish in color.
destroys most of the species of Aloe vera plants. Too much water in the soil makes the leaves pale
The Aloe vera plants consist mostly of 95% water and sunlight again restores the color. Too much
238 Journal of Research in Biology (2011) 3: 237-241
Parthipan et al.,2011

Plate 1. Epiphytic nature of Aloe barbadensis Mill. on tree trunks of Grevillea robusta Cunn.

water is not good for survival of the plant. The leaf is also used for rock gardening or rockery (Binu
tissues having a gel known as aloe gel. The leaves Thomas et al., 2011).
of the plant appear to be sword type and have small Aloe barbadensis Mill. is used in alternative
harmless spines on the entire edge of the plant. The medicines and in home first aid
leaves of Aloe barbadensis Mill. is made up of four A brief account of Aloe barbadensis Mill.
layers such as Rind- the outer part of protective reported to be used in folk medicine in different
layer, Sap- a layer of bitter fluid which helps to parts of India as well as mentioned in the literature.
protect the plant from animals, Mucilage Gel- the
inner part of the leaf that is filleted out to make DISCUSSION AND CONCLUSION
Aloe gel and Inner gel- It contains 8 essential Aloe barbadensis Mill. usually found in
Amino acids (Shen et al., 2001). The root and the terrestrial habitat but the studies on the diversity
leaves both are fibrous as it holds enough water in it and distribution of chasmophytic plants from
and the leaves have dots on the entire surface. The coimbatore district of Tamil Nadu shows that the
plant bears fruit which are triangular in shape and plant like Aloe barbadensis Mill. also lives in rock
has many seeds in it crevices (Binu Thomas et al., 2009). The present
Importance of Aloe barbadensis Mill. in various study on the habitat variability of Aloe barbadensis
aspects Mill. reveals that, it also found on tree trunks. The
Using as Indoor Plant epiphytic nature of the highly medicinal plant like
Aloe barbadensis Mill needs direct sunlight for Aloe barbadensis Mill. was proved through the
growth. Some people keep it as indoor plant but the present observation. This plant also shows a
plant has to be kept in sunlight every alternate day xerophytic nature. The tissues of the plant body
(Kapoor and Sharga, 1993). stores large amount of water, thereby it can
Used for rock gardening withstand at any environmental conditions.
The xerophytic species like Aloe barbadensis Mill.

Journal of Research in Biology (2011) 3: 237-241 239
Parthipan et al.,2011
No. The Various Medicinal Uses of Aloe barbadensis Mill. Referrals
1 It is helpful in curing any type of burns, skin wounds and scalds. Aloe Grindlay and Reynolds,
barbadensis Mill. lotion suggests effectiveness for treating seborrheic 1986; Janardhanan, 1963.
dermatitis when applied to the skin
2 It is also very useful in the treatment of vaginal infections, blisters, sties, Voglar and Ernst, 1999.
herpes, insect bites, allergic reactions, urticaria, rashes, athlete’s foot,
conjunctivitis, dry skin, fungus and sores
3 It is beneficial in recovering fast after the surgery Shekhawat and Anand, 1984.
4 Aloe barbadensis Mill. is also used in treating frostbite, psoriasis, warts, Okyar et al., 2001.
eczema, shingles, acne, preventing scarring and wrinkles from aging,
sunburns, rosacea and screening out X-ray radiation
5 The leaf juice has the property to enhance the immune system in our body Hu et al., 2003.
6 It also assists cancer patients by stimulating the development of non- Paulsen, et al., 2005.
cancerous cells and white blood cells
7 Aloe Gel may treat recurrent ulcers, reduce pain and increase the amount of Habeeb et al., 2007.
time between the appearances of new ulcers
8 It may aid healing of mild to moderate skin burns and ulcers. The Aloe Gel Bhalla et al,1982.
has a dramatic ability to heal wounds, ulcers and burns by putting a
protective coating on the affected areas and speeding up the healing rate
9 Dried latex from the inner lining of Aloe leaves has been used traditionally as Janardhanan, 1963.
a laxativ, wash hair
10 The extracts from Aloe barbadensis Mill. in a hydrophilic cream may be an Rai, 1985.
effective treatment of genital herpes in men
11 The leaf pulp of the plant has been used to cure boils,spleen disorders, as Bhalla et al, 1982; Sebastian
vermicidal breast tissue hardening; constipation, rheumatism, jaundice, fever, and Bhandhari, 1989; Rai,
piles, gonorrhea, liver and menstrual complaints; sexual utility. 1985; Bhatt and Sabnis,
12 The leaf mucilage either alone or with honey is given orally twice in a day to Khanna and Ramesh kumar,
cure liver disorders. It is also given orally for sunstroke and for improving 2000.

The Critical screening of literature reveals References
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terrestrial habitat with various ecological Diversity and distribution of herbaceous vascular
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Frame G. 2003. Generalist flowers, Biodiversity Paulsen E, Korsholm L and Brandrup F. 2005. A
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Ltd., London. (Repr. ed. vols. 1-3.1957). of child wara District ( M . P. ) J .E co n.T ax . Bot . ,
Grindly D and Reynolds T. 1986. The Aloe
barbadensis Mill.Phenomenon: A review of the Rao RR. 1994. Biodiversity in India (Floristic
properties and modern uses of the leaf parenchyma aspects) Bishen Singh Mahendra Pal Singh, Dehra
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Journal of Research in Biology (2011) 3: 237-241 241
Journal of Research in Biology
An International Online Open Access
Original Research paper
Publication group

Studies on Methanotrophs from Lonar Lake
Journal of Research in Biology

Authors: ABSTRACT:
Tambekar DH, Patil RV
and Pawar AL.
Methanotrophic bacteria were isolated and characterized from sediment
from alkaline Lonar Lake. Four bacterial strains were isolated using minimal salt media
to study the methanotrophs of Lonar Lake and selected bacterial strains were further
Institution: characterized, screened on the basis of the temperature, and salt tolerance. Bacterial
Post Graduate Department of isolates were subjected to morphological, biochemical characterization and 16S rRNA
Microbiology, sequencing. Isolates were related to the phylum proteobacteria contains different
S.G.B. Amravati University, genera such as Acinetobacter baumannii, Pseudomonas aeruginosa, Achromobactor
Amravati 444602 (India). xylosoxidans and Ochromobactrum tritici. These results clearly showed that the Lonar
lake ecosystem harbors unexplored methanotrophs which can be used to control
global warming as well methanol remediation.

Corresponding author:
Tambekar DH

Email: Lonar Lake, methanotrophs, Acinetobacter, Pseudomonas, Achromobactor and Ochromobactrum.

Article Citation:
Web Address:
Tambekar DH, Patil RV and Pawar AL.
Documents/RA0064.pdf. Studies On Methanotrophs from Lonar Lake
Journal of research in Biology (2011) 3: 230-236.

Received: 19 Jul 2011 /Accepted: 25 Jul 2011 /Published: 29 Jul 2011

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Tambekar et al.,2011

INTRODUCTION Oxidation of methane to methanol by
The alkaline Lonar Lake, in Central India, methanotrophic bacteria is mediated by the
situated in the village at Lonar, Buldhana district, particulate membrane bound form (pMMO) or the
Maharashtra ranks third in the world based on soluble cytoplasmic form (sMMO) of methane
diameter and its high (pH 10.5) alkalinity (Taiwade, monooxygenase. The conversion of methanol to
1995). It is a closed system without outlets and formaldehyde is catalyzed in extant methanotrophs/
regular influents are responsible for its existence. methylotrophs by the methanol dehydrogenase
Based on the geological studies, it is postulated that (MDH) enzyme. Methanol plays an important role
the Lake originated as a meteorite impact crater in global warming, and its atmospheric
around 50-53 thousand years ago (Jhingran and concentration has been increasing over many
Rao, 1954; Nandy and Deo, 1961). decades and very much difficult to degrade
The diameter around the Lake is about 1.75 (Whittenbury et al, 1970). The present study
Km and water enters the Lake through rain, ground planned to isolate methanotrophic bacteria present
water seepage and the springs situated in the cliffs in Lonar lakes which can degrade the industrial
at the edge of the Lake. It does not receive any pollutant methanol and utilize methane by which
industrial discharges. Alkalinity of the Lake is the global warming can be reduced.
attributed to the high content of sodium carbonate
and hence was used previously as a source of MATERIALS AND METHODS:
washing soda (Thakker and Ranade, 2002,). The Four sediments samples were collected from
water in the crater is very salty. It is 10 times saltier selected sites of Lonar Lake with the help of
than drinking water. Salts and Minerals like scooper in sterile polythene bag. They were labeled
sodium, chloride, carbonates, fluorides and and transported to the laboratory and stored at 40C
bicarbonates (TDS around are found and as this until further analysis.
water do not drain away these substances get Medium composition: The one liter of medium
collected beneath the surface (Nathani, 2010, containing NaNO3 2.5 g, KCL 0.1g, KH2PO4 3g,
Shoemaker, 1963; Bhandari, 1984). In such K2HPO4 0.01g, MgSO4 0.5g, FeSO4 0.116g,
conditions one cannot think of any living H3BO3 0.232g, CuSO4 0.41g, MnSO4 0.008g, (NH4)
organisms, but microorganisms like Arthrospora, 6 Mo7O24, 0.008g, and ZnSO4 0.174g, 20 ml
proteobacteria and algae are found abundant (Malu methanol and pH 10 was prepared for isolation of
et al, 2002). It revealed that Lake water is alkaline methanotrophs (Haddad et al, 2009).
(pH 10.3) and characterized by high concentration Isolation of bacterial strains: Sediment samples
of salts (9060 mg/l), chloride (3492 mg/l), salinity were inoculated in minimal salt media containing
(6391 mg/l), alkalinity (3751 mg/l), total hardness 2% methanol as carbon source. All flasks were
(480 mg/l), calcium hardness (118 mg/l), incubated at 370C in Rotary shaker (100rpm) for 3
magnesium hardness (361 mg/l), sulphate (21 mg/ days and repeated subculturing was made in the
l), phosphate (0.44 mg/l), nitrate (3.7 mg/l) and same medium. After repeated subculturing, the
dissolved oxygen (0.0034 mg/l). The Lonar Lake is bacterial growth was subcultured on same solid
unique in the world for its alkalinity and salinity of minimal salt medium for isolation of
the water but it was seen that chlorides and salinity methanotrophs. Well isolated and differentiated
of the Lake water is decreasing day by day colonies from these minimal media were transferred
(Tambekar et al, 2010). on Nutrient agar slant and maintained for further
Methanotrophic bacteria are a group of study.
organisms with the ability to use compounds with Morphological, biochemical identification of
no carbon-carbon bonds (C1 compounds) as single isolates: Bacterial strains were examined for colony
sources of carbon and energy, thus playing a role in and cell morphology, motility, Gram staining and
global carbon cycling. A wide range of C1 standard biochemical test (catalase, oxidase, IMViC
compounds are consumed by methanotrophs in the and fermentation of sugar such as lactose, dextrose,
environment, including methane, methanol, mannitol, xylose and arabinose, nitrate reduction,
methylated amines, methylated glycines, urease activity, methyl, hydrolysis of starch etc).
halomethanes, and methylated sulfur species. Phylogenetic analysis: 16S rRNA sequencing was
Methanotrophs are a unique group of performed at NCCS, Pune. Nearly-full-length 16S
Methylotrophic bacteria, which utilize methane as rRNA gene sequences were submitted to CHECK-
sole carbon and energy source (Olivier et al, 2005). CHIMERA, available on the Ribosomal Database
231 Journal of Research in Biology (2011) 3: 230-236
Tambekar et al.,2011

Project release 10.26, in order to identify chimeras. methanotrophs. A preliminary study was carried
Phylogenetic analyses were performed using the out to isolate the bacterial strains from sediment
ARB software package. The 16S rRNA gene using Minimal salt broth. Data of four sediment
phylogenetic analyses were performed by the sample as per the characterization are conformed up
maximum-likelihood method, using 1,285 to 1,392 to generic the level.
nucleotide positions. The functional genes were Four different isolated strains were
translated into amino acid sequences, and these identified by using standard procedures. The
were included in phylogenetic analyses using the experimental outcome of morphological and
neighbor-joining method (Dayhoff PAM model). biochemical characterization proved that all four
stains are Gram negative and identified as
RESULTS AND DISCUSSION: Acinetobacter, Pseudomonas, Achromobactor,
Methanotrophic bacteria are a group of Ochromobactrum which prove to survive in
organisms with the ability to use compounds with extremophilic condition of high salt concentration
no carbon-carbon bonds (C1 compounds) as single and high pH (10.5). Present study reported that 4
sources of carbon and energy, thus playing a role in methanotrophic gram negative, bacterial isolates
global carbon cycling. Traditional microbial from the sediments of the Lonar Lake. Among
techniques such as enrichment and isolation on four isolates Acinetobacter baumannii was oxidase
defined culture media have revealed that negative and other three Pseudomonas aeruginosa,
methanotrophic bacteria occur in a variety of Achromobacter xylosoxidans and Ochromobactrum
environments, such as freshwater, marine, and tritici were positive. Catalase, indol, methyl red,
terrestrial habitats, including habitats characterized voges-proskauer were negative and citrate
by extreme conditions of temperature, salinity, or utilization was positive in all isolates. On the basis
pH. Active members of the bacterial community in of sugar fermentation, Acinetobacter baumannii
the sediment of Lonar Lake with special emphasis fermented dextrose the other isolates can not
on C1 utilizers (Methanotrophs) were identified by ferment the sugars; nitrate reductase test was
employing two complementary culture-dependent positive for Achromobacter xylosoxidans and
and independent approaches: morphological and negative for all other. Urease production was absent
standard biochemical identification and 16S rRNA for all isolates. Among four bacterial strains
analysis probing C1 substrates methanol. The Acinetobacter baumannii showed clear zone of
objectives of this study were to isolate, identify starch hydrolysis indicate amylase production. The
methanol utilizing bacterial from Lonar Lake and present studies on optimization of various growth
study further to evaluate their potential as parameters such as temperature, salt concentration

Table 1: Morphological, cultural, biochemical characteristics and 16S rRNA identification of bacteria isolated
from sediments of Lonar Lake

Journal of Research in Biology (2011) 3: 230-236 232
Tambekar et al.,2011

and the strains showed better growth at 42°C and aerugi nosa ( Lonar –B) , Achromobact er
6.5% salt concentration (Table 1). All the isolates xylosoxidans (Lonar–C) and Ochromobactrum
were analyzed by 16S rRNA analysis and found, tritici (Lonar–D) (Table 2). A related work was
Acinetobacter baumannii (Lonar–A), Pseudomonas carried out by Jurjen Heyer (2005). It was evidently
Table 2a: 16S rRNA analysis (Blast analysis), Dendrogram showing phylogenic relationship of methanotrophs isolated
from Lonar Lake
Lonar – A:

Acinetobacter baumannii Lonar-A
Acinetobacter baumannii EU760625
Acinetobacter baumannii EU760626
Acinetobacter baumannii EU760627
Acinetobacter baumannii EU760628
Acinetobacter sp. GQ178052
Acinetobacter sp. GQ178054
Acinetobacter baumannii FN563422
Acinetobacter baumannii HQ180180
Acinetobacter baumannii FJ860866

Lonar B:

clone P5D18-571
Pseudomonas sp. DG2b
Uncultured bacterium clone P5D18-561
Uncultured bacterium clone P5D18-526
Uncultured bacterium clone P5D15-458
Uncultured bacterium clone P5D4-567
Uncultured bacterium clone P2D11-424
Uncultured bacterium clone P2D11-425
Uncultured bacterium clone P2D11
Pseudomonas aeruginosa DQ989018
Pseudomonas aeruginosa Lonar-B

233 Journal of Research in Biology (2011) 3: 230-236
Tambekar et al.,2011

Table 2b: 16S rRNA analysis (Blast analysis), Dendrogram showing phylogenic relationship of methanotrophs
isolated from Lonar Lake
Lonar – C

Achromobacter xylosoxidans(2)
Achromobacter xylosoxidans FM999735
Uncultured Achromobacter sp.(7)
Uncultured Achromobacter sp.(6)
Uncultured Achromobacter sp.(5)
Uncultured Achromobacter sp.(4)
Uncultured Achromobacter sp.(3)
Uncultured Achromobacter sp.(2)
Uncultured Achromobacter sp.
Achromobacter xylosoxidans
Achromobacter xylosoxidans Lonar- C

Lonar – D:

Uncultured Ochrobactrum sp. clone RBE...
Bacterium IPN9
Bacterium IPN2
Ochrobactrum tritici
Bacterium IPN20
Bacterium H2SBIOF
Ochrobactrum tritici Lonar-D
Ochrobactrum intermedium
Ochrobactrum intermedium AF526510

found that methanotrophic bacteria grow at 300C different seasons from saline and hyperalkaline
and 8.7% salt concentrations. Surakasi et al, (2010) Lonar Lake using 16S rRNA gene library analysis
also reported the phylogenFetic diversity of and demonstrated the presence of Arthrospira
bacterial communities in microbial mats of two (Cyanobacteria), Fusibacter (LAI-1 and LAI-59)

Journal of Research in Biology (2011) 3: 230-236 234
Tambekar et al.,2011

and Tindallia magadiensis (LAI-27) in post- Heyer J, Berger U, Hardt M and Dunfield PF.
monsoon and Planococcus rifietensis (LAII-67), 2005. Methylohalobius crimeensis gen. nov., sp.
Bordetella hinzii (LAII-2) and Methylobacterium nov., a moderately halophilic, methanotrophic
variabile (LAII-25) in pre-monsoon. They claimed bacterium isolated from hypersaline lakes of
the first time detection of these putative Crimea. Int J syst Evol Microbial., 1817-1826.
methanotrophs in surface mats of Lonar Lake
Methanol was primarily assimilated by Jhingran AG and Rao KV. 1954. Lonar Lake and
Acinetobacter baumannii, Pseudomonas its salinity. Geol Surv India 85:313-334.
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Ochromobactrum tritici species from the family 2002. Diversity of Lonar Lake. J Aquat Bio., 15:16-
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