Professional Documents
Culture Documents
Original Research
Squalidae Squalus cubensis 86 F+M 38.5-079.3 0255-2750 0.008 0.0021 - 0.0276 2.912 2.5912 - 3.2334 0.162 0.89 I
Squatinidae Squatina dumeril 67 F 29.2-110.4 0177-10744 0.012 0.0029 - 0.0395 2.954 2.6645 - 3.2436 0.145 0.93 I
23 Mi 62.0-086.4 1389-5188 0.001 0.0002 - 0.0047 3.435 3.1134 - 3.7569 0.155 0.98 A (+)
190 Mm 79.9-098.0 4423-8902 2.574 0.8432 - 7.8550 1.706 1.4566 - 1.9551 0.127 0.7 A (-)
Triakidae Mustelus canis 75 F+M 67.0-126.0 0851-1397 0.0003 0.0000 - 0.0011 3.589 3.2703 - 3.9072 0.160 0.93 A (+)
Mustelus higmani 547 F 28.0-088.4 0057-1673 0.006 0.0015 - 0.0043 3.085 2.9540 - 3.2160 0.067 0.89 I
125 Mi 34.7-053.3 0113-0425 0.001 0.0002 - 0.0038 3.302 2.9603 - 3.6434 0.174 0.86 A (+)
293 Mm 39.8-058.3 0227-0624 0.18 0.0736 - 0.4392 1.997 1.7778 - 2.2167 0.112 0.72 A (-)
Mustelus norrisi 730 F 37.2-072.2 0172-1191 0.003 0.0016 - 0.0046 3.082 2.9509 - 3.2136 0.067 0.86 I
477 M 37.3-059.3 0170-0709 0.009 0.0042 - 0.0181 2.765 2.5780 - 2.9521 0.095 0.8 A (-)
n=sample size; Lmin-max=minimal and maximal length; Wmin-max=minimal and maximal weight; F=females; Fi=immature females; M=males; Mi=immature males; Mm=mature males; e=embryos;
a=intercept; CL=confidence limits; b=slope; SE=Standard error; R2=coefficient of correlation; I=isometric; A(+)=Allometric positive; A(-)=Allometric negative; *= Disc width (DW) was the length
used (all the rest Total Length).
1460
Tagliafico et al., 2014
and that the organisms analyzed in this work ranged (n = 21) for LWR. In this work, statistic differences
between 34.6 and 106.4 cm of TL, and between 113 and between sexes are reported (ANOVA, F (1,964) = 7.88,
5499 g of weight, which includes organisms bigger and P<0.005), even between mature and immature males
heavier than those analyzed by Gmez and Bashirulah (ANOVA, F (1,417) = 21.43, P<0.001), perhaps due to a
(1984). biggest sample size (n = 965). Additionally, a previous
For Mustelus higmani, Etchevers (1975) did not study on C. limbatus report a b-value of 3.028 for both
find differences between males (n = 13) and females sexes (Tavares, 2009), however in this work differences
1461 Journal of Research in Biology (2014) 4(7): 1458-1464
Tagliafico et al., 2014
between sexes were found (ANOVA, F (1,97) = 34.31, From around 32470 species of fishes contained
P<0.001) and as consequences two different LWR were in FishBase, LWR studies were only available for less
made; also, different patterns of growth were reported for than 12% (3587 species) (Froese et al., 2014), and if the
both sexes: females shows allometric positive growth same analysis is performed by country or region, the
(b=3.3), whereas males allometric negative growth numbers become even smaller; which is the case in
(b=2.4). Different LWR for the same species can be Caribbean waters, where information on the LWRs is
attributed to sampling different populations or changes in limited to a few species.
the environmental conditions over time (Froese, 2006).
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Original Research
Studies on the reproductive biology and seed biology of Aconitum nagarum Stapf:
a threatened medicinal plant of North East India
Authors: ABSTRACT:
Journal of Research in Biology
maintained in the polybag till they were brought in to the Seed biology
laboratory. The anther lobe was removed from the style The plant and mature fruits were collected from
with the help of forceps and blade and the anther lobe the forest of Dzukou valley, Khonoma, Nagaland, India
was put on a slide. The anther was smashed uniformly by at an elevation of 2648 m ASL from the grassy bamboo
adding a drop of glycerine and spread evenly. The slide slope. Aconitum nagarum reached peak flowering from
was covered with the cover slip. The slide was kept the first week of October and seed setting starts from the
under the microscope and pollen present in 10 second week of October. Therefore harvesting of seeds
microscopic fields were counted. Total number of pollen can be done from the third week of October. On
was determined by multiplying the pollen present in the contrary, in Shirui hills, Manipur, at an elevation of
mean microscopic field and total number of microscopic 2427m asl, peak flowering starts from the first week of
field per slide. november and seed setting was from the second week of
Distribution pattern of the plant and its associated november. During the second week of november few
species flowers were seen but are very rare. Matured seeds could
The distribution of Aconitum nagarum in North- be collected from the third week of november.
Eastern parts of India (Nagaland and Manipur) was taken Seed collection and processing
into account for the comparative study of plant For any study of this nature may be affected by
distribution pattern. A study was conducted to various factors like seed collection technique, processing
understand the rule of associated species on the growth, of seeds and post harvest method etc. In the present
reproduction and survival of A. nagarum. study, seed collection and processing protocol developed
Figure 1. a. Aconitum nagarum plant growing in the hilly slope; b. Tuber of A. nagarum; c. Floral bud of
A. nagarum; d. A. nagarum flower at full bloom; e. Immature fruits; f. Mature dry fruits; g. Germinated
seed showing the radical and h. Rooted seedlings formed from the germinated seeds in the poly bag.
by Deb et al., (2012) was followed with suitable study. A part of the seeds were processed and sowed
modifications as per laboratory condition and immediately after the harvest while others were treated
experiment. In the present study, mature seeds were differentially at 4C in a refrigerator for 0, 24, 48, 72, 96
harvested randomly from the natural habitat during hours and sowed as described below:
2011-2013 along with the plant stalk. The stalks were 1. A set of stratified seeds were sowed in filter paper in a
wrapped in newspapers and covered with polythene bag humidity chamber of 90 mm in diameter and kept in a
and transported to the laboratory within 1-2 days. The laboratory (25C).
collected fruits were dried by spreading uniformly over 2. Another set of processed seeds (stratified seeds) were
o
the old newspaper for 1-2 days in the laboratory at 25 C. sowed in the potting mix and kept in an incubator at the
The dried fruits were removed from the stalk and seeds constant temperature of 30C.
are taken out of the carpel. The seeds were then stored 3. While another set of stratified seeds were sowed in
in poly bag in the laboratory for further experiments. seed bed (poly bag) and maintained in a poly house.
The processed seeds were washed with 4. To test the post harvest tolerance of the seed for
Labolene (1:100, v/v) (a commercial laboratory various periods, the processed seeds were stored at 25C
detergent) and rinsed under running tap water and finally (in the laboratory) in sealed poly bags before they were
with distilled water. The seeds were made into different sowed in the seed bed for seed tolerance experiment.
groups for germination experiment. 5. The seedling morphology and seedling mortality rate
Preparation of potting mix was also studied.
The potting mix for the experimental purpose To study the emergence, survival and growth of
was made following Deb et al., (2012) by mixing soil seedlings of Aconitum nagarum under each condition,
and chopped coconut coir at 1:1 ratio. The garden soil 4 replicates of 13 seeds each (for filter paper test, N=52
was crushed into fine powder, sun dried and mixed with seeds/test) and 20 seeds each (for seed bed germination,
the coconut coir in the ratio of 1:1 and put in a plastic pot N=80 seeds/test) were used. In each poly bag, the soil
and transparent poly bags. The poly bag and plastic pot mixture was packed and 20 seeds were sowed. In filter
were made perforated for better aeration. They were kept paper test 13 seeds were sowed. The seed beds were
moist before sowing the seeds for germination. watered at regular intervals. The experimental design
Experimental process was completely randomized. The data were collected
The protocol developed by Deb et al., (2012) daily based on seed germination, seedling morphology;
was followed with suitable modification in the present seedling mortality; percent response etc. For the study
Table 1. Distribution pattern of Aconitum nagarum at different location of Nagaland and Manipur
Site GPS Coordination Altitude Distribution Locality
(mASL)
Nagaland
Southern Dzukou valley N25 34 30.4, E 94 02 43.3 2400 Common Valley and hill slope
Western Dzukou valley N 25 36 44.8, E 094 00 03.4 2648 Common Valley and hill slope
Mount Saramati N 26 2 26.7, E97 6 97 13 2000-3841 Common Valley and hill slope
Japfu Hills N 25 35 86.3, E 094 04 047 3020 Common Hill slope
Manipur
Shirui Hils N 25 06 39.6, E 094 27 13.3 2427 Less common Hill slope and top of
hills
Dzukou valley N 25 34.40.5, E 094 04 48.9 2550 Common Valley and hill slope
the seedlings were maintained in the respective polybags seeds/treatment). The differently stratified seeds were
and watered at regular interval and allowed to grow un sowed in the potting mix and incubated at 30C in the
till becoming normal plantlets. Once the seedling showed incubator. The seeds were monitored at regular intervals
normal functioning like rooted plantlets, emergence of for seed germination, seedling morphology and seedling
normal leaves etc., the seedlings were transferred to the mortality etc.
poly house. Once the seedlings were established in the Germination test in seed beds (poly bags)
poly house, the seedlings exhibited differential growth The differentially stratified seeds were placed on
and many seedlings died. potting mix in a perforated polybag of 150 mm in
Filter paper test at room temperature (25C) diameter. Each treatment consists of four replicates with
The seeds were treated at 4C in a refrigerator for 20 seeds (N=80 seeds/treatment). The plants are
different periods (0, 24, 48, 72, 96 h). The seeds were monitored regularly for germination. Germination
then placed on moist filter paper in a humidity chamber percentages were calculated after eight weeks of seed
of 90 mm diameter and kept for germination at the culture.
laboratory (25C). The seeds were kept moist throughout Post harvest storage tolerance test for
the study period. The germination process gets Aconitum nagarum
completed by the emergence of radical followed by leaf The seeds are stored at a temperature of 25C.
with seedling formation. A total of 13 seeds were used The seeds were tested for the post harvest storage
for each treatment with 4 replicates (N=52 seeds/ tolerance. Every month sets of processed seed were
treatment). The seedlings were transferred to poly house sowed in the seed bed. Their germination rate for each
for further seedling growth studies. The experimental experiment was carried out and the results obtained were
design followed was based on Deb et al., (2012). recorded monthly. The result obtained was compared and
Germination test in incubator checked to see how far Aconitum nagarum seeds can
The stratified seeds (stratified at 4C for 0, 24, tolerate storage. All the above experiments were based
48, 72 and 96 h) were placed on potting mix to test on the works reported by Deb et al., (2012) with
the role of stratification on germination. Each treatment Cinnamomum tamala.
consists of four replicates with 20 seeds each (N=80
Seedling mortality and seedling morphology In the present study, it was observed that the
The seedling mortality was observed by plants of Aconitum nagarum growing at Dzukou valley
transplanting the seedling grown from the differently started budding from September second week onwards
treated seeds in the poly house and percent seedling (Figure 1c) with peak flowering in October first week
survival was recorded till the plant mature or till its (Figure 1d). The flowers are blue in colour, in slender
survival period which ever is earlier. raceme, petals and filaments glabrous, carpel 5, and
The transplanted seedlings in the poly house bisexual. The flowers bloom acropetally i.e. flower starts
were also checked for changes/modifications in the blooming from the base of the inflorescence to the tip of
seedling, which was observed after the transplantation of the inflorescence. Thus the fruits also mature acropetally.
the germinated seeds (like modification in the leaf, roots The anthesis was observed between 6.00 - 6.30 AM.
etc). Anther dehisced longitudinally from 7.00 AM till
9.30 AM. The number of anther was 49 per flower.
RESULTS AND DISCUSSION During the study on floral phenology, it was found that
Associated species, floral biology and morphology there is a strong correlation between anther development
Present study was conducted in Nagaland and and the stigma development (Table 2). The flower colour
Manipur at the altitude between 2000 m to 3841 m above changes as the plant fully dehisced. The flowering
mean sea level (Table 1) in six different geographical duration per flower varies from 4-6 days followed by
areas. In all the study areas the populations were found in fruit formation. Fruits mature within 10-15 days. Fruit
the hill slopes of the valley. In the present study, an formation starts from the second week of October
interesting feature was observed as common associated (Figure 1e) and by the third week of October, fruits
species growing in all the study areas; they are, mature and the plant dries up (Figure 1f). While, in
Sinarundinella species, Gaultheria species and Fragaria Shirui hills of Manipur at an elevation of 2427 m ASL
species. In all the areas A. nagarum was growing healthy 25 06' 39. 6" N and 094 27' 13.3" E peak flowering of
where these associated species were available. This Aconitum nagarum was observed by the first week of
information on associated species could be used for the November and by the second week flowering decreases
identification of new niches of this species for with the formation of fruits. By the third week, fruits are
rehabilitation of the species. mature and the plants were all dried up. In local dialect
of the Shirui village, it is known as the summer blue. The
Table 3. Difference in the floral phenology of Aconitum nagarum at Dzukou valley, Nagaland and Shirui hills, Manipur
number of flowers per plant varies from 8-28. The most morphology appears to be species specific. Seedling
common pollinator was found to be honey bee. Seeds per survival on the seed beds/forest floor is governed by the
carpel varies from 9-13 i.e., mean seeds per flower was availability of light, water and nutrients (Kitajima, 2007).
45-65 and per plant was 270-540 (Table 3). Pollen per The requirement of plant species differ greatly with
anther varies from 1000-2000 which means an average reference to their habit preference, temperature
of 98000 pollen grains per flower. In the present study it requirement, and post harvest storage, specific
was found that all the flowers to fruits ratio was not 1:1, pre-treatment for seed germination, seedling emergence
it was 3:2 i.e., one third of the flowers did not support and survival. Many plant species exhibit differential
fruit formation. An average of 21.35 flowers developed correlation with reference to vegetation cover and light
per inflorescence while of the 21.35 flowers 13.65 requirements, temperature etc. (Kwit and Platt, 2003,
flowers ended with fruit formation and remaining Pages et al., 2003). Storage containers have also great
flowers did not form any fruits. influence on the germination of seeds (Verma et al.,
Seed biology 2009). Seed treatment with chemical and low
In the present study it was found that emergence temperature enhances seed germination (Pandey et al.,
of radicals from the germinated seeds, percent 2000).
germination, morphology of seedling and seedling In the present study different techniques were
establishment are influenced by various factors. The light adapted for seed germination. In the filter paper test,
requirement of seeds for germination and seedling maximum germination was achieved from the seed
Table 4. Effect of stratification on the seed germination of Aconitum nagarum on filter
paper (in a humidity chamber of 90 mm diameter)
Stratification period (h) at 4C % response (SE)* Types of plant response
0 67.00 (0.20) Healthy roots
24 15.00 (0.25) Healthy roots
48 38.00 (0.30) Root healthy, hairy at the zone of maturation
72 15.40 (0.20) Root healthy, hairy at the zone of maturation
96 15.00 (0.25) Elongated healthy roots
* SE: Standard error from mean; Data represents the mean of three replicates.
Initiation of the roots was considered as breaking of dormancy.
Table 5. Effect of stratification on the seed germination of Aconitum nagarum in seed bed (Polybag)
Treatment type Avg. time taken to % response (SE)* Types of response
germinate (days)
Without stratification 29 06.7 (0.2) Healthy rooted seedlings
Stratified for 24h at 4C before 29 10.0 (0.2) Healthy rooted seedlings with
sowing cotyledonary leaf
Stratified for 48h at 4C before 28 03.3 (0.3) Healthy rooted seedlings
sowing
Stratified for 72h at 4C before 25 03.3 (0.1) Healthy rooted seedlings
sowing
Stratified for 96h at 4C before 23 20.0 (0.2) Healthy rooted seedlings with
sowing cotyledonary leaf
* SE: Standard error from mean; Data represents the mean of three replicates.
Emergence of the root was considered as breaking of dormancy.
Table 6. Effect of post harvest storage (at 25C) on seed germination and viability of
Aconitum nagarum on seed bed
Storage duration at Time for first sign of % germination Types of response
25C (months) germination (days) (SE)*
0 10-30 6.7 (0.2) Healthy rooted seedlings
1 30-60 6.5 (0.7) Healthy rooted seedlings
2 60-90 6.0 (0.6) Healthy seedling with stunted growth
3 90-110 5.5 (0.5) Delayed germination
4 110-130 4.5 (0.6) Delayed germination
5 130-160 2.2 (0.3) Delayed germination with stunted
growth
6 0 0 No germination
* Standard error from mean.
Data represent the mean of three replicate without any stratification.
stratified for 48 h at 4C followed by 96 h when recalcitrant seeds appear to be species specific (Fu et al.,
maintained in the laboratory at 25C with minimum days 1990, Oliveira and Valio, 1992, Barbedo and Cicero,
taken to germinate. Under this condition 38% and 2000, Tommasi et al., 2006, Sharma and Gaur, 2012).
15.38% seed germination recorded after 13 days and 10 Tommasi et al., described that Ginkgo biloba seeds could
days of sowing respectively (Table 4 and Figure 1g). be stored at 4C for one year but when stored at 25C,
Seeds without stratification exhibited very poor seeds died after six months (Tommasi et al., 2006).
germination (6.7%), on comparison to filter paper test During the present study a similar response was recorded
then stratified seeds sowed on seed beds (prepared in where seeds stratified at 4C germinated better over
poly bags supported better germination). seeds stored at 25C.
Seeds sowed in the poly bags exhibited 20% Post harvest storage tolerance of seeds of
germination within 23 days from the seeds stratified for Aconitum nagarum
96 h at 4C (Table 5). But there was no seed germination Though, the seed preservation practices are as
recorded from the seeds maintained in the incubator at old as agricultural civilization but organized and
30C across the stratification period. Seed germination systemic storage facilities have been developed only in
was achieved within 29-30 days with emergence of roots the 20th century. There are over 1500 seed or gene banks
with cotyledonary leaves while true leaves are formed present world over and accessions are increasing
within 58-60 days (Figure 1h). In the present study it regularly. Longevity of seeds is not universal and is of
was found that seed germination rate and germination species specific. After harvest the viability of seeds
time were greatly influenced by pre-treatment of seeds. decline with time, seeds may exhibit differential
There was significant difference in the germination germination and seedling morphology and field
period and germination rate with the stratified and non- establishment (Walters, 2004). So, for some plant
stratified seeds. During the present study, investigation species, using relatively fresh seeds give superior
was carried out on the relationships among rate of cold germination over stored seeds.
stratification to determine whether the seeds require pre During the present study, efforts were put to
treatment of low temperature for germination. The examine the post harvest storage tolerance of the seeds.
finding in the present studies thus support that seeds of The seeds were stored at 25C for 0-6 months and seeds
Aconitum nagarum prefer low temperature for seed were sowed in the seed beds at one month interval (Table
germination. The lowest temperature tolerance by the 6). Data collected in the present study exhibited gradual
Kwit C and Platt WJ. 2003. Disturbance history Singh KP, Bhavana KP and Dhakre 2010.
influences regeneration of non-pioneer understory trees. Reproductive biology of Evolvulus alsinoides L.
Ecology, 84(10): 2575-2581. (Medicinal herb). Intl. J. Bot., 6(3): 304-309.
Mo EF. 2014. India's Fifth National Report to the Srivastava N, Sharma V and Kamal B. 2010.
Convention on biological diversity. Ministry of Advancement in research on Aconitum species
Environment and Forests, Government of India, p.142. (Ranunculaceae) under different area: A Review.
Biotechnology. 9(4): 411-427.
Moza KM and Bhatnagar AK. 2007. Plant
reproductive biology studies crucial for conservation. Tommasi F, Paciolla C, De Pinto MC and De Gara L.
Curr. Sci., 92(9): 1207. 2006. Effects of storage temperature on viability,
germination and antioxidant metabolism in
Oliveira LMQ and Valio IFM. 1992. Effects
Ginkgo biloba L. seeds. Pl. Physiol. Biochem., 44(5-6):
of moisture content on germination of seeds of
359-368.
Hancornia speciosa Gom. (Apocynaceae). Annals Bot.,
69(1): 1-5. Verma NK, Verma AK and Kumar D. 2009. Effect of
storage container on seed germination and viability in
Pages JP, Pache D, Joud N, Magnan N and
Aconitum heterophyllum and Podophyllum hexandrum -
Michalet R. 2003. Direct and indirect effects of shade on
endangered medicinal plants species of Himalayan
four forest tree seedlings in the French Alps. Ecology, 84
region. Biological Forum -An Intl. J., 1(1): 8-11.
(10): 2741-2750.
Walters C. 2004. Appendix 2 in Ex situ conservation,
Pandey H, Nandi SK, Nadeem M and Palni LMS.
supporting species survival in the wild. Guerrant, Havens
2000. Chemical stimulation of seed germination in
and Maunder, Island Press.
Aconitum heterophyllum wall and A. balfourii Stapf.:
important Himalayan species of medicinal value. Seed Zhang F, Peng SL, Luo F and Ding LS. 2005. Two
Sci. Technol., 28(1): 39-48. New Norditerpenoid Alkaloids of Aconitum nagarum.
Chinese Chem. Let., 16(8): 1043-1046.
Sharma E and Gaur AK. 2012. Aconitum balfourii
Stapf: A rare medicinal herb from Himalayan Alpine. J.
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Original Research
Sanindhar Shreedhar
Gaikwad1* and Nitin Water current plays vital role in the development of an aquatic ecosystem. It
Anandrao Kamble2 performs various activities in the aquatic media, which in turns replenish the nutrients
and alter biotic conditions of the water bodies. In order to elucidate the exact role of
water current on life of aquatic fauna, present investigation was carried out. Members
of phylum mollusca are world wide distributed and include the commercially
Institution: important group of organisms. These creatures are continuously exposed to waters
1. Research Scholar, rapidly altering conditions and have the ability to withstand with this challenging
Department of Zoology, atmosphere. So, for the present investigation, three freshwater uninoid molluscs
Shivaji University, Kolhapur Lamellidens marginalis, Lamellidens corrianus and Pyressia corrugata were selected.
- 416 004, (MS) India These molluscs were exposed to monitor or regulate aquatic conditions. Comparative
assessment among these molluscan species, showed the impact of water current
2. Assistant Professor, on their growth, physiology and biometric relationships. Uninoid mollusc
Department of Zoology, Lamellidens corrianus proved its dominancy at availed atmospheric conditions and
Shivaji University, Kolhapur
hence noted ideally suitable for commercial rearing.
- 416 004, (MS) India.
Figure 1. Temperature assessed during the study period. Figure 2. pH assessed during the study period.
20 to 24C and 7.7 to 8.4 respectively (Figure 1 and 2). Highest average growth rate was remarked for
Whereas highly significant variations were L. corrianus along with the significant variation in case
noticed for DO (P < 0.0232) and CO2 (P < 0.0048), of control and experimental group whereas L. marginalis
which varied between 0.7 to 2.1 mg/lit and 7 to 12 mg/lit and P. corrugata were noted with moderate non-
for control as well as experimental groups (Figure 3 significant level of growth rates (Figure 5).
and 4). Respiratory rate
Growth rate Oxygen consumption capacity i.e. respiratory
Growth of the individual is measured by their rate was noted with narrow range of fluctuation in case
length and weight relationship. In molluscs, after sexual of all the individuals. Highest respiratory activity was
maturity lengthwise increment or growths get restricted showed by L. corrianus while P. corrugata was observed
to 1 or 2 mm per year only. Hence, by keeping in view with least level of respiratory activity. L. marginalis was
the economic importance of mature individuals for remarked with moderate respiratory rate and showed
present investigation average weight gain by the significant variation (P < 0.0058) amongst compared
individuals during the experimentation was noted as control and experimental groups (Figure 6).
growth rate. The results obtained as mean growth rate for Biometric relationships
control and experimental groups of the individuals were Parameters representing biometric relationships
summarized in the Table 1 and 2. showed significant differences amongst compared
molluscan species.
Table 1. Mean growth achieved by the compared molluscan
species of the control group.
Figure 3. Dissolved Oxygen assessed Figure 4. Free CO2 assessed during the study period.
during the study period.
Body Condition Index (BCI) - Mean BCI richest BCI, whereas non-significant moderate BCI was
evaluated for control and experimental group during the noted for L. marginalis and P. corrugata (Figure 7).
study was tabulated in the Table 3 and 4 respectively. Shell Component Index (SCI) - Mean SCI
Maximum picks of average BCI was remarked estimated for both the groups were represented in the
for experimental group, whereas slightly altered BCI was Table 5 and 6.
represented by control groups. L. corrianus showed
Figure 5. Growth rate noted during the Figure 6. Oxygen consumption noted during the
investigation period. investigation period.
Figure 7. BCI achieved by comparing molluscan Figure 8. Shell Component Index achieved by
individuals during the investigation comparing molluscan species during the investigation.
justified by the less production of faecal matter and showed almost doubled growth increment than that of
debris in case of control group organisms, whereas control group individuals. Whereas in case of control
experimental individuals produces tremendous amount of group though they are provided with, satisfactory diet
faecal debris. Carbon dioxide revealed exactly similar due to less favourable biotic and abiotic conditions the
trend as that of oxygen, because excess organic activities growth of the individuals, get retarded as proved by
and physiological processes enhances CO2 concentration, Yoo et al., (1986) for P. fucata. These observations were
which was assured by the higher temperature and pH supported by the evaluated condition indices. Significant
concentration of the experimental groups as previously differences among control and experimental group high
put forth by Widdow (1973) for M. edulis species. light the role of water current along with species habitat
Above-mentioned abiotic and biotic conditions specificity at the laboratory conditions. BCI and MYI
had its impact over the biometric relationships of the reaches to its maximum limit in case of experimental
cultured molluscan species. Satisfactory growth of the individuals representing magnificent enhancement in the
individual is a multitude of favourable biotic and abiotic tissue weight i.e. growth, which may be because of onset
interactions. Experimental groups were accomplished of breeding season as mentioned by the Narasimham,
with such delightful interactions along with ample (1988) for the species A. rehombea. Control individuals
amount of dietary, which provided maximum showed moderate growth, which may be the impact or
opportunities for the growth of individuals. Hence, result of stressed body physiology. SCI was significantly
altered than other condition indices, denoting advantage of water current in maintenance of the aquatic
comparatively doubled Shell Component Index for animals. In vitro treatment enhances health and
control individuals, whereas experimental group showed reproductive capacity of animals. Obtained results
downward pattern of SCI. Overall assessment of these showed magnificent increment in the regeneration
condition indices confirm maximum channelization or capacity of animals. The technique enlightens advance
transformation of energy for the tissue or gonadal bioscience practices in animal culture with significant
development in case of experimental groups. applications.
A good condition value indicates accumulation
of nutrients reserve to accomplish successful ACKNOWLEDGEMENT
reproduction (Bligh and Dyar, 1984, Dare and Edwards, The authors expressed their gratitude to Science
1975 and Aldrich and Crowley, 1986). While comparing and Engineering Research Board, New Delhi for offering
among the individuals of analyzed species, L. corrianus the grants under Young Scientist Fast track Major
was found as most favourable individual to counteract Research Project. We also expressed our gratitude
with the availed atmospheric conditions at the time of towards Head Department of Zoology, Shivaji
rearing. It was noticed with richest growth rate and BCI University, Kolhapur for infrastructural support in the
representing its dominancy in experimental conditions. progress of work.
While in case of unfavourable atmospheric conditions its
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Original Research
Ravichandran Ramanibai*,
Sivalingam Govindan and
Thamotharan Balakrishnan
We spotted the presence of blue buttons washed ashore during the month of
Institution: December 2013 in Pulicat lagoon. These rare sited organisms were observed during
Unit of Aquatic Biodiversity, our regular faunal field visit along the Pulicat lagoon.
Department of Zoology,
University of Madras,
Guindy campus, Chennai,
Tamil Nadu -600 025, India.
Dates:
Web Address: Received: 21 Jul 2014 Accepted: 19 Aug 2014 Published: 07 Nov 2014
http://jresearchbiology.com/
documents/RA0465.pdf
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
3D which showed the presence of a disc in the middle of Garcia-Barrientos R, Ramos-Puebla A, Hernandez-
P. porpita; this helps the organism to float in the water Samano A, Minor-Perez H and Legarreta GI. 2009.
and is golden brown in colour measuring around 1.5 Jellyfish (Stomolophus meleagris) tentacles proteins and
inches width. The mouth present below the float is to their Proteolysis Endogenous World Academy of
engulf prey along with water and its ingredients. The Science, Engineering and Technology. 3(6): 618-620.
second part is known as hydroid colony which posses
Herring PJ. 1971. Stability of the blue pigment of
bright colour tentacles. With the help of tentacles and the
Velella and Porpita (Coelenterata: Siphonophora),
float it moves along and across the water body. One can
Comp. Biochem. Physiol. Part B: Comp. Biochem., 39
observe and admire blue buttons for their beautiful
(4): 10391043.
colour but better to avoid its contact which causes skin
irritation; above all it is one of the good suppliers of Pandya KM, Parikh KV, Dave CS and Mankodi PC.
bioactive compounds from the sea. 2013. Occurrence of Hydrozoans from the saurashtra
Coast of Gujarat, India. Res. J. Mar. Sci., 1(4):1-3.
CONCLUSION
Raskoff KA. Sommer FA. Hamner WM and Cross
The suspended hydrozoan contains nematocyst in
KM. 2003. Collection and culture techniques for
their tentacles that hideaway biochemical compounds.
gelatinous zooplankton. Biol. Bull., 204:6880.
We suggest that it is a good source to work in the field of
bioactive compounds active against human pathogens.
ACKNOWLEDGEMENT
We thank DST (SERB), New Delhi for their
funding support.
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uncommon Pelagic Jellyfish species in Maltese Advantages
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Complete Peer review
Fredrick SW and Ravichandran S. 2010. Anti Affordable Charges
Quick processing
microbial activity of the Cnidarian Blue Button Extensive indexing
Porpita porpita (Linneaus, 1758), Midle east Journal You retain your copyright
of Scientific Research 5 (5) : 355-358. submit@jresearchbiology.com
www.jresearchbiology.com/Submit.php
Original Research
Quincy Bart,
Jenna Indarsingh,
Hamraji Jugmohan and Nine insecticides were evaluated for their toxicity (LC50) and 50% lethal times
Ayub Khan* (LT50) against 3rd instar Spodoptera frugiperda larvae. Two groups of insecticides were
identified based on LC50 and LT50 values. Bright 30EC was the most toxic (LC50 =
0.0006 g/g) while Fastac 5EC was the least toxic (LC50 = 0.6046g/g) among all the
insecticides tested. Haemolymph protein changes from insecticide treated larvae were
Institution: also determined. The total haemolymph protein content in insecticide treated larvae
Department of Life Sciences was generally lower than the control. Additionally, the number of protein bands
University of the West present in electrophoresis gels of insecticide treated larvae was also lower than that
Indies, St. Augustine of untreated larvae. The implications of these results are discussed.
TRINIDAD, West Indies
Dates:
Web Address: Received: 18 Oct 2014 Accepted: 25 Oct 2014 Published: 12 Nov 2014
http://jresearchbiology.com/
documents/RA0486.pdf This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
INTRODUCTION mesh cloth. Food was supplied via a wax paper strip
The fall armyworm, Spodoptera frugiperda (F) (2cm x 15cm) coated with honey that was mounted to the
(Lepidoptera:Noctuidae) is a serious pest of corn, top of the cage allowing it to hang down. A large
sorghum and several other grasses in the Neotropics. bouquet of fresh corn leaves was placed in a glass vial
S. frugiperda is an avid flyer which can be found with a cotton wool plug around the rim of the vial to
between south-eastern United States to Argentina. A prevent moths from drowning. The bouquet was replaced
light coloured inverted Y marking is found on the front after the old one had wilted. Cages were checked daily
of its head and its raised, dark shiny spots that occur for dead moths and oviposition. Eggs were collected
dorsally on the body distinguishes it from other daily from the corn leaves and placed in test tubes for
armyworm species (Sparks, 1979). This pest can cause larval emergence. Neonate larvae were transferred to
significant reduction in crop yield and as much as 50% mesh covered plastic containers that had a fresh supply
losses in corn in Brazil have been documented (Cruz of corn leaves. On the third day after hatching, larvae
et al., 1999; Carvalho et al., 2010). Synthetic insecticides were placed individually in test tubes with the aid of a
are the most commonly used form of control for this pest small artists brush (No. 3/0). Neonate larvae were fed
with a wide variety being utilized (Tavares et al., 2010). with corn leaves until 3rd instar (approximately 10 days)
Associated with the widespread, frequent use of and then used in insecticide bioassays.
synthetic insecticides is the development resistance and Insecticide bioassay
S. frugiperda has been recorded as resistant to several Nine commercial insecticides with different
insecticide groups including organophosphates, active ingredients were obtained from the University of
carbamates and pyrethroids (Yu, 1991). the West Indies Field Station, Trinidad for use in
The effect of synthetic insecticides on the insecticide bioassays. These insecticides were:
haemolymph proteins of S. frugiperda has not been Abamectin (abamectin), Boxer 30EC (etofenprox),
previously studied apart from those involving Bright 25EC (carbosulfan), Fastac 5EC
Bacillus thuringiensis (Valdez-Lira et al., 2012). The (-cypermethrin), Flip 800DF (fipronil), Karate 5EC
purpose of this study was to determine the LC50 and LT50 (-cyhalothrin), Malathion 50 EC (malathion), Neem X
for nine synthetic insecticides against S. frugiperda and 0.4EC (azadirachtin) and Supertak 10EC
to determine insecticide-induced changes in haemolymph ( cypermethrin).
proteins in S. frugiperda with the aim to better A corn (Zea mays) leaf dip bioassay was used for
understand the physiological mechanisms for the each population of S. frugiperda. Each bioassay
insecticide induced protein changes. comprised five concentrations for each insecticide
(4%, 0.4%, 0.04%, 0.004%, and 0.0004%) and a control.
MATERIALS AND METHODS Young corn leaves were cut into 7 cm x 7 cm segments.
Insect culture Each segment was dipped into their respective
An initial stock of S. frugiperda larvae was insecticide concentration solution for 30s, held vertically
collected on corn (Zea mays) from the University of the to permit excess solution to drip off and then placed on
West Indies Field Station, Trinidad. Larvae were taken paper towel to air dry for 30 minutes. Each treated leaf
back to the laboratory and reared on corn leaves until segment was placed in a 9 cm petri dish with moistened
adult emergence. Adult moths were placed in an insect filter paper lining the bottom. S. frugiperda 3rd instar
sleeve cage (30 cm x 30 cm x 30 cm) covered with a fine larvae were starved for 5 h prior to being placed on
1492 Journal of Research in Biology (2014) 4(7): 1491-1497
Bart et al., 2014
leaves of each petri dish. Five replicates were maintained minutes, then gradually increased to 14,000 rpm for 2
for the treatment of each insecticide. The control minutes. Each sample (35l) was mixed separately with
comprised of leaves treated only with distilled water. 35l sample buffer (1000l of 50% glycerol, 800l of
Petri dish lids were covered with fine gauze to allow for running buffer and 200l of 0.1% bromophenol blue)
ventilation and prevent fumigant action of the and 30l placed in separate lanes together with 20 l
insecticides. Each petri dish was sealed around the edge each of the following standards: alpha-lactalbumin
with clear tape to prevent escape of larvae. Larval (MW= 14.2kDa), carbonic anhydrase (29.0kDa), bovine
mortality was assessed every 2 h for 24 h. Larvae erythrocytes (45.0kDa), albumin from chicken egg white
unresponsive to a gentle prod with a toothpick within 5s (66.0kDa) and albumin from bovine serum (66.43kDa).
were regarded as dead. Data were corrected for control The samples were allowed to run for 1 h at 180V after
mortality using Abbotts (1925) formula. Mortality data which plates were washed with deionized water to
were subjected to probit analysis using EPA Probit remove the gels. Gels were placed into 150cm Pyrex
program Version 1.4. petri dishes with 100ml of Coomassie blue stain on a
Protein bioassay Labnet Rocker 25 for 45 minutes to ensure proper and
Based on LC50 values obtained for each even stain penetration. Gels were then de-stained with
th
insecticide, 4 instar S. frugiperda larvae were subjected 30% methanol: 10% acetic acid for 1h and then rinsed
to sub-lethal doses on each insecticide for 24 h. Live with deionized water (Labban et al., 2012). Bands on the
larvae exposed to a particular insecticide after 24 h were gel were then observed under a fluorescent light and
collected and crushed in an Eppendorf tube, centrifuged scanned using a UVP Gel Doc-It 300 imaging system
and the supernatant collected and analyzed for total and then analyzed using VisionWorksLS Analysis
protein content using Lowry et al., (1951) method and Software.
also separated using Polyacrylamide Gel Electrophoresis
(PAGE). 7.5% separating gel was prepared from 30% RESULTS AND DISCUSSION
acrylamide-BIS, 10% ammonium persulfate and There were two distinct groups of insecticides
tetramethylethylenediamine (TEMED). The mixture was based on toxicity (LC50) to 3rd instar larvae of
then swirled to ensure thorough mixing. The solution S. frugiperda (Table 1). The first group comprised
TM
was pipetted into Gel Wrap Gasket maker and left at Boxer, Malathion, Flip, Bright and Supertak
room temperature for 45 minutes to polymerize. A 4% among which there were no significant differences
stacking gel was prepared using 30% acrylamide-BIS (P>0.05). The second group comprised Fastac,
with 10% ammonium persulfate and TEMED and left at Neem-X, Abamectin and Karate among which there
room temperature for 45 minutes to polymerize and then were no significant differences (P>0.05) but were
refrigerated at 4C overnight. significantly different (P>0.05) from all members of the
Fourth instar S. frugiperda larvae were exposed first group. Bright 30EC was the most toxic (LC50 =
to the lowest concentration (0.0004%) of each insecticide 0.0006g/g) while Fastac 5EC was the least toxic (LC50
for 24 h before protein extraction took place. Larvae = 0.6046g/g) among all the insecticides evaluated. The
were crushed to a smooth texture in micro-centrifuge active ingredient in both Fastac 5EC and
tubes containing 100 l of deionized water. All samples Supertak 10EC is -cypermethrin, however their LC50
were thoroughly mixed for 5s with the aid of a Vortex values differed significantly (P>0.05) with
Genie 2 machine and centrifuged at 10,000 rpm for two Supertak 10EC being approximately 62 times more
Values followed by the same letter are not significantly different from each other
based on Tukey-Kramer Multiple comparisons test
toxic to 3rd instar S. frugiperda larvae than Fastac 5EC Spodoptera litura in Pakistan (Ahmad et al., 2005).
(Table 1) and apart from the doubling in concentration, Although Bright 25EC was the most toxic insecticide
may have been as a result of other components tested (LC50 = 0.0006mg/ml), the 50% lethal time (LT 50
(adjuvants) in the formulation. Mesnage et al., (2014) = 6.63 h) was high, indicating that it would take a
conducted studies on other pesticides using human cell population of S. frugiperda larvae approximately 6.63 h
lines also concluded that adjuvants listed as inert to achieve 50% mortality at a concentration of
ingredients in pesticides can amplify the toxicity to 1000 0.0006mg/ml (Tables 1 and 2). However, Flip 800 DF
-fold. (fipronil) which had a LC50 of 0.0028mg/ml was not
Among the insecticides tested, Flip 800DF took significantly different (P>0.05) from the LC50 of Bright
the shortest time to cause 50% mortality (LT 50 = 2.05 h), 25EC but had a LT50 = 2.05h (Table 2).
while Abamectin took the longest (LT 50 = 18.18 h) The total haemolymph protein content of
which was significantly different (P<0.05) from all the larvae treated with seven of the nine insecticides was
other insecticides tested (Table 2). Abamectin also took significantly lower (P<0.05) than that of the control,
the longest to achieve 50% mortality when used against while Bright (632.79g/ml) and Abamectin
Table 2. Lethal time (LT50) of insecticides to 3rd instar Spodoptera frugiperda larvae
Values followed by the same letter are not significantly different from each other based
on Tukey-Kramer Multiple comparisons test
(617.04g/ml)) were significantly higher (P<0.05) than Table 3 Total haemolymph protein content of
the control. Total haemolymph protein content ranged insecticide treated Spodoptera frugiperda 3rd instar larvae
insecticides tested. As indicated by Nath et al., (1997) insecticides. Pakistan Entomologist 27(1): 67-70.
the insect may have reduced proteins to their amino acid
Carvalho EV, Gonclaves AH, Affrri FS, Dott MA
components to enable their entry to the Tricarboxylic
and Peluzio JM. 2010. Influencia da lagarta-do-cartucho
Acid Cycle (TCA) as compensation for stress induced
(Spodoptera frugiperda J.E. Smith), sobre hibridos de
lower energy levels.
milho no sul do Tocantins-Brasil. Revista Verde de
The number of protein bands generally decreased
Agroecologia e Desenvolvimento Sustentvel 5(5): 152-
in insecticide treated haemolymph compared with the
157.
control. The control in Gel 1 had seven bands which
ranged from 315.35 g/ml to 20.13 g/ml, while Bright, Cruz I, Figueiredo MLC, Oliveira AC and
Supertak, Neem X and Abamectin had proteins of Vasconcelos CA. 1999. Damage of Spodoptera
molecular weights ranging from (315.35 25.39 g/ml), frugiperda (Smith) in different maize genotypes
(315.35 24.56 g/ml), (474.59 38.99 g/ml) and cultivated in soil under three levels of aluminium
(556.92 34.18 g/ml) respectively (Figure 1). The saturation. International Journal of Pest Management 45
control in Gel 2 had six bands which ranged from 495.03 (4): 293-296.
g/ml to 29.46 g/ml, while Boxer, Malathion, Fastac
Labban O, Jugmohan H, Khan A, Matthew J and
and Flip had proteins of molecular weights ranging from
Wisdom S. 2012. Haemolymph composition of
(407.96 13.63 g/ml), (421.33 12.21 g/ml), (76.30
Ancylostomia stercorea Zeller (Lepidoptera:Pyralidae)
18.95 g/ml) and (310.18 39.23 g/ml) respectively
larvae with particular reference to proteins and amino
(Figure 2). Karate insecticide was unusual in that there
acids. Journal of Research in Biology 2(3): 178-183.
were no visible protein bands present and may have been
as a result of the staining technique. Lowry OH, Rosebrough NJ, Farr AL and Randall
RJ. 1951. Protein measurement with the Folin-Phenol
CONCLUSION reagent. Journal of Biological Chemistry 193(1): 265-
The synthetic insecticides used in the present 275.
study caused significant reduction in both total
Mesnage R, Defarge N, de Vendmois J and Sralini
haemolymph protein content and number of proteins in
GE. 2014. Major pesticides are more toxic to human
S. frugiperda 3rd instar larvae. It is postulated that this
cells than their declared active principles. BioMed
may be as a result of the need for amino acids and/or
Research International 2014 Article ID 179691. 8.
their components to aid in detoxification of these
synthetic insecticides via the TCA cycle. Nath BS, Suresh A, Varma BM and Kumar RPS.
1997. Changes in protein metabolism in hemolymph and
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(Lepidoptera:Noctuidae) by some new chemistry
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