Journal of Research in Biology
An International Scientific Research Journal
Length-Weight Relationships of 21 species of Elasmobranchii from
Margarita Island, Venezuela
Authors: ABSTRACT:
Journal of Research in Biology
Tagliafico A1, Rago N2 and
Rangel MS1
Institution:
1. Escuela de Ciencias
Aplicadas del Mar,
Universidad de Oriente,
Boca de Ro, Isla de
Margarita, Venezuela.
2. Oceanografa y pesca,
Fundacin de Ciencias
Naturales La Salle, Isla de
Margarita, Venezuela.
Corresponding author:
Tagliafico A
Email Id:
tagliaficoa@gmail.com
Web Address:
http://jresearchbiology.com/
documents/RA0471.pdf
Journal of Research in Biology
An International
Scientific Research Journal
ISSN No: Print: 2231 6280; Online: 2231- 6299
Original Research
Length-Weight Relationships (LWR) were estimated for Elasmobranchii
caught by the artisanal fishing fleet of Margarita Island, Venezuela. A total of 3604
organisms belonging to 21 species (14 sharks and 7 batoids) were analyzed. Growth
type, minimum and maximum length and weight are summarized. The estimates for
the b parameter of the LWR ranged between 1.706 and 3.955, with a mean of
3.034. To the best of our knowledge, no information currently exists on the LWR
of Heptranchias perlo, Squalus cubensis, Squatina dumeril, Gymnura micrura,
Myliobatis freminvillei and Mobula hypostoma. This article stands as a pioneer
towards the growth research in these elasmobranchs.
Keywords:
Elasmobranch, shark, batoids, ray, Caribbean.
Article Citation:
Tagliafico A, Rago N and Rangel MS
Length-Weight Relationships of 21 species of Elasmobranchii from Margarita Island,
Venezuela
Journal of Research in Biology (2014) 4(7): 1458-1464
Dates:
Received: 06 Aug 2014 Accepted: 28 Aug 2014 Published: 10 Oct 2014
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.
1458-1464 | JRB | 2014 | Vol 4 | No 7
www.jresearchbiology.com

INTRODUCTION
The knowledge of how fish grow is essential for
stock assessment objectives, since the growth of every
single fish is precisely the source of information of every
catch obtained by the fishery (Pauly, 1983). Even though
there is variation between the weights of organisms of a
certain length, the Length-Weight Relationship (LWR)
estimates the mean body weight with a high grade of
confidence, depending of its coefficient of correlation.
LWR could be considered as a trustworthy tool to
estimate weight of organisms knowing its length
(Cubillos, 2005).
In Venezuela, for Elasmobranchii, there are
published data of LWR only on Mustelus higmani
(Etchevers, 1975), Rhizoprionodon porosus (Gmez and
Bashirulah, 1984), Carcharhinus limbatus, C. perezi,
C. falciformis, Gimglymostoma cirratum (Tavares, 2009)
and Aetobatus narinari (Tagliafico et al., 2012). This
work attempts to extend the knowledge of this biological
aspect of the commercial elasmobranchii caught in
Margarita Island.
MATERIALS AND METHODS
From January 2006 to December 2007,
elasmobranches catches landed at three artisan fishing
ports of the Margarita Island, Venezuela (Juan Griego,
La Pared and El Tirano) and two fishing markets (Los
Cocos and Conejeros) were sampled weekly. The
specimens were caught by artisanal fishery using surface
and bottom gillnets. Detailed descriptions of vessels and
fishing gears are accounted by Iriarte (1997) and
Gonzlez et al., (2006).
The specimens were identified using descriptions
by Compagno (1984) Cervign and Alcal (1999) and
Compagno et al., (2005). Lengths and weights were
obtained by using an ichthyometer (measured to the
nearest millimeter) and an electronic balance (measured
to the nearest gram), respectively. The total length (TL)
was used for all the sharks and some batoids
1459
Tagliafico et al., 2014
(Rhinobatos percellens and Narcine brasiliensis); for the
rest of the batoids, the disc width (DW) was used
(Cervign and Alcal, 1999).
The parameters a and b of LWR were estimated
with the statistic program STATGRAPHIC PLUS 5.1,
using the potential equation W=aLb; were W is the total
weight shown in grams, L the total length or disc width
according to the species shown in centimeters, a is the
coefficient related with the body shape, and b is an
exponent related with the type of growth (isometric when
is equal to 3, or allometic when is different to 3)
(Beverton and Holt, 1996). To determinate if the
estimated values of b for every LWR were significantly
deviated from isometric growth (b=3), a students t-test
was performed.
The slopes and intercepts of the LWR were
compared between males and females, and mature and
immature with ANOVA; in case of differences,
separated relationships by sex or mature state were
established, according to the case. For few infrequent
species, it was established a unique relationship for
males and females, both mature and immature.
RESULTS AND DISCUSSIONS
A total of 3604 organisms belonging to 21
elasmobranchs: 14 sharks (Figure 1) and 7 batoids
(Figure 2) were analyzed. Table 1 shows the obtained
coefficients of the equations of the LWR of each species.
Low number of specimens analyzed can be acceptable
for rare species (Froese, 2006), which is the case of
Mobula hypostoma.
According to Gmez and Bashirulah (1984)
significant differences occur in LWR between
males (n=136) and females (n=151) for
Rhizoprionodon porosus. In the present study, there are
no differences between sexes (ANOVA, F (1,214),
P=0.01657), analyzing a total of 215 organisms
(76 females and 139 males). This discrepancies can be
due to differences in the period and zones of sampling,
Journal of Research in Biology (2014) 4(7): 1458-1464
 Table 1. Length-Weight Relationships (W=aLb) of 21 species of Elasmobranchii caught by the artisanal fishery of Margarita Island, Venezuela.

Family Species L(cm) W (g) ES Growth

n Sex a 95% CL a b 95% CL b r2
Lmin-max Wmin-max (b) type
Hexanchidae Hexanchus nakamurai 15 F+M 84.6-106.0 1418-5499 0.00005 0.0000 - 0.3717 3.955 1.9904 - 5.9187 0.909 0.77 A (+)
Heptranchidae Heptranchias perlo
21 F 69.6-117.5 0964-4706 0.0005 0.0001 - 0.0028 3.392 3.0196 - 3.7646 0.178 0.97 A (+)
Tagliafico et al., 2014

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 (-)

Journal of Research in Biology (2014) 4(7): 1458-1464

Carcharhinidae Carcharhinus acronotus 14 F+M 52.1-083.7 0425-3260 0.003 0.0001 - 0.0439 3.148 2.4674 - 3.8280 0.312 0.95 I
Carcharhinus brevipinna 29 F+M 56.7-079.8 1200-3800 0.003 0.0003 - 0.0239 3.181 2.6878 - 3.6732 0.240 0.93 I
Carcharhinus falciformis 20 F+M 64.3-094.0 1758-4848 0.078 0.0026 - 2.3202 2.385 1.6133 - 3.1567 0.367 0.84 I
Carcharhinus limbatus 35 F 63.6-096.4 1361-6500 0.001 0.0004 - 0.0045 3.344 3.0764 - 3.6112 0.131 0.98 A (+)
62 M 54.5-109.6 2098-6600 0.105 0.0439 - 0.2522 2.346 2.1424 - 2.5495 0.102 0.95 A (-)
Rhizoprionodon lalandii 202 F 38.3-074.1 0170-2041 0.001 0.0004 - 0.0021 3.357 3.1767 - 3.5375 0.092 0.93 A (+)
143 M 39.0-095.3 0199-4260 0.005 0.0029 - 0.0095 2.944 2.7938 - 3.0891 0.075 0.96 I
Rhizoprionodon porosus 215 F+M 34.6-106.4 0113-5499 0.002 0.0009 - 0.0024 3.25 3.1305 - 3.3699 0.061 0.96 A (+)
Sphyrnidae Sphyrna lewini 11 F+M 37.2-093.0 0425-4350 0.011 0.0003 - 0.3393 2.789 1.9336 - 3.6451 0.378 0.93 I
Narcinidae Narcine brasiliensis 9 F+M 21.0-042.0 0113-7370 0.016 0.0008 - 0.2808 2.875 2.0276 - 3.7155 0.357 0.95 I
Rhinobatidae Rhinobatos percellens 62 F+M 45.5-075.7 0340-1729 0.0004 0.0001 - 0.0014 3.531 3.2233 - 3.8364 0.154 0.95 A (+)
Dasyatidae Dasyatis gutata 36 F+M 36.0.007-99 1786-27000 0.048 0.0144 - 0.1582 2.906 2.6020 - 3.2107 0.15 0.96 I
Dasyatis americana 21 F 46.2-091.0 2637-24295 0.173 0.0269 - 1.1051 2.56 2.1024 - 3.0171 0.219 0.94 A (-)
24 M 35.0-074.0 0539-12389 0.002 0.0000 - 0.0331 3.716 2.9702 - 4.4616 0.36 0.91 A (+)
Gymnuridae Gymnura micrura 6 F+M 14.6-026.4 0024-1620 0.003 0.0006 - 0.0142 3.313 2.7885 - 3.8364 0.189 0.99 I
Mobulidae Mobula hypostoma (e) 3 F+M 32.6-071.4 0386-4800 0.004 0.0000 - 226.01 3.296 0.4192 - 6.1736 0.226 0.99 I
Myliobatidae Myliobatis freminvillei 63 F+M 24.0-118.0 0227-12190 0.021 0.0062 - 0.0699 2.896 2.5981 - 3.1929 0.149 0.93 I

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

Figure 1. Sharks species analyzed: A) Carcharhinus acronotus; B) Mustelus norrisi; C) Carcharhinus

falciformis; D) Rhizoprionodon lalandei; E) R. porosus; F) Squalus cubensis; G) Hexanchus nakamurai;
H) Heptranquias perlo; I) Sphyrna lewini; J) Squatina dumeril; K) Mustelus canis; L) M. higmani;
M) Carcharhinus brevipinna and N) C. limbatus. (Scales between species are not real).

and that the organisms analyzed in this work ranged
between 34.6 and 106.4 cm of TL, and between 113 and
5499 g of weight, which includes organisms bigger and
heavier than those analyzed by Gmez and Bashirulah
(1984).
For Mustelus higmani, Etchevers (1975) did not
find differences between males (n = 13) and females
1461
(n = 21) for LWR. In this work, statistic differences
between sexes are reported (ANOVA, F (1,964) = 7.88,
P<0.005), even between mature and immature males
(ANOVA, F (1,417) = 21.43, P<0.001), perhaps due to a
biggest sample size (n = 965). Additionally, a previous
study on C. limbatus report a b-value of 3.028 for both
sexes (Tavares, 2009), however in this work differences
Journal of Research in Biology (2014) 4(7): 1458-1464
Tagliafico et al., 2014

Figure 2. Batoids species analyzed: A) Narcine brasiliensis; B) Mobula hypostoma; C) Rhinobatos

percellens; D) Myliobatis freminvillei; E) Gymnura micrura; F) Dasyatis guttata and G) Dasyatis
americana. (Scales between species are not real).

between sexes were found (ANOVA, F (1,97) = 34.31,
P<0.001) and as consequences two different LWR were
made; also, different patterns of growth were reported for
both sexes: females shows allometric positive growth
(b=3.3), whereas males allometric negative growth
(b=2.4). Different LWR for the same species can be
attributed to sampling different populations or changes in
the environmental conditions over time (Froese, 2006).
From around 32470 species of fishes contained
in FishBase, LWR studies were only available for less
than 12% (3587 species) (Froese et al., 2014), and if the
same analysis is performed by country or region, the
numbers become even smaller; which is the case in
Caribbean waters, where information on the LWRs is
limited to a few species.

Journal of Research in Biology (2014) 4(7): 1458-1464 1462


CONCLUSION
To the best of our knowledge, no information
currently exists in Venezuela on the LWR for 17 of
the species studied (only R. porosus, M. higmani,
C. limbatus and C. falciformis have been previously
studied), and no LWR information is currently
worldwide available for Heptranchias perlo,
Squalus cubensis, Squatina dumeril, Gymnura micrura,
Myliobatis freminvillei and Mobula hypostoma (Froese
and Pauly 2011).
The coefficients of correlation of all the
regressions in this study ranged between 0.7 and 0.99; all
statistically significant (P<0.0001) and can be used to
estimate weight from the length of individuals with
similar length intervals.
ACKNOWLEDGEMENTS
The authors thank all the fisherman and sellers
for the patience in the measurement of the specimens.
Asdrbal Lrez and INIA for logistic help. Richard
Parkinson and the anonymous referees provided valuable
comments.
REFERENCES
Beverton R and Holt S. 1996. On the Dynamics of
Exploited Fish Populations. Chapman and Hall, London
p 533.
Cervign F and Alcal A. 1999. Los peces marinos de
Venezuela. Fundacin Museo del Mar. Caracas Vol 5
p 231.
Compagno LJV. 1984. FAO species catalogue. Vol. 4.
Sharks of the world. An annotated and illustrated
catalogue of shark species known to date. Part 1,
Hexanchiformes to Lamnifor mes. Part 2,
Carcharhiniformes, FAO Fish. Synop 4 (125):251-655.
Compagno L, Dando M and Fowler S. 2005. Sharks of
the world, (Princeton Field Guides). Princeton University
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Press, New Jersey p 368.
Cubillos L. 2005. Biologa pesquera y Evaluacin de
Stock. Laboratorio de Evaluacin de Poblaciones
Marinas & Anlisis de Pesqueras, Departamento de
Oceanografa, UDEC, Concepcin 1-198.
Etchevers S. 1975. La relacin longitud peso de 7 peces
de inters comercial en el Nororiente de Venezuela. Bol.
Inst Oceanogr Venezuela Univ. Oriente 14(2): 243-246.
Froese R. 2006. Cube law, condition factor and weight-
length relationships: history, meta-analysis and
recommendations. J Appl Ichthyol., 22(4): 241-253.
Froese R and D Pauly. Editors. 2011. FishBase. World
Wide Web electronic publication. www.fishbase.org,
(06/2014 ).
Froese R, Thorson JT and Reyes RB. 2014. A
Bayesian approach for estimating length-weight
relationships in fishes. J. Appl. Ichthyol., 30: 78-85.
Gmez Fermin E and Bashirulah AKM. 1984.
Relacin longitud-peso y hbitos alimenticios de
Rhizoprionodon porosus Poey 1861 (Fam.
Carcahrhinidae) en el oriente de Venezuela. Bol. Inst.
Oceanogr. Venezuela Univ. Oriente 23 (1&2): 49-54.
Gonzlez LW, Eslava N and Guevara F. 2006.
Catlogo de la pesca artesanal del Estado Nueva Esparta,
Venezuela. Cordinacin de publicaciones del Rectorado
de la Universidad de Oriente, Cuman p 222.
Iriarte L. 1997. Embarcaciones, artes y mtodos de
pesca del estado Nueva Esparta. Fundacin La Salle de
Ciencias Naturales, Estacin de Investigaciones Marinas
de Margarita, Monografa Punta de Piedras, Estado
Nueva Esparta No. 42: p 349.
Pauly D. 1983. Algunos mtodos simples para la
evaluacin de recursos pesqueros tropicales. FAO Doc.
Tec. Pesca, No. 234. p 49.
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Tagliafico A, Rago N, Rangel MS and Mendoza J.

2012. Exploitation and reproduction of the spotted eagle
ray (Aetobatus narinari) in the Los Frailes Archipelago,
Venezuela. Fish. Bull. 110:307316.

Tavares R. 2009. Anlisis de abundancia, distribucin y

tallas de tiburones capturados por pesca artesanal en el
Parque Nacional Archipilago de los Roques, Venezuela.
Interciencia, 34(7): 463-470.

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 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

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

Tabitha Langhu and

Chitta Ranjan Deb* Present study was undertaken to study the reproductive behaviours and seed
biology of Aconitum nagarum. As per the present study, the species starts flowering
from october first week onwards. The flowers are blue in colour, arranged as slender
raceme, petals and filaments glabrous, carpel five and bisexual. The flowers bloom
Institution: acropetally and anthesis was observed between 6.00 - 6.30 AM. Anther dehisced
Department of Botany, longitudinally from 7.00 AM till 9.30 AM. The number of anthers were found to be 49
Nagaland University, per flower. It was observed that flower colour changes as the plant goes on fully
Lumami 798 627, dehisced. The flowering duration per flower varied from 4-6 days followed by fruit
Nagaland, India formations and matures within 10-15 days. The average flowers per plant varied from
8-28 and common pollinator was found to be bees. Mean seeds per plant was
~270-540 and pollen per anther was approximately 1000 - 2000. The seeds exhibited
~38% germination from seeds stratified at 4C for 96 h.

Corresponding author: Keywords:

Chitta Ranjan Deb Aconitum nagarum, Floral biology, Medicinal plant, Reproductive biology.

Email Id: Article Citation:

Tabitha Langhu and Chitta Ranjan Deb
Studies on the reproductive biology and seed biology of Aconitum nagarum Stapf:
a threatened medicinal plant of North East India
Journal of Research in Biology (2014) 4(7): 1465-1474

Web Address: Dates:

http://jresearchbiology.com/ Received: 27 Aug 2014 Accepted: 12 Sep 2014 Published: 21 Oct 2014
documents/RA0475.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.

1465-1474 | JRB | 2014 | Vol 4 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Among 34 biodiversity hotspots of the world,
India is a home for four of them extending to the
neighboring countries the Western Ghats/Sri Lanka,
the Himalaya, the North-Eastern region and the Nicobar
Island (MoEF, 2014). India is also one of the 17 mega
biodiversity countries and has 26 recognized endemic
centres that account for about one third of the flowering
plants. North East India is a centre of mega-biodiversity
and is equally rich in flora and fauna and contain more
than one-third of the countrys total biodiversity. The
region is the home for number of plant species which are
endemic to the region. But the populations of these
economically important plant species are down sized in
their natural habitats due to various factors including
reproductive bottlenecks. Reproductive bottleneck
includes failure of pollination, pre and post fertilization
barriers leading to no or poor seed set, poor reproductive
vigour due to inbreeding depression and low germination
rate imposed constraints on the multiplication and
survival of the species. Therefore, any conservation
approach has to be based on an in-depth study of plant
reproductive biology which provides information on the
ability of seed germination, seedling viability, age of
plant at which they reaches reproductive phase,
reproductive longevity, seed setting ability etc., Such
studies would provide fruitful insights in planning
various programmes specific to different habitats (Silva
and Silingardi, 2001, Abera et al., 2008; Singh et al.,
2010). Studies on the phenology of medicinal plants are
the basic knowledge to be obtained for the right season
for collecting plants and propagules and for establishing
the appropriate growth environment for propagation
purpose (Moza and Bhatnagar, 2007, Abera et al., 2008).
Aconitum nagarum Stapf. is an endemic
medicinal plant of North-Eastern part of India and grows
in grassy sloppy mountain (Figure 1a). The species is of
great medicinal importance for its tuber (Figure 1b). The
alkaloid produced from the tubers are used in curing
1466
Langhu and Deb, 2014
wide range of diseases and also used as arrow poison.
The diterpenoid alkaloids from A. nagarum have been
isolated by different workers (Dong et al., 2000, Zhang
et al., 2005, Ji and Wang, 2006). The alkaloids are used
in the treatment of antipyretic, anti-rheumatic, paralysis
and snake bite (Srivastava et al., 2010). The species has
also an antibacterial activity against several bacteria
(Sinam et al., 2012). Due to habitat destruction, over
collection for herbal drugs etc., the species has entered
into threatened category in their natural habitat. Besides
different anthropogenic activities, decrease in the
population size is also because of their abnormal
reproductive behaviors. The present study was
undertaken to study the reproductive behaviors and seed
biology of this species.
MATERIALS AND METHODS
Floral biology
The study was conducted in Dzukou valley at
Khonoma village, Nagaland, at an elevation of 2684 m
above sea level (ASL) 2536'44.8" N and 9400'03.4" E
and Shirui hills of Ukhrul district, Manipur at an
elevation of 2427 m ASL, 25 06' 39. 6"N and 094 27'
13.3"E among the grassy bamboo slope. The
reproductive phenology and floral morphology viz, time
of budding, time of anthesis and stigma receptivity,
different stages of anther development, anther
dehiscence, fruit and seed setting etc., were studied.
Floral phenology of A. nagarum at Dzukou valley,
Nagaland and Shirui hills, Manipur were comparatively
studied. In order to estimate flower production, total
number of flowers per plant was counted manually in the
selected plants. Seeds per pod were counted to quantify
production of pods. Anther counts were done on
randomly selected flowers. Pollen counts were made on
20 anthers from different flowers. The anthers were
collected during the onset of blooming season
successively for three years. The anthers were collected
from the flowers and kept in moist cotton pad and
Journal of Research in Biology (2014) 4(7): 1465-1474
Langhu and Deb, 2014

maintained in the polybag till they were brought in to the
laboratory. The anther lobe was removed from the style
with the help of forceps and blade and the anther lobe
was put on a slide. The anther was smashed uniformly by
adding a drop of glycerine and spread evenly. The slide
was covered with the cover slip. The slide was kept
under the microscope and pollen present in 10
microscopic fields were counted. Total number of pollen
was determined by multiplying the pollen present in the
mean microscopic field and total number of microscopic
field per slide.
Distribution pattern of the plant and its associated
species
The distribution of Aconitum nagarum in North-
Eastern parts of India (Nagaland and Manipur) was taken
into account for the comparative study of plant
distribution pattern. A study was conducted to
understand the rule of associated species on the growth,
reproduction and survival of A. nagarum.
Seed biology
The plant and mature fruits were collected from
the forest of Dzukou valley, Khonoma, Nagaland, India
at an elevation of 2648 m ASL from the grassy bamboo
slope. Aconitum nagarum reached peak flowering from
the first week of October and seed setting starts from the
second week of October. Therefore harvesting of seeds
can be done from the third week of October. On
contrary, in Shirui hills, Manipur, at an elevation of
2427m asl, peak flowering starts from the first week of
november and seed setting was from the second week of
november. During the second week of november few
flowers were seen but are very rare. Matured seeds could
be collected from the third week of november.
Seed collection and processing
For any study of this nature may be affected by
various factors like seed collection technique, processing
of seeds and post harvest method etc. In the present
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.

Journal of Research in Biology (2014) 4(7): 1465-1474 1467

 Langhu and Deb, 2014

by Deb et al., (2012) was followed with suitable
modifications as per laboratory condition and
experiment. In the present study, mature seeds were
harvested randomly from the natural habitat during
2011-2013 along with the plant stalk. The stalks were
wrapped in newspapers and covered with polythene bag
and transported to the laboratory within 1-2 days. The
collected fruits were dried by spreading uniformly over
o
the old newspaper for 1-2 days in the laboratory at 25 C.
The dried fruits were removed from the stalk and seeds
are taken out of the carpel. The seeds were then stored
in poly bag in the laboratory for further experiments.
The processed seeds were washed with
Labolene (1:100, v/v) (a commercial laboratory
detergent) and rinsed under running tap water and finally
with distilled water. The seeds were made into different
groups for germination experiment.
Preparation of potting mix
The potting mix for the experimental purpose
was made following Deb et al., (2012) by mixing soil
and chopped coconut coir at 1:1 ratio. The garden soil
was crushed into fine powder, sun dried and mixed with
the coconut coir in the ratio of 1:1 and put in a plastic pot
and transparent poly bags. The poly bag and plastic pot
were made perforated for better aeration. They were kept
moist before sowing the seeds for germination.
Experimental process
The protocol developed by Deb et al., (2012)
was followed with suitable modification in the present
study. A part of the seeds were processed and sowed
immediately after the harvest while others were treated
differentially at 4C in a refrigerator for 0, 24, 48, 72, 96
hours and sowed as described below:
1. A set of stratified seeds were sowed in filter paper in a
humidity chamber of 90 mm in diameter and kept in a
laboratory (25C).
2. Another set of processed seeds (stratified seeds) were
sowed in the potting mix and kept in an incubator at the
constant temperature of 30C.
3. While another set of stratified seeds were sowed in
seed bed (poly bag) and maintained in a poly house.
4. To test the post harvest tolerance of the seed for
various periods, the processed seeds were stored at 25C
(in the laboratory) in sealed poly bags before they were
sowed in the seed bed for seed tolerance experiment.
5. The seedling morphology and seedling mortality rate
was also studied.
To study the emergence, survival and growth of
seedlings of Aconitum nagarum under each condition,
4 replicates of 13 seeds each (for filter paper test, N=52
seeds/test) and 20 seeds each (for seed bed germination,
N=80 seeds/test) were used. In each poly bag, the soil
mixture was packed and 20 seeds were sowed. In filter
paper test 13 seeds were sowed. The seed beds were
watered at regular intervals. The experimental design
was completely randomized. The data were collected
daily based on seed germination, seedling morphology;
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

1468 Journal of Research in Biology (2014) 4(7): 1465-1474

Langhu and Deb, 2014

the seedlings were maintained in the respective polybags
and watered at regular interval and allowed to grow un
till becoming normal plantlets. Once the seedling showed
normal functioning like rooted plantlets, emergence of
normal leaves etc., the seedlings were transferred to the
poly house. Once the seedlings were established in the
poly house, the seedlings exhibited differential growth
and many seedlings died.
Filter paper test at room temperature (25C)
The seeds were treated at 4C in a refrigerator for
different periods (0, 24, 48, 72, 96 h). The seeds were
then placed on moist filter paper in a humidity chamber
of 90 mm diameter and kept for germination at the
laboratory (25C). The seeds were kept moist throughout
the study period. The germination process gets
completed by the emergence of radical followed by leaf
with seedling formation. A total of 13 seeds were used
for each treatment with 4 replicates (N=52 seeds/
treatment). The seedlings were transferred to poly house
for further seedling growth studies. The experimental
design followed was based on Deb et al., (2012).
Germination test in incubator
The stratified seeds (stratified at 4C for 0, 24,
48, 72 and 96 h) were placed on potting mix to test
the role of stratification on germination. Each treatment
consists of four replicates with 20 seeds each (N=80
seeds/treatment). The differently stratified seeds were
sowed in the potting mix and incubated at 30C in the
incubator. The seeds were monitored at regular intervals
for seed germination, seedling morphology and seedling
mortality etc.
Germination test in seed beds (poly bags)
The differentially stratified seeds were placed on
potting mix in a perforated polybag of 150 mm in
diameter. Each treatment consists of four replicates with
20 seeds (N=80 seeds/treatment). The plants are
monitored regularly for germination. Germination
percentages were calculated after eight weeks of seed
culture.
Post harvest storage tolerance test for
Aconitum nagarum
The seeds are stored at a temperature of 25C.
The seeds were tested for the post harvest storage
tolerance. Every month sets of processed seed were
sowed in the seed bed. Their germination rate for each
experiment was carried out and the results obtained were
recorded monthly. The result obtained was compared and
checked to see how far Aconitum nagarum seeds can
tolerate storage. All the above experiments were based
on the works reported by Deb et al., (2012) with
Cinnamomum tamala.

Table 2. Floral display of Aconitum nagarum

Parameters Observation
Inflorescence Alternate raceme
Number of inflorescence/plant 8-28 nos.
Flower type Hermaphrodite
Anthesis 6-6.30A.M
Mode of anther dehiscence Longitudinal
No. of anther/flower 49
No. of pollen grains/anther 1000-2000
Stigma type Pentacarpellary
Ovary type Pentalocular
Seed Obpyramidal, brown
Seed/plant 270-540 nos.
Root Hearth shape, dark brown
Data are compiled from successive two years of study/observations.

Journal of Research in Biology (2014) 4(7): 1465-1474 1469

 Langhu and Deb, 2014

Seedling mortality and seedling morphology
The seedling mortality was observed by
transplanting the seedling grown from the differently
treated seeds in the poly house and percent seedling
survival was recorded till the plant mature or till its
survival period which ever is earlier.
The transplanted seedlings in the poly house
were also checked for changes/modifications in the
seedling, which was observed after the transplantation of
the germinated seeds (like modification in the leaf, roots
etc).
RESULTS AND DISCUSSION
Associated species, floral biology and morphology
Present study was conducted in Nagaland and
Manipur at the altitude between 2000 m to 3841 m above
mean sea level (Table 1) in six different geographical
areas. In all the study areas the populations were found in
the hill slopes of the valley. In the present study, an
interesting feature was observed as common associated
species growing in all the study areas; they are,
Sinarundinella species, Gaultheria species and Fragaria
species. In all the areas A. nagarum was growing healthy
where these associated species were available. This
information on associated species could be used for the
identification of new niches of this species for
rehabilitation of the species.
In the present study, it was observed that the
plants of Aconitum nagarum growing at Dzukou valley
started budding from September second week onwards
(Figure 1c) with peak flowering in October first week
(Figure 1d). The flowers are blue in colour, in slender
raceme, petals and filaments glabrous, carpel 5, and
bisexual. The flowers bloom acropetally i.e. flower starts
blooming from the base of the inflorescence to the tip of
the inflorescence. Thus the fruits also mature acropetally.
The anthesis was observed between 6.00 - 6.30 AM.
Anther dehisced longitudinally from 7.00 AM till
9.30 AM. The number of anther was 49 per flower.
During the study on floral phenology, it was found that
there is a strong correlation between anther development
and the stigma development (Table 2). The flower colour
changes as the plant fully dehisced. The flowering
duration per flower varies from 4-6 days followed by
fruit formation. Fruits mature within 10-15 days. Fruit
formation starts from the second week of October
(Figure 1e) and by the third week of October, fruits
mature and the plant dries up (Figure 1f). While, in
Shirui hills of Manipur at an elevation of 2427 m ASL
25 06' 39. 6" N and 094 27' 13.3" E peak flowering of
Aconitum nagarum was observed by the first week of
November and by the second week flowering decreases
with the formation of fruits. By the third week, fruits are
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

Parameter Dzukou valley Shirui hills

Budding September October
Flowering time October 1st week November 1st week
Anthesis 6-6.30A.M 6-6.30 A.M
Seed setting Oct 2nd week Nov 2nd week
Seed maturity Oct 3rd week Nov 3rd week
Sprouting of plants March/April April/May
Duration of flowering 4-5 days 5-6 days
Altitude and GPS Coordinates of study area 2684m ASL, N 2536'44.8 2427 m ASL, N 25 06' 39. 6" latitude and E
Latitude E09400'03.4 094 27' 13.3" longitude
Longitude
Temperature during flowering at Dzuku valley-16C
Temperature during flowering at Sirui Hills-17C

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Langhu and Deb, 2014

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.

Journal of Research in Biology (2014) 4(7): 1465-1474 1471

 Langhu and Deb, 2014

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
maintained in the laboratory at 25C with minimum days
taken to germinate. Under this condition 38% and
15.38% seed germination recorded after 13 days and 10
days of sowing respectively (Table 4 and Figure 1g).
Seeds without stratification exhibited very poor
germination (6.7%), on comparison to filter paper test
then stratified seeds sowed on seed beds (prepared in
poly bags supported better germination).
Seeds sowed in the poly bags exhibited 20%
germination within 23 days from the seeds stratified for
96 h at 4C (Table 5). But there was no seed germination
recorded from the seeds maintained in the incubator at
30C across the stratification period. Seed germination
was achieved within 29-30 days with emergence of roots
with cotyledonary leaves while true leaves are formed
within 58-60 days (Figure 1h). In the present study it
was found that seed germination rate and germination
time were greatly influenced by pre-treatment of seeds.
There was significant difference in the germination
period and germination rate with the stratified and non-
stratified seeds. During the present study, investigation
was carried out on the relationships among rate of cold
stratification to determine whether the seeds require pre
treatment of low temperature for germination. The
finding in the present studies thus support that seeds of
Aconitum nagarum prefer low temperature for seed
germination. The lowest temperature tolerance by the
recalcitrant seeds appear to be species specific (Fu et al.,
1990, Oliveira and Valio, 1992, Barbedo and Cicero,
2000, Tommasi et al., 2006, Sharma and Gaur, 2012).
Tommasi et al., described that Ginkgo biloba seeds could
be stored at 4C for one year but when stored at 25C,
seeds died after six months (Tommasi et al., 2006).
During the present study a similar response was recorded
where seeds stratified at 4C germinated better over
seeds stored at 25C.
Post harvest storage tolerance of seeds of
Aconitum nagarum
Though, the seed preservation practices are as
old as agricultural civilization but organized and
systemic storage facilities have been developed only in
the 20th century. There are over 1500 seed or gene banks
present world over and accessions are increasing
regularly. Longevity of seeds is not universal and is of
species specific. After harvest the viability of seeds
decline with time, seeds may exhibit differential
germination and seedling morphology and field
establishment (Walters, 2004). So, for some plant
species, using relatively fresh seeds give superior
germination over stored seeds.
During the present study, efforts were put to
examine the post harvest storage tolerance of the seeds.
The seeds were stored at 25C for 0-6 months and seeds
were sowed in the seed beds at one month interval (Table
6). Data collected in the present study exhibited gradual

1472 Journal of Research in Biology (2014) 4(7): 1465-1474

Langhu and Deb, 2014
decline in the germination response after one month of
storage and from the third month, germination rate
declined significantly. The germination rate declined
from 6.7% to 4.5% in the fourth month and in the sixth
month, there was no germination i.e., seeds lost viability
completely The findings of the present study clearly
showed that the seeds of A. nagarum are recalcitrant in
nature. The seed in the natural habitat does not get the
appropriate environment immediately after maturation
for germination which in turn affects the seed
propagation of the species in the natural habitat.
Seedling morphology and seedling mortality
The first sign of seed germination was the
emergence of radical from the seed coat followed by a
pseudo cotyledonary leaf formation. The cotyledonary
leaf was replaced by a true leaf. The true leaf starts
emerging from the base of the pseudo leaf. As the true
leaf progress in size, the pseudo leaf turn yellowish in
appearance and slowly wither giving way for the true
leaf to succeed after 58-60 days of seed germination.
The seedling mortality was observed by
transplanting the seedling in the poly house. Monthly
basis survey was conducted to access the seedling
mortality in the poly house. Seedling survival was very
high till the month of April/May and starts decreasing by
the last week of June/July. The growth of the plant
became stunted. The seedling establishment was very
low though seed germination was high in the forest as
well as in the poly house.
CONCLUSION
The plant reproductive study is crucial for
conservation strategies of this endemic plant. Present
study suggests that the seeds of A. nagarum are of
recalcitrant in nature and demand cold stratification prior
to germination. For seed propagation of A. nagarum, one
should take good care of the seed after collection to
avoid desiccation and storage of seeds beyond 5-6
weeks.
Journal of Research in Biology (2014) 4(7):1465-1474
ACKNOWLEDGEMENT
Authors are thankful to the Department of
Biotechnology, Ministry of Science and Technology,
Government of India, New Delhi for financial support
through research grant to Prof. Chitta Ranjan Deb.
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seeds of Inga uruguensis, a tropical species with
recalcitrant seeds. Seed Sci. Technol., 28(3): 793-808.
Deb MS, Jamir NS and Deb CR. 2012. Studies on seed
biology of Cinnamomum tamala Nees. (Lauraceae): a
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XL. 1990. Physiological studies on desiccation, wet
storage and cryopreservation of recalcitrant seeds of
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Ji H and Wang FP. 2006. Structure of lasiansine from
Aconitum nagarum var. lasiandrum. J. Asian Nat.
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Pugnaire, F. I. and Valladares, F.) Functional Plant
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Michalet R. 2003. Direct and indirect effects of shade on
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 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

In vitro assessment of water current on growth and biometric relationship

among molluscs
Authors: ABSTRACT:
Journal of Research in Biology

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.

Corresponding author: Keywords:

Sanindhar Shreedhar Biometric relationships, Freshwater, Malacofauna, Water current.
Gaikwad

Email Id: Article Citation:

Sanindhar Shreedhar Gaikwad and Nitin Anandrao Kamble
In vitro assessment of water current on growth and biometric relationship among
molluscs
Journal of Research in Biology (2014) 4(7): 1475-1486

Web Address: Dates:

http://jresearchbiology.com/ Received: 03 Sep 2014 Accepted: 02 Oct 2014 Published: 21 Oct 2014
documents/RA0479.pdf
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Journal of Research in Biology 1475-1486| JRB | 2014 | Vol 4 | No 7

An International
Scientific Research Journal
www.jresearchbiology.com

INTRODUCTION
Water is a crucially important parameter for the
sustenance of aquatic life. Palatability of the water
majorly depends on its physicochemical properties,
which maintains the water quality (Mustapha and
Omotosho, 2005 and Saxena et al., 2011). Water quality
denotes the total health of that area (Sala et al., 2000).
Amongst the water resources available on the earth
surface, palatable resources are confined only to the
freshwater (Aggarwal and Arora, 2012). When
discussing about the freshwater resources, rivers and
lakes cannot be exempted (Gupta et al., 2011 and
Jonnalagada and Mhere, 2001). These lentic and lotic
habitats contribute the major portion of the palatable
water resources and found as dwelling place for variety
of animals (Alam and Pathak, 2010, Mandal and Das,
2011 and Jayalakshmi et al., 2011). Nowadays, these
valued resources are continuously being contaminated
and resulted to deterioration, which become a serious
concern for scientific community (Sudhira and Kumar,
2003 and Singh, 2007). The only reliable way to
overcome this problem is the water filtration, which is a
really challenging task in large scale. River has the
potential to clean this unwanted contaminations by its
continuously flowing water current (Adeyemo et al.,
2008). Water current has a key role in maintaining
equilibrium physicochemical properties of the river,
which in turns regulates the floral and faunal diversity of
the area.
Amongst the invertebrates, molluscan fauna
comprises second largest population of the world and
distributed in the every possible habitats or niche except
aerial habitats (Kotpal, 1973). Due to such a wide
distribution and commercial value, they provide many
opportunities to the researchers to analyze their biology
(Stauffer, 1937, Dreyer and Castle, 1941, Laxmilatha,
2008, Peretz and Adkins, 1982 and Cubillo, 2012).
Along with its key role as model, it also exhibits as
bioindicator of the pollution (Gupta and Singh, 2011 and
1476
Gaikwad and Kamble, 2014
Zhou et al., 2008). Hence, it has become necessary to
investigate them in order to describe their correlation
with the water quality. Biometric relationships are
crucially important in this concern, as they are directly
associated with the growth of individuals in particular
atmosphere or habitat. These parameters help to
determine the impact of water current along with its
quality on the physiology of molluscs. Numbers of
researchers have focused on the biometric relationships
of the molluscs, as a tool to investigate the impact of
variety of parameters on the growth and reproduction.
Previously, Cataldo et al., (2001) expressed the
importance of molluscs C. flumina as bioindicator by
evaluating its biometry against the available water
quality. Shriver et al., (2002), described the effect of
eutrophic driven changes on the growth, condition,
reproductive potential and mortality of A. irradians.
Recently, Kolliyil et al., (2006) mentioned the effect of
habitat alteration on the pearl oyster P. fucata with the
help of biometry. Nevertheless, all these investigation
detailed the impact of environmental parameters on the
individual molluscan species, but failed to explain the
exact role or impact of water current on composite
molluscan community. Comparative assessment of these
biometric relations among different species of molluscs
has never been elucidated. Hence, by keeping in view the
crucial importance of the water current, present
investigation has conducted so as to consider our
knowledge regarding the biometric association of the
different species of molluscs living all together in the
aquatic ecosystem.
MATERIALS AND METHODS
Mussel sampling
For the present investigation, three freshwater
Uninoidae molluscs, L. corrianus, L. marginalis and
P. corrugata were selected due to their importance as
bioindicator and socially adapting behaviour. Mature
individuals with shell length ranges between 70 mm to
Journal of Research in Biology (2014) 4(7): 1475-1486
Gaikwad and Kamble, 2014
90 mm for L. corrianus, 60 mm to 80 mm for
L. marginalis and 40 mm to 60 mm for P. corrugata
were collected from all along the marginal area of the
river Panchganga. Total of 90 individuals i.e. 30
individuals per species were utilized for the experimental
analysis.
Experimental Design
Specimens were taken to the laboratory and kept
in plastic container of 50-liter size up to 48 h for
acclimatization. After acclimatization, 10 individuals of
each species were stored in separate containers. Out of
such six containers; three containers were provided with
water circulating systems i.e. with one inlet and one
outlet tubes and termed as experimental groups. Aerator
was provided for the proper oxygen supply to avoid the
suffocation. Filters were attached to the containers to
check the fecal matter. Bottom of the containers were
covered with gravels or sand particles of more than
0.5 mm size to provide the natural anchoring bed for the
molluscs. A continuous water current of the 3.4 ml/sec
was regulated to create the exactly resembling
environment of the river. For remaining three containers
only exception of water circulating system was made to
provide the controlled habitat. These containers were
treated as control groups. Water from these three
containers were routinely replaced. For the well
flourishment of the reared molluscs, a routine supply of
the planktonic mass was conducted. The experimentation
was continued for the period of two months. In order to
assess the exact impact of the water current, the
experimentations were repeated thricely from January
2013 to June 2013.
Data analysis
Respiratory assessment
The respiration rate of the selected molluscan
species was assessed fortnightly by using the amount of
oxygen consumed by the individuals of the species, in
order to notice their normal physiology as a previously
described method of Resgalla et al., (2006).
Journal of Research in Biology (2014) 4(7): 1475-1486
Hydrological parameters assessment
During the period of investigation, in order to
keep checking on the water quality, hydrological
parameters such as Temperature, pH, DO (Dissolved
oxygen) and free CO2 (Carbon dioxide) were assessed by
using standard methodologies of APHA (2005).
Biometric relationships
In order to elucidate the exact impact of the
water current on the growth, different condition indexes
viz. Body Condition Index (BCI), Meat Yield Index
(MYI) and Shell Component Index (SCI) were
evaluated. Growth was noted as weight gain by the
individual, as shell lengths growth was lowered after
maturity. The BCI was calculated by applying the
Davenport and Chen (1987), Rainer and Mann (1992)
and Rahim et al., (2012) formula. Shell Component
Index (SCI) and Meat Yield Index (MYI) were evaluated
by using Pekkarinen (1983) and Freeman (1974); Yildiz
(2011) methods respectively. Formulae for these indexes
were as follows:
Meat dry weight
BCI = 100
Total weight
Shell wet weight
SCI = 100
Total weight
Meat wet weight
MYI = 100
Total weight
Total weight gain = Final weight Initial weight
The data were analyzed with ANOVA by using
Kruskal Wallis multiple comparison test, to determine
the level of significance. The results obtained as mean of
the triplicate were interpreted as an average values with
mean S.D in graphical and tabular format.
RESULTS
During the entire period of investigation,
hydrological parameters like temperature, and pH did not
described any significant differences and ranged between
1477
 Gaikwad and Kamble, 2014

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).
Whereas highly significant variations were
noticed for DO (P < 0.0232) and CO2 (P < 0.0048),
which varied between 0.7 to 2.1 mg/lit and 7 to 12 mg/lit
for control as well as experimental groups (Figure 3
and 4).
Growth rate
Growth of the individual is measured by their
length and weight relationship. In molluscs, after sexual
maturity lengthwise increment or growths get restricted
to 1 or 2 mm per year only. Hence, by keeping in view
the economic importance of mature individuals for
present investigation average weight gain by the
individuals during the experimentation was noted as
growth rate. The results obtained as mean growth rate for
control and experimental groups of the individuals were
summarized in the Table 1 and 2.
Table 1. Mean growth achieved by the compared molluscan
species of the control group.
Highest average growth rate was remarked for
L. corrianus along with the significant variation in case
of control and experimental group whereas L. marginalis
and P. corrugata were noted with moderate non-
significant level of growth rates (Figure 5).
Respiratory rate
Oxygen consumption capacity i.e. respiratory
rate was noted with narrow range of fluctuation in case
of all the individuals. Highest respiratory activity was
showed by L. corrianus while P. corrugata was observed
with least level of respiratory activity. L. marginalis was
remarked with moderate respiratory rate and showed
significant variation (P < 0.0058) amongst compared
control and experimental groups (Figure 6).
Biometric relationships
Parameters representing biometric relationships
showed significant differences amongst compared
molluscan species.

Weight gain in g for Control group

Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
5.27 6.75 2.26
3.94 3.97 3.35
0.45 4.56 1.01
8.93 5.88 1.74
1.00 5.15 2.76
8.16 2.20 1.99
20.72 4.46 4.53
4.84 3.71 3.43
2.87 2.87 2.02
4.18 6.34 2.51

1478 Journal of Research in Biology (2014) 4(7): 1475-1486

Gaikwad and Kamble, 2014

Figure 3. Dissolved Oxygen assessed Figure 4. Free CO2 assessed during the study period.
during the study period.

Table 2. Mean growth achieved by the compared molluscs

of the experimental group.

Weight gain in g for Experimental group

Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
16.92 06.34 07.55
05.13 06.31 03.05
14.38 05.04 05.07
09.99 19.07 01.36
30.48 20.03 00.27
12.49 10.07 00.28
17.09 18.05 12.76
06.64 07.25 07.06
05.55 05.33 08.12
07.86 06.17 07.97

Body Condition Index (BCI) - Mean BCI
evaluated for control and experimental group during the
study was tabulated in the Table 3 and 4 respectively.
Maximum picks of average BCI was remarked
for experimental group, whereas slightly altered BCI was
represented by control groups. L. corrianus showed
richest BCI, whereas non-significant moderate BCI was
noted for L. marginalis and P. corrugata (Figure 7).
Shell Component Index (SCI) - Mean SCI
estimated for both the groups were represented in the
Table 5 and 6.

Table 3. Estimated BCI for control group individuals.

Body Condition Index of control group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
1.453744 0.58548 2.207479
1.804176 3.364993 3.016661
1.381264 3.605256 1.258103
1.525716 2.017336 2.435835
1.250187 1.811960 1.122779
1.473294 2.422558 1.670242
1.908157 3.002183 2.223226
2.167902 1.249793 1.690028
1.340942 1.722309 1.341314
1.331899 1.655757 1.612251

Journal of Research in Biology (2014) 4(7): 1475-1486 1479

 Gaikwad and Kamble, 2014

Figure 5. Growth rate noted during the Figure 6. Oxygen consumption noted during the
investigation period. investigation period.

Table 4. Estimated BCI for experimental group individuals.

Body Condition Index of experimental group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
1.717426 2.009174 1.704364
1.680832 0.947290 1.321680
1.931249 0.934551 2.641986
1.198415 4.990991 1.484685
2.906365 4.751566 1.762833
1.241548 2.089872 1.910265
1.919226 4.605892 3.580756
1.888711 2.520781 3.228804
3.062775 1.950860 3.003290
1.959476 2.877358 3.398894
Average values obtained for SCI with the help of groups was described in the Table 7 and 8.
Kruskal Wallis test revealed significant differences Average differences in the question for control
(P < 0.05) during the investigation period. Maximum and experimental groups were very highly significant
SCI was achieved by P. corrugata, while least was (P < 0.001). The differences were highest oscillating
counted for L. corrianus. L. marginalis showed moderate around doubled for L. marginalis while L. corrianus and
SCI while comparing both the groups (Figure 8). P. corrugata were noted with moderate differences
Meat Yield Index - Assessment of Meat Yield (Figure 9).
Index of the reared molluscs as mean in case of both the

Table 5. SCI observed during the investigation for control

group individuals
SCI of Control group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
77.71001 87.77729 83.86395
72.19069 81.12000 80.83522
87.13850 80.59058 85.80750
90.11091 87.66959 85.02491
87.36543 87.62167 86.88411
42.23457 85.68455 84.98980
86.58500 83.81993 85.75603
79.53250 86.47710 86.02370
84.37946 83.86895 85.23634
88.12045 83.73996 85.35105

1480 Journal of Research in Biology (2014) 4(7): 1475-1486

Gaikwad and Kamble, 2014

Figure 7. BCI achieved by comparing molluscan Figure 8. Shell Component Index achieved by
individuals during the investigation comparing molluscan species during the investigation.

et al., 2005 and Gibbs et al., 1991). In the environment

Figure 9. MYI achieved by the compared molluscan
species during the investigation.
DISCUSSION
Drastically altering environmental conditions had
its strong influence on the growth and reproduction of
the molluscs (Bayne et al., 1983, Hawkins and Bayne,
1992, Griffiths et al.,1987 and Seed Suchanek, 1992).
Both biotic and abiotic parameters play a major role in
the overall development of the molluscs (Gascoigne
as per the nature of the habitat, the molluscs are evolved
to sustain with prolific growth and reproduction (Sahi,
2006). In our experimentation, we observed that
hydrological parameters have its significant impact on
the molluscan development as previously mentioned by
Yukihira et al., (2002) for P. margaritifera and
P. maximum. Oxygen was remarked as most essential
parameter for the survival and normal growth of the
individuals and tends to be more soluble in case of
running water. Ample amount of oxygen accelerates the
various physiological activities, which in turn enhances
the respiratory rate of the animals (Bayne, 1967). Control
group individuals showed slightly stunted respiratory
rate, which may be the result of less oxygen
concentration along with restricted physiological
activities as mentioned by Bayne and Thompson, (1970),
Gabbott, (1976) and Bayne et al., (1983). It was also

Table 6. SCI observed during the investigation for

experimental group individuals.
Shell Component Index of control group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
81.30651 77.55957 83.10370
79.88342 69.57480 81.34172
84.49993 63.11569 82.78805
86.56812 77.24327 82.63982
86.11364 77.29858 82.93289
86.24293 76.76349 82.23638
83.30588 76.22295 80.62310
82.46388 76.64173 81.88153
80.36785 84.74201 81.69309
81.41228 73.16038 80.54702

Journal of Research in Biology (2014) 4(7): 1475-1486 1481

 Gaikwad and Kamble, 2014

Table 7. MYI representing tissues build up in the compared

molluscan individuals.
Meat Yield Index of control group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
22.29 12.23 16.14
27.81 18.88 19.16
12.86 19.41 14.19
12.33 12.33 14.97
12.64 12.38 13.11
13.33 14.32 15.01
13.42 16.18 14.97
13.52 13.52 15.69
03.73 16.13 14.76
11.88 16.26 14.64

justified by the less production of faecal matter and
debris in case of control group organisms, whereas
experimental individuals produces tremendous amount of
faecal debris. Carbon dioxide revealed exactly similar
trend as that of oxygen, because excess organic activities
and physiological processes enhances CO2 concentration,
which was assured by the higher temperature and pH
concentration of the experimental groups as previously
put forth by Widdow (1973) for M. edulis species.
Above-mentioned abiotic and biotic conditions
had its impact over the biometric relationships of the
cultured molluscan species. Satisfactory growth of the
individual is a multitude of favourable biotic and abiotic
interactions. Experimental groups were accomplished
with such delightful interactions along with ample
amount of dietary, which provided maximum
opportunities for the growth of individuals. Hence,
showed almost doubled growth increment than that of
control group individuals. Whereas in case of control
group though they are provided with, satisfactory diet
due to less favourable biotic and abiotic conditions the
growth of the individuals, get retarded as proved by
Yoo et al., (1986) for P. fucata. These observations were
supported by the evaluated condition indices. Significant
differences among control and experimental group high
light the role of water current along with species habitat
specificity at the laboratory conditions. BCI and MYI
reaches to its maximum limit in case of experimental
individuals representing magnificent enhancement in the
tissue weight i.e. growth, which may be because of onset
of breeding season as mentioned by the Narasimham,
(1988) for the species A. rehombea. Control individuals
showed moderate growth, which may be the impact or
result of stressed body physiology. SCI was significantly

Table 8. Meat Yield Index representing tissues build up in the

compared molluscan individuals
Meat Yield Index of experimental group
Lamellidens corrianus (g) Lamellidens marginalis (g) Parreysia corrugata (g)
18.69 22.44 16.89
20.11 30.42 18.65
15.05 17.75 17.21
13.43 22.75 17.36
13.88 22.07 17.09
13.75 23.23 17.76
16.69 23.77 19.37
17.53 23.35 18.11
19.63 15.25 18.35
18.58 26.83 19.45

1482 Journal of Research in Biology (2014) 4(7): 1475-1486

Gaikwad and Kamble, 2014
altered than other condition indices, denoting
comparatively doubled Shell Component Index for
control individuals, whereas experimental group showed
downward pattern of SCI. Overall assessment of these
condition indices confirm maximum channelization or
transformation of energy for the tissue or gonadal
development in case of experimental groups.
A good condition value indicates accumulation
of nutrients reserve to accomplish successful
reproduction (Bligh and Dyar, 1984, Dare and Edwards,
1975 and Aldrich and Crowley, 1986). While comparing
among the individuals of analyzed species, L. corrianus
was found as most favourable individual to counteract
with the availed atmospheric conditions at the time of
rearing. It was noticed with richest growth rate and BCI
representing its dominancy in experimental conditions.
While in case of unfavourable atmospheric conditions its
growth and BCI may slightly be hampered, but remains
constant when compared with the other two species and
hence denoted its ideal suitability in composite
community structure. L. marginalis was noted as least
adapted species during the experimental period due to its
slower growth rate and BCI in case of both the groups,
which should be the reason to have its least population
and endangeredness in the natural ecosystems as stated
by Cataldo and Boltovskoy, (1998) for C. fluminea.
P. corrugata was evaluated as suitable species to rear in
natural and artificial environments as it denotes moderate
growth rate for provided experimental conditions.
However, due to its smaller size than other two reared
species the growth rate was comparatively similar as that
of the field conditions, which was previously described
by Mudigere and Seetharamaiah, (2009) for P. corrugata
and hence mentioned as well adapted form in the
composite community structure.
CONCLUSION
Water current has proved dominancy over the
controlled laboratory conditions, which confirms the
Journal of Research in Biology (2014) 4(7): 1475-1486
advantage of water current in maintenance of the aquatic
animals. In vitro treatment enhances health and
reproductive capacity of animals. Obtained results
showed magnificent increment in the regeneration
capacity of animals. The technique enlightens advance
bioscience practices in animal culture with significant
applications.
ACKNOWLEDGEMENT
The authors expressed their gratitude to Science
and Engineering Research Board, New Delhi for offering
the grants under Young Scientist Fast track Major
Research Project. We also expressed our gratitude
towards Head Department of Zoology, Shivaji
University, Kolhapur for infrastructural support in the
progress of work.
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 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Notes on the occurrence of Porpita porpita (Blue button)

from Pulicat Lagoon
Authors: ABSTRACT:
Journal of Research in Biology

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.

Corresponding author: Keywords:

Ravichandran Ramanibai Hydrozoans, Porpita porpita, Pulicat lagoon

Email Id: Article Citation:

Ravichandran Ramanibai, Sivalingam Govindan and Thamotharan Balakrishnan
Notes on the occurrence of Porpita porpita (Blue button) from Pulicat Lagoon
Journal of Research in Biology (2014) 4(7): 1487-1490

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.

1487-1490 | JRB | 2014 | Vol 4 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Ramanibai et al., 2014

INTRODUCTION reproduction P. porpita is hard, slightly convex disc,

Blue buttons were seen in Pulicat lagoon during golden brown, gas-filled float in the centre, blue, yellow
the post-monsoon season of the year 2013. Attracted by hydroids, which look like tentacles having stinging cells
their fabulous blue colour with various sizes while called nematocysts in it. The gas filled centre helps them
isolated first and then identified them as Porpita porpita, to float on the surface (Pandya et al., 2013). In the
(Fredrick and Ravichandran, 2010). Blue buttons come present study, we have analysed the morphological
under class Hydrozoa and possess colony polyps which features along with the brief descriptions of P. porpita.
act as their defence organ (Pandya et al., 2013). Systematics of Porpita porpita
In general few jellyfishes are considered as
Kingdom Animalia
primitive organisms belonging to the phylum Cnidaria
Sub kingdom Eumetazoa
(Raskoff, 2003, Barzansky et al., 1975). They are known
Phylum Cnidaria
for producing toxic substances which leads to skin
Sub phylum Medusozoa
irritation (Garcia-Barrientos et al., 2009). At the time of
Class Hydrozoa
our collection, we felt mild skin irritation. Very few
Order Anthomedusae
informations about blue buttons are available in this
Family Porpitidae
lagoon.
Genus Porpita
Porpita porpita the blue button, is mainly found
Species porpita
on the surface of the sea. Pandya et al. (2013) reported
on customary sea currents and in the progress of air
P. porpita shows its movement. The extracted blue MATERIALS AND METHODS
carotene proteins of Porpita species are very sensitive to Collection of blue buttons
conditions of temperature and salt concentration, The organisms were observed and identified
exhibiting reversible hypsochromic shifts in absorption during a regular field visit at Pulicat lagoon in
maxima with increasing temperature and decreasing salt Tamilnadu. Blue buttons were collected during the
concentration (Herring, 1971). Deidun (2010) reported month of December 2013 from the Pulicat lagoon, with
the epipelagic zone of oceanic surroundings which carry the help of hand net (Figure 1) and transported to the
P. porpita to shore by the waves; polymorphic settlement laboratory and preserved in 4% formalin. The organisms
awakening different personage zooids and each were photographed with Sony Cyber shot camera.
specialized for a different function such as eating and

Figure 1 Collection of blue button

1488 Journal of Research in Biology (2014) 4(7): 1487-1490

Ramanibai et al., 2014

Figure 2 Satellite map of Pulicat (Source: Google Earth)

The Pulicat lagoon (Figure 2) extend between RESULTS AND DISCUSSION

13 66 N 8023E and join with the back water area of P. porpita watching was done throughout
Bay of Bengal. It is the second largest brackish water Dec-2013 at Pulicat lagoon. They were observed in large
2
lagoon having an area of approximately 600 Km . The numbers and one of the reasons for their amount in plane
samples were collected from five different locations maybe due to their reproductive period, ever since
from Pulicat lagoon. different colony of miniature and huge sizes were
Based on the morphological features the observed in all locations (Figure 3A).
collected organisms were identified as Porpita porpita, Figure 3B shows the P. porpita photograph
(Fredrick and Ravichandran, 2010). collected on the sites of Pulicat lagoon. Dorsal and
Ventral view of P. porpita were shown in Figure 3C and

Figure 3 Photographs of Porpita porpita

Journal of Research in Biology (2014) 4(7): 1487-1490 1489

 Ramanibai et al., 2014

3D which showed the presence of a disc in the middle of
P. porpita; this helps the organism to float in the water
and is golden brown in colour measuring around 1.5
inches width. The mouth present below the float is to
engulf prey along with water and its ingredients. The
second part is known as hydroid colony which posses
bright colour tentacles. With the help of tentacles and the
float it moves along and across the water body. One can
observe and admire blue buttons for their beautiful
colour but better to avoid its contact which causes skin
irritation; above all it is one of the good suppliers of
bioactive compounds from the sea.
CONCLUSION
The suspended hydrozoan contains nematocyst in
their tentacles that hideaway biochemical compounds.
We suggest that it is a good source to work in the field of
bioactive compounds active against human pathogens.
Garcia-Barrientos R, Ramos-Puebla A, Hernandez-
Samano A, Minor-Perez H and Legarreta GI. 2009.
Jellyfish (Stomolophus meleagris) tentacles proteins and
their Proteolysis Endogenous World Academy of
Science, Engineering and Technology. 3(6): 618-620.
Herring PJ. 1971. Stability of the blue pigment of
Velella and Porpita (Coelenterata: Siphonophora),
Comp. Biochem. Physiol. Part B: Comp. Biochem., 39
(4): 10391043.
Pandya KM, Parikh KV, Dave CS and Mankodi PC.
2013. Occurrence of Hydrozoans from the saurashtra
Coast of Gujarat, India. Res. J. Mar. Sci., 1(4):1-3.
Raskoff KA. Sommer FA. Hamner WM and Cross
KM. 2003. Collection and culture techniques for
gelatinous zooplankton. Biol. Bull., 204:6880.

ACKNOWLEDGEMENT
We thank DST (SERB), New Delhi for their
funding support.

REFERENCES
Barzansky BH, Lenhoff M and Bode H. 1975. Hydra
mesoglea: similarity of its amino acid and neutral sugar
composition to that of vertebrate basal lamina. Comp.
Biochem. Physiol. Part B: Comparative Biochemistry. 50
(3): 419424.

Deidun A. 2010. Notes on the recent occurrence of Submit your articles online at www.jresearchbiology.com
uncommon Pelagic Jellyfish species in Maltese Advantages
Coastal waters. Naturalista Sicil. S. (3-4): 375-384. Easy online submission
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

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 Journal of Research in Biology ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

Original Research

Insecticide induced changes in haemolymph protein profiles of

Spodoptera frugiperda (F) (Lepidoptera:Noctuidae)
Authors: ABSTRACT:
Journal of Research in Biology

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

Corresponding author: Keywords:

Ayub Khan Spodoptera frugiperda, insecticides, haemolymph proteins, induced changes

Email Id: Article Citation:

Quincy Bart, Jenna Indarsingh, Hamraji Jugmohan and Ayub Khan
Insecticide induced changes in haemolymph protein profiles of Spodoptera frugiperda (F)
(Lepidoptera:Noctuidae)
Journal of Research in Biology (2014) 4(7): 1491-1497

Web Address:
http://jresearchbiology.com/
documents/RA0486.pdf
Dates:
Received: 18 Oct 2014 Accepted: 25 Oct 2014 Published: 12 Nov 2014
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.

1491-1497 | JRB | 2014 | Vol 4 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
The fall armyworm, Spodoptera frugiperda (F)
(Lepidoptera:Noctuidae) is a serious pest of corn,
sorghum and several other grasses in the Neotropics.
S. frugiperda is an avid flyer which can be found
between south-eastern United States to Argentina. A
light coloured inverted Y marking is found on the front
of its head and its raised, dark shiny spots that occur
dorsally on the body distinguishes it from other
armyworm species (Sparks, 1979). This pest can cause
significant reduction in crop yield and as much as 50%
losses in corn in Brazil have been documented (Cruz
et al., 1999; Carvalho et al., 2010). Synthetic insecticides
are the most commonly used form of control for this pest
with a wide variety being utilized (Tavares et al., 2010).
Associated with the widespread, frequent use of
synthetic insecticides is the development resistance and
S. frugiperda has been recorded as resistant to several
insecticide groups including organophosphates,
carbamates and pyrethroids (Yu, 1991).
The effect of synthetic insecticides on the
haemolymph proteins of S. frugiperda has not been
previously studied apart from those involving
Bacillus thuringiensis (Valdez-Lira et al., 2012). The
purpose of this study was to determine the LC50 and LT50
for nine synthetic insecticides against S. frugiperda and
to determine insecticide-induced changes in haemolymph
proteins in S. frugiperda with the aim to better
understand the physiological mechanisms for the
insecticide induced protein changes.
MATERIALS AND METHODS
Insect culture
An initial stock of S. frugiperda larvae was
collected on corn (Zea mays) from the University of the
West Indies Field Station, Trinidad. Larvae were taken
back to the laboratory and reared on corn leaves until
adult emergence. Adult moths were placed in an insect
sleeve cage (30 cm x 30 cm x 30 cm) covered with a fine
1492
Bart et al., 2014
mesh cloth. Food was supplied via a wax paper strip
(2cm x 15cm) coated with honey that was mounted to the
top of the cage allowing it to hang down. A large
bouquet of fresh corn leaves was placed in a glass vial
with a cotton wool plug around the rim of the vial to
prevent moths from drowning. The bouquet was replaced
after the old one had wilted. Cages were checked daily
for dead moths and oviposition. Eggs were collected
daily from the corn leaves and placed in test tubes for
larval emergence. Neonate larvae were transferred to
mesh covered plastic containers that had a fresh supply
of corn leaves. On the third day after hatching, larvae
were placed individually in test tubes with the aid of a
small artists brush (No. 3/0). Neonate larvae were fed
with corn leaves until 3rd instar (approximately 10 days)
and then used in insecticide bioassays.
Insecticide bioassay
Nine commercial insecticides with different
active ingredients were obtained from the University of
the West Indies Field Station, Trinidad for use in
insecticide bioassays. These insecticides were:
Abamectin (abamectin), Boxer 30EC (etofenprox),
Bright 25EC (carbosulfan), Fastac 5EC
(-cypermethrin), Flip 800DF (fipronil), Karate 5EC
(-cyhalothrin), Malathion 50 EC (malathion), Neem X
0.4EC (azadirachtin) and Supertak 10EC
( cypermethrin).
A corn (Zea mays) leaf dip bioassay was used for
each population of S. frugiperda. Each bioassay
comprised five concentrations for each insecticide
(4%, 0.4%, 0.04%, 0.004%, and 0.0004%) and a control.
Young corn leaves were cut into 7 cm x 7 cm segments.
Each segment was dipped into their respective
insecticide concentration solution for 30s, held vertically
to permit excess solution to drip off and then placed on
paper towel to air dry for 30 minutes. Each treated leaf
segment was placed in a 9 cm petri dish with moistened
filter paper lining the bottom. S. frugiperda 3rd instar
larvae were starved for 5 h prior to being placed on
Journal of Research in Biology (2014) 4(7): 1491-1497
Bart et al., 2014
leaves of each petri dish. Five replicates were maintained
for the treatment of each insecticide. The control
comprised of leaves treated only with distilled water.
Petri dish lids were covered with fine gauze to allow for
ventilation and prevent fumigant action of the
insecticides. Each petri dish was sealed around the edge
with clear tape to prevent escape of larvae. Larval
mortality was assessed every 2 h for 24 h. Larvae
unresponsive to a gentle prod with a toothpick within 5s
were regarded as dead. Data were corrected for control
mortality using Abbotts (1925) formula. Mortality data
were subjected to probit analysis using EPA Probit
program Version 1.4.
Protein bioassay
Based on LC50 values obtained for each
th
insecticide, 4 instar S. frugiperda larvae were subjected
to sub-lethal doses on each insecticide for 24 h. Live
larvae exposed to a particular insecticide after 24 h were
collected and crushed in an Eppendorf tube, centrifuged
and the supernatant collected and analyzed for total
protein content using Lowry et al., (1951) method and
also separated using Polyacrylamide Gel Electrophoresis
(PAGE). 7.5% separating gel was prepared from 30%
acrylamide-BIS, 10% ammonium persulfate and
tetramethylethylenediamine (TEMED). The mixture was
then swirled to ensure thorough mixing. The solution
TM
was pipetted into Gel Wrap Gasket maker and left at
room temperature for 45 minutes to polymerize. A 4%
stacking gel was prepared using 30% acrylamide-BIS
with 10% ammonium persulfate and TEMED and left at
room temperature for 45 minutes to polymerize and then
refrigerated at 4C overnight.
Fourth instar S. frugiperda larvae were exposed
to the lowest concentration (0.0004%) of each insecticide
for 24 h before protein extraction took place. Larvae
were crushed to a smooth texture in micro-centrifuge
tubes containing 100 l of deionized water. All samples
were thoroughly mixed for 5s with the aid of a Vortex
Genie 2 machine and centrifuged at 10,000 rpm for two
Journal of Research in Biology (2014) 4(7): 1491-1497
minutes, then gradually increased to 14,000 rpm for 2
minutes. Each sample (35l) was mixed separately with
35l sample buffer (1000l of 50% glycerol, 800l of
running buffer and 200l of 0.1% bromophenol blue)
and 30l placed in separate lanes together with 20 l
each of the following standards: alpha-lactalbumin
(MW= 14.2kDa), carbonic anhydrase (29.0kDa), bovine
erythrocytes (45.0kDa), albumin from chicken egg white
(66.0kDa) and albumin from bovine serum (66.43kDa).
The samples were allowed to run for 1 h at 180V after
which plates were washed with deionized water to
remove the gels. Gels were placed into 150cm Pyrex
petri dishes with 100ml of Coomassie blue stain on a
Labnet Rocker 25 for 45 minutes to ensure proper and
even stain penetration. Gels were then de-stained with
30% methanol: 10% acetic acid for 1h and then rinsed
with deionized water (Labban et al., 2012). Bands on the
gel were then observed under a fluorescent light and
scanned using a UVP Gel Doc-It 300 imaging system
and then analyzed using VisionWorksLS Analysis
Software.
RESULTS AND DISCUSSION
There were two distinct groups of insecticides
based on toxicity (LC50) to 3rd instar larvae of
S. frugiperda (Table 1). The first group comprised
Boxer, Malathion, Flip, Bright and Supertak
among which there were no significant differences
(P>0.05). The second group comprised Fastac,
Neem-X, Abamectin and Karate among which there
were no significant differences (P>0.05) but were
significantly different (P>0.05) from all members of the
first group. Bright 30EC was the most toxic (LC50 =
0.0006g/g) while Fastac 5EC was the least toxic (LC50
= 0.6046g/g) among all the insecticides evaluated. The
active ingredient in both Fastac 5EC and
Supertak 10EC is -cypermethrin, however their LC50
values differed significantly (P>0.05) with
Supertak 10EC being approximately 62 times more
1493
 Bart et al., 2014

Table 1. Toxicity of insecticides to 3rd instar Spodoptera frugiperda larvae

Insecticide Probit line LC50 mg/ml (95% CI)* S.E. 2

Boxer 30EC Y = 0.78x + 7.21 0.0014 (0.0002, 0.0089)a 2.55 1.94

Malathion 50EC Y = 0.88x + 6.72 0.0111 (0.0023, 0.0549)ad 2.26 1.38
Flip 800DF Y = 1.32x + 8.37 0.0028 (0.0008, 0.0098)a 1.91 0.43
Bright 25EC Y = 0.60x + 6.95 0.0006 (0.0001, 0.0064)a 3.44 0.27
Supertak 10EC Y = 0.43x + 5.87 0.0098 (0.0007, 0.1421)ac 3.90 1.18
Fastac 5EC Y = 0.55x + 5.12 0.6046 (0.0525, 6.9626)b 3.48 0.57
Neem-X 0.4EC Y = 0.61x + 5.42 0.2052 (0.0255, 1.6487)b 2.89 0.22
Abamectin Y = 0.46x + 5.17 0.4192 (0.0259, 6.7786)b 4.14 0.01
Karate 5EC Y = 0.71x + 2.50 0.1339 (0.0230, 0.7781)bcd 0.01 0.21

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
(Table 1) and apart from the doubling in concentration,
may have been as a result of other components
(adjuvants) in the formulation. Mesnage et al., (2014)
conducted studies on other pesticides using human cell
lines also concluded that adjuvants listed as inert
ingredients in pesticides can amplify the toxicity to 1000
-fold.
Among the insecticides tested, Flip 800DF took
the shortest time to cause 50% mortality (LT 50 = 2.05 h),
while Abamectin took the longest (LT 50 = 18.18 h)
which was significantly different (P<0.05) from all the
other insecticides tested (Table 2). Abamectin also took
the longest to achieve 50% mortality when used against
Spodoptera litura in Pakistan (Ahmad et al., 2005).
Although Bright 25EC was the most toxic insecticide
tested (LC50 = 0.0006mg/ml), the 50% lethal time (LT 50
= 6.63 h) was high, indicating that it would take a
population of S. frugiperda larvae approximately 6.63 h
to achieve 50% mortality at a concentration of
0.0006mg/ml (Tables 1 and 2). However, Flip 800 DF
(fipronil) which had a LC50 of 0.0028mg/ml was not
significantly different (P>0.05) from the LC50 of Bright
25EC but had a LT50 = 2.05h (Table 2).
The total haemolymph protein content of
larvae treated with seven of the nine insecticides was
significantly lower (P<0.05) than that of the control,
while Bright (632.79g/ml) and Abamectin

Table 2. Lethal time (LT50) of insecticides to 3rd instar Spodoptera frugiperda larvae

Insecticide Probit line LT50 (h) (95% CI)* S.E. 2

Boxer 30EC Y = 3.36x + 2.82 4.45 (3.20, 6.20)a 1.18 0.95

Malathion 50EC Y = 5.42x + 2.33 3.12 (2.28, 4.27)a 1.17 0.30
Flip 800DF Y = 3.77x + 3.83 2.05 (1.28, 3.27)ac 0.17 0.17
Bright 25EC Y = 2.01x + 3.35 6.63 (3.55, 12.40)ad 1.38 0.65
Supertak 10EC Y = 2.19x + 3.67 4.04 (2.34, 6.98)a 1.32 1.16
Fastac 5EC Y = 2.59x + 3.10 5.40 (3.39, 8.62)ad 1.27 0.56
Neem-X 0.4EC Y = 1.27x + 4.00 6.12 (2.60, 14.41)a 1.55 0.69
Abamectin Y = 2.26x + 2.15 18.18 (10.59, 31.20)b 1.32 0.38
Karate 5EC Y = 1.61x + 3.94 4.59 (2.19, 9.64)a 1.46 1.02

Values followed by the same letter are not significantly different from each other based
on Tukey-Kramer Multiple comparisons test

1494 Journal of Research in Biology (2014) 4(7): 1491-1497

Bart et al., 2014

Figure 1 Electrophoresis Gel 1 of haemolymph proteins from

Spodoptera frugiperda exposed to different insecticides

Figure 2 Electrophoresis Gel 2 of haemolymph proteins from

Spodoptera frugiperda exposed to different insecticides

(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

from 147.46g/ml (Karate 5EC) to 632.79g/ml Treatment Total haemolymph protein

(Bright 25EC) (Table 3). Both (Nath et al., 1997 and content Mean SE (g /ml)*
Control 393.70 2.51a
Usmani and Knowles, 2001) reported that the total Karate 5EC 147.46 3.86 b
protein content in larval haemolymph of insects Boxer 30EC 244.10 1.97 c
Malathion 50EC 303.51 4.21 d
decreased significantly compared to the control when Fastac 5EC 230.50 1.67 c
exposed to organophosphate and pyrethroid insecticides. Flip 800DF 186.83 1.24 e
Bright 25EC 632.79 2.23 f
A similar trend was observed in the present study with Supertak 10EC 352.19 2.62 g
larvae of S. frugiperda. This haemolymph protein decline Neem-X 0.4EC 286.33 3.11 h
Abamectin 617.04 1.97 i
may be as a result of increased protein breakdown which *Values followed by the same letter are not significantly
may be required to detoxify the components of the different from each other based on Tukey test (P>0.05)

Journal of Research in Biology (2014) 4(7): 1491-1497 1495

 Bart et al., 2014

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
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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.
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