CURRENT STATUS AND CHALLENGES FOR CONSERVATION AND SUSTAINABLE USE OF BIODIVERSITY

336 Current status and challenges for conservation and sustainable use of biodiversity Discussion bug; D. cingulatus causes developmental abnormalities such as prolongation of A typical heteropteran insect as Dysdercus nymphal periods, ecdysial stasis, formation cingulatus, the life cycle consists of egg, of adultoids and adults with varied degrees nymph and adult. Eggs are oval in shape and of wing deformities. The studies of seed and the first instar nymph emerges from it in root extracts of medicinal plant, about 5-6 days. There are five different Ashwagandha to prepupae of S.litura and nymphal instars having the duration of 2-3, P.ricini suggest that the similar results 3-4, 4-5 and 4-5 days respectively. The total produced by the administration of JHAs and life span of the insect extends to 35-40 days. hence mimic their action by way of The above findings totally agree with the interfering with the neuroendocrine system earlier findings of Jalaja and Prabhu (1977). (Sandeep Kumar Gaur et al., 2019). The present study on normal fifth instar Changing electrophoretic protein bands nymph of Dysdercus cingulatus was to observed in treated nymphs are quite confirm whether the Andrographis convincing. Treated insects indicate that the paniculata plant extract has got any JH like A.paniculata extract elicited some effect on activity. It has been shown by Prabhu (1977) protein synthesis in D.cingulatus nymphs. A that the juvenalizing effect of plant extracts significant increase in the fat body protein are not mediated through corpus allatum concentration of supernumerary nymphs (CA) and the effects are likely to be due to indicated that the A.paniculata extract the JH mimic contained in the plant extract influence protein synthesis in D. cingulatus. rather due to synergism (Staal, 1972). This phytochemical biomolecule may act as a Fat body electrophoretic pattern showed special repressor agent which inhibits emergence of new protein bands in the morphogenetic processes at any life stage intermediate that are absent in the normal between the fertilized egg and fully adult. This suggests that fat body is the differentiated adult form (Slama et al., centre of synthesizing a number of proteins 1974). secreted into the haemolymph. The fat body tissue plays a key role in insect juvenile Hebsy Bai and George Koshy (2004) hormone synthesis and they are later reported that the juvenomimetic activity of released in to the haemolymph. The ethanol extracts of fresh leaves and seeds of transcriptome analysis of the Aedes aegypti Thevetia neriifolia on red cotton bug, related to vitellogenesis represent the fat Dysdercus cingulatus showed increased body transcriptome at 24hr post blood meal larval mortality, emergence of malformed that encode vitellogenesis. These data in adults and reduced fecundity of the bugs. agreement with increase of vitellogenic Saradamma (1989) also observed a similar protein expression in mosquitoes (Kokoza et effect on the fifth instar nymphs of D. al., 2001). It has been already established cingulatus treated with ether and benzene that vitellogenin synthesized by the fat extracts of T. neriifolia. Pandey and Tiwari bodies in response to a blood meal, secreted (2011) reported that different concentrations in to the hemolymph that accumulates in of Neem based insecticides on red cotton ovaries, where they are deposited in the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.331-338 © Principal, Sree Narayana College, Kollam, Kerala, India

Juvenile Hormone Mimic from Medicinal Plant Andrographis Paniculata (Burm F.) 337 oocytes during development (Raikhel et al., Antifeedant, larvicidal and pupicidal 2002, Riberio 2003). Proteome analysis of silkworm (Bombyx mori L.) fat bodies activities of Atlantia monophylla (L) Correa reported that chaperonin Cpn60 and other heat shock proteins are also observed in the against Helicoverpa armigera Hubner silk glands, which actively synthesize a large amount of proteins (Hideyuki Kajiwara et (Lepidoptera: Noctuidae). al., 2009). Chemosphere.,75:355-359. Conclusion Bede, J.C., Goodman, W.G. and Tobe, S.S. 1999. Developmental distribution of insect Juvenile hormone III in the sedge, Cyperus iria L. Phytochemistry, 52: 1269-1274. Juvenile hormone is of crucial importance in Bede, J.C. and Tobe, S.S. 2001. Biosynthetic the regulation of development, pathway of insect JH III in the cell metamorphosis and reproduction in insects. suspension cultures of the sedge Cyperus Juvenoids or JH analogues eliciting JH iria. Plant physiology, 127: 584-593. hormonal effects that disrupt those vital functions of the insect which are controlled Chieka Minakuchi and Lynn M. Riddiford, or influenced by JH. The plant kingdom can 2006. Insect juvenile hormone action as a be considered as a rich source of chemicals potential target of pest management. J. with the potentials for development of Pestic. Sci., 31(2): 77- 84. successful pest control agents. These potent chemical agents known as allelochemicals Gilbert, L.I., Granger, N.A. and Roe, R.M. operate between diverse species of plant and 2000. The juvenile hormones: historical facts animal communities. The use of plant and speculation on future research allelochemicals in pest management program directions. Insect Biochem and Mol.Biol., 30 has considerable intuitive appeal as it (8-9): 617-644. satisfies the need to protect specific crops while avoiding damage to non-target Goodman, W. G. and Granger, N. A. 2005. organisms. The plant derived products are Comprehensive molecular Insect Science. considered to be attractive in pest control Vol.3, Gilbert L.I, Iatrou K, Gill S.S, Eds. programs as they are species specific, easily Elsevier: Oxford, 319-408. biodegradable and nontoxic to humans and are environmentally compatible. Hebsy Bai and George Koshy, 2004. Juvenomimetic activity of extracts of References Thevetia neriifolia Juss. to Dysdercus cingulatus F.(Hemiptera: Pyrrhocoreidae). J. Tropical Agriculture., 42(1-2): 45-47. Alvarez, C.O., Neske, A., Popich, S. and Hideyuki kajiwara, Astue Imamaki, Bardon, A. 2007. Toxicity effect of Masatoshi Nakamura, Kazuei Mita, Qingyu annonaceous acetogenins from Annona xia and Masami Ishizaka, 2009. Proteome cherimolia (Magnoliales: Annonaceae) on analysis of silkworm fat body. J. Spodoptera frugiperda (Lepidoptera: Electrophoresis., 53(2 ): 19-26. Noctuidae). J. Pest. Sci., 80: 63-67. Jagajothi, A. and Martin, P. 2010. Efficacy Baskar, K.S., Kingsley, E.S., Vendan of Andrographolide on pupal-adult Paulraj, M.G. and Ignacimuthu, S. 2009. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.331-338 © Principal, Sree Narayana College, Kollam, Kerala, India

338 Current status and challenges for conservation and sustainable use of biodiversity transformation of Corcyra cephalonica Ahmed, A., Dittmer, N. and Attardo, G. stainton. J. of Biopesticides., 3(2): 508-510. 2002. Molecular biology of mosquito vitellogenesis: from basic studies to genetic Jalaja, M. and Prabhu, V.K.K. 1977. engineering of anti-pathogen immunity. Endocrine control of vitellogenesis in the red Insect Biochem. and Mol. Biol., 32: 1275- cotton bug, Dysdercus cingulatus Fabr. 1286. (Heteroptera: Pyrrhocoridae) Entomon, 2: 17-19. Riberio, J. M. 2003. A catalogue of Anopheles gambiae transcripts significantly Jeyasankar, A. N., Raja and Ignacimuthu, S. more or less expressed following a blood 2010. Antifeedant and growth inhibitory meal. Insect Biochem. and Mol. Biol., 33: activities of Syzygium lineare Wall 865-882. (Myrtaceae) against Spodoptera litura Fab. (Lepidoptera: Noctuidae). Current research. Sandeep Kumar Gaur and Krishnakumar, J.of Biologi. Sci., 2(3): 173-177. 2019. A comparative bioefficacy of seed and root extracts of a medicinal plant, Withania Kokoza, V. A., Martin, D., Mienaltowski, M. somnifera when administered to prepupae of J., Ahmed, A., Morton, C. M. and Raikhel, lepidopteran insects Spodoptera litura and A. S. 2001. Transcriptional regulation of the Pericallia ricini. The journal of Basic and mosquito vitellogenin gene via a blood meal- Applied Zoology, 80 (1): 37. triggered cascade. Gene., 274: 47-65. Saradamma, K. 1989. Biological activity of Malarvannan, S., Giridharan, S., Sekar, S., different plant extracts with particular Prabavathy, V.R. and Nair, S. 2008. reference to their insecticidal, hormonal and Bioefficacy of crude and fraction of Argemone antifeeding actions. Ph. D Thesis. Kerala maxicana against tobacco caterpillar, Agricultural University, Thrissur, p. 252. Spodoptera litura (Fab.) (Noctuidae: Lepidoptera). J. Biopest. Pp: 55-62. Slama, K., Romanuk, N. and Sorm, F. 1974. Insect hormone and Bioanalogues. Springer, Muraleedharan, D. 1988. Insect anti- New York. hormones from plants and their pest control prospects. Phytophaga 2: 153-157. Slama, K. and Williams. C.M. 1966. Paper factor as an inhibitor of embryonic Pandey, J.P. and Tiwari, R.K. 2011. Neem development of European bug, Pyrrhocoris based insecticides interaction with apterus, Nature., 210: 329-330. development and fecundity of red cotton bug, Dysdercus cingulatus Fab. Seok Hee Lee, Hyun woo oh, Ying fang International J. of Agricultural Research., 6 (2015). Identification of plant compounds (4): 335- 346. that disrupt the insect JH receptor complex. PNAS 112 (6) 1733-1738. Prabhu, V.K.K. 1977. Insect JH in plants and the problem of host specification in insects. ToyomiKotaki, Testsuro shinada, Kanako Pp. 69-74. In Insect and host specificity. (ed. kaihara, Yasufumi ohfune and Hideharu Ananthakrishnan T.N) Macmillan. India. Numata, 2009. Structural determination of a new JH from a Heteropteran insect. Organic Raikhel, A. S., Kokoza, V. A., Zhu, J., letters, 11(22):5234-5237 Martin, D., Wang, S. F., Li C, Sun, G., Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.331-338 © Principal, Sree Narayana College, Kollam, Kerala, India

339 Current status and challenges for conservation and sustainable use of biodiversity ISBN 978-93-5396-871-7 CHAPTER 49 SCREENING OF VARIOUS PLANT EXTRACTS AS BIOPESTICIDES AGAINST RHYNCHOPHORUS FERRUGINEUS AND ITS ENZYMATIC ALTERATIONS. Chandana J S* and Ajitha V S P.G. and Research Department of Zoology, University College, Thiruvananthapuram, Kerala, India *Correspondence E-mail: [email protected] ABSTRACT Experiments were carried out to evaluate the insecticidal potential of some medicinal plants such as Ocimum sanctum, Saraca asoka and Asparagus racemosus etc, against the serious coconut pest Rhynchophorus ferrugineus (Red Palm Weevil). Plant extracts were prepared using solvents like acetone, ethanol and water. Different doses of these plant extracts were applied to the fourth instar larvae of R.ferrugineus. The results showed the plant extracts had direct changes on red palm weevil larvae and among them ethanol extracts of A.racemosus showed significant morphological changes and cause enzymatic alterations in treated insects and the effect was dose dependent. LD 50 value was assessed using probit analysis. The effect of the plant A.racemosus was found to be most significant causing highest mortality compared to other plants and sublethal concentration of this extract elicited significant changes in the enzymatic parameters in treated insects compared to control, sometime it may be lead to several physiological disruption in insects body. Key words: Rhynchophorus ferrugineus, Biopesticide, Biochemicals, Asparagus racemosus. Introduction understanding of plant allele chemical mechanisms of activity offer new prospects The red palm weevil (RPW), Rhynchophorus for using the substances in crop protection. The present study highlights the potential of ferrugineus Olivier (Coleoptera: the ethanol extract of Asparagus racemoses against the insect pest of Coconut palm Curclionidae) is a major pest on coconut Rhynchophorus ferrugineus. palms (Cocos nucifera).In the context of Digestion is a process in which ingested macromolecules by insects break down to agricultural pest management, botanical smaller ones to be absorbable via epithelial cells of midgut. Several enzymes based on pesticides are best suited for use in organic food materials have critical roles in this process. Any disruption in their activity food production in industrialized countries disables insects to provide their nutrients for biological requirements. Several studies but can play a much greater role in the demonstrated the effect of botanical insecticides on feeding parameters of insects production and post harvest protection of food in developing countries. Botanicals have been in use for a long time for pest control. The compounds offer many environmental advantages. However, their uses during the 20th century have been rather marginal compared with other bio-control methods of pests and pathogens. Improvement in the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

340 Current status and challenges for conservation and sustainable use of biodiversity by demonstrating food consumption [CR = processes amongst others (Neurath, 1984). I/DT], approximate digestibility of consumed The proteases are classified according to their food [%AD = 100(I–F)/I], efficiency of mechanism of catalysis: (1) serine proteases; converting the ingested food to body (2) cysteine proteases; (3) aspartic proteases, substance [%ECI = 100 G/I], efficiency of and (4) metalloproteases (Bode and Huber, converting digested food to body substance 1992). For an efficient management of pest [%ECD = 100G/(I–F)] and consumption control through proteinase inhibitor index [CI = I/W] (Shekari et al., 2008). The transgenes, it is imperative to know the type fact underlying these changes is inhibitory of enzymes present in the gut of insects and effects of botanical insects on digestive pests. The two major protease classes in the enzymes (Zibaee and Bandani, 2010a). digestive systems of phytophagous insects are the serine and cysteine proteases (Haq et Amylase of insects al., 2004). Murdock et al. (1987) carried out an elaborate study of the midgut enzymes of Agricultural crops are facing several losses various pests belonging to Coleoptera, while due to pest. Pesticides, bio pestcides, plant Srinivasan et al.(2008) have reported on the defense compounds are different ways to midgut enzymes of various pests belonging protect the agricultural crop losses. The to Lepidoptera. Serine proteases are known to enzyme inhibitors act on insect gut digestive dominate the larval gut environment and α -amylases and proteases, which play a key contribute to about 95 % of the total role in the digestion of plant starch and digestive activity in Lepidoptera, whereas proteins. Insects like weevils are highly the Coleopteran species have a wider range dependent on starch for their energy supply. of dominant gut proteases. In this review, we summarize the effects of pesticides, need of bio pesticides, different Proteases have a crucial role in food types and sources of plant α - amylase digestion by insects. Different types of inhibitors their interaction with insect α - proteases are necessary to do this because the amylases and subsequent insect mortality. amino acid residues vary along the peptide chain (Terra and Ferriera, 2005). There are Plant defenses against insect herbivores are three subclasses of proteinases involved in mediated, in part, by enzymes that impair digestion classified according to their active digestive processes in the insect gut. Little is site group (and hence by their mechanism): known about the evolutionary origins of serine, cysteine, and aspartic proteinases. The these enzymes, their distribution in the plant oligopeptides resulting from proteinase kingdom, or the mechanisms by which they action are attacked from the N-terminal end act in the protease- rich environment of the by aminopeptidases and from the C-terminal animal digestive tract (Chen et al., 2007).The end by carboxypeptidases. Studies by transgenic expression of insecticidal proteins Johnson et al. (1990), Senthil-Nathan et al. such as α-amylase and protease inhibitors is (2006) and Zibaee and Bandani (2010a) also being evaluated as a potential protective inferred that Botanical insecticides may strategy against insects (Schuler et al., 1998). interfere with the production of certain types of proteases and disable them to digest Proteases of insects ingested proteins. The proteases are a major group of hydrolytic In the majority of enzymes, the presence of enzymes in insects and are involved in the plant extract decreased the value of Vmax digestive processes, proenzyme activation, and increased Km. Since Km has an inverse liberation of physiologically active peptides, complement activation, and inflammation Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

Screening of various plant extracts as biopesticides against rhynchophorus ferrugineus … 341 relationship with the substrate concentration material was reduced to a viscous dark green required to saturate the active sites of the residue and the crude extracts were further enzyme, this indicates decreased enzyme evaporated. affinity for the substrate (Wilson and Goulding, 1986). In other words, Km is the Bioactivity of plant extracts on measurement of the stability of the enzyme- R.ferrugineus substrate complex and a high Km would indicate weak binding while a low Km would Newly moulted fourth instar larvae of indicate strong binding (Stryer, 1995). In this R.ferrugineus were introduced in separate type of inhibition, plant extracts can bind to bottles. They were placed in bottles the enzyme at the same time as the enzyme containing coconut husk dipped in different binds to the substrate, and this binding affects concentration of plant extracts which were the binding of the substrate and vice versa dried in room temperature after soaking for 6 (Stryer, 1995; Zibaee and Bandani, 2010a). hours. The percentage of mortality was checked and the larvae were maintained till Materials and Methods pupation. The untreated control was maintained for each treatment. Six replicates Insect culture were maintained for each experiment. The insect were collected from local affected Effect of plant extracts on biochemical coconut palms. The collected insects were parameters paired and kept in small plastic containers for mating and provided with sugarcane pieces. Biochemical analysis can be analysed After hatching, the newly emerged larvae using appropriate protocol. were transferred in to the plastic bottle containing coconut husk. At the early stage Preliminary phytochemical screening of larval development only one larva was (Harborne, 1973; Trease and Evans 1989) of placed in each bottle to avoid cannibalism. ethanol extracts of Asparagus racemosus leaf Actively feeding 4th instar larvae were extract. selected for the experiment. Estimation of Acetyl choline esterase Plants selected for the study activity. Estimation of digestive enzymes activity using standard protocol. Three plants namely Ocimum sanctum, Saraca asoka and Asparagus racemosus  α – Amylase were collected from local areas and identified at Botany Department, University college,  Proteases and Acetylcholine-esterase TVPM. Activity. Preparation of extracts Statistical analysis Fresh leaves of three medicinal plants were collected, washed and shade dried. The dried One way ANOVA, followed by Duncan’s plant materials were ground to fine powder analysis.(P< 0.05) LD 50 - probit analysis using a mechanical grinder and proceeded for using IBM SPSS version 20. soxhlet extraction for 8-10 hrs; according to Karmegam et al. (1997) method using Results and Discussion different solvents such as ethanol, acetone and water. The weighed quantity of the plant By conducting the screening experiment, the most effective plant extract was found to be Asparagus in ethanol, (LD 50) comparing to all other plants (Fig.1). so further detailed Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

342 Current status and challenges for conservation and sustainable use of biodiversity analysis were conducted only with this extracts of Asparagus racemosus, mortality particular plant extracts. When the fourth was increased in a dose dependent manner in instar larvae were treated with ethanol leaf Rhynchophorus ferrugineus (Fig. 2, 3, 4). Fig.1 Increase in mortality with increased concentration of the extract. Larval mortality percentage is highly significant (p < 0.05) in high concentration (8000ppm). Fig.2 Corrected mortality percentage of Rhynchophorus ferrugineus IV instar larvae after treating with Asparagusracemosus Increase in mortality with increased concentration of the extract. Larval mortality percentage is highly significant (p < 0.05) in high concentration. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

Screening of various plant extracts as biopesticides against rhynchophorus ferrugineus … 343 Fig.3 Percentage pupation of Rhynchophorus ferrugineus IVth instar larvae after treating with Asparagus racemosus % of pupation was shown to decrease with increased concentration of the extract. Mean value of pupal mortality is significantly different in different concentration(p < 0.05). Fig.4 Percentage adult emergence of Rhynchophorus ferrugineus IV treating ih Asparagus racemosus. instar larvae after Adult emergence was also significantly reduced with increased concentration of the extract. Mean value of adult emergence is significantly different in different concentration(p< 0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

344 Current status and challenges for conservation and sustainable use of biodiversity Effect on growth and development of Table.2 Estimation of protease larvae Sample protease(µg/ml) Duration of larval- pupal stages and adult ( Mean ± S E) emergence at selected doses of LD50. When treated with Asparagus ethanol extracts at Control 0.00723±.1435 concentration 8000ppm and Sample 0.00182±.0231 LD50 value 1041.08 on fourth instar larvae they shows abnormalities. Fig.6 Estimation of protease o Showed moulting disturbances. Mean values of different superscript letter (a) significantly different (p<0.05) o Duration of moulting time increased. In our findings the digestive enzyme such as o The size and weight reduced after the α-amylase and protease content reveal that the amount decreased in treated larvae treating. when compared to control larvae (Table.1,2;Fig.5,6). The methanolic and o Incomplete metamorphosis. aqueous extract of Taraxacum officinale showed potential percentage of α-amylase o Moulting distruptrion. inhibition (Amin et al., 2015). Reduction of α-amylase and protease enzyme activity Table 1. Estimation of α-amylase could be due to a cytotoxic effect of different extracts on epithelial cells of midgut that Sample Concentration of α- synthesize α -amylase ((Ryan, 1990; Franco Amylase(µg/ml) (Mean ± S E) et al., 2002;Jbilou et al., 2008). In certain studies elevation or reduction in enzyme Control 417.10709±.1723 level is associated with metabolic alteration Treated 282.29021±.0512 in insects which leads to the death of insects after insecticidal Poisoning. Fig. 5 Concentration of α Amylase Table 3. Estimation of acetylcholine activity Mean values of different superscript letter (a) significantly different (p<0.05) Sample AchE(µg/ml) ( Mean ± S E) Control 1.039±.2356 Treated 0.5535±.0567 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

Screening of various plant extracts as biopesticides against rhynchophorus ferrugineus … 345 Fig.7 Estimation of Acetylcholine activity Discussion Mean values of different superscript letter (a) In these control insects seen 72%of pupation, significantly different (p<0.05) but the concentration is increased the effect on pupation is decreased because of treating Acethylcholine esterase (achei) activity with the sub lethal concentration of extracts. So this result shows the effect and AChE is a key enzyme that terminates nerve importance of ethanol leaf extracts of impulses by catalyzing the hydrolysis of Asparagus racemosus. The larval stages are neurotransmitter, acetylcholine, in the more sensitive to the toxic effect than pupae nervous system of various organisms and adults. The higher concentrations were (Oehmichen and Besserer, 1982; Grundy and highly effective as they produced 80% Still, 1985; Wang et al. 2004). Zibaee and mortality in larval stage itself. Moreover, Bandani (2010b) demonstrated that A. annua there was a delay in pupa to adult moult after extract inhibited the AChE activity in higher treatment with this extracts doses which coincided with other reports about effect of botanical insecticides on This study has provide a fundamental AChE inhibition. The alteration of AChE was understanding on the morphology and observed in the cockroach, Periplaneta biochemical parameters of RPW larva. This americana L., at 4 ppm of AZA, (Shafeek et knowledge may be the first step to further al. 2004) and the snail, Limnaea acuminate study to finding the possibility of new Lamarck, at 40% and 80% concentrations treatment to handle the infestation of RPW at of neem oil (Singh and Singh, 2000). It was the most destructive stage of its life cycle. It also observed that 25 g of distilled water is possible that the insecticidal property extracts of the botanicals Punica granatum present in the selected plant extract may L., Thymus vulgaris L., and Artemisia arrest various biochemical as well as absinthium L., significantly inhibited the metabolic activities. The result of this AChE activity of nematodes at 100% preliminary study thus indicates that ethanol concentrations (Korayem et al. 1993). Senthil leaf extract of A. racemosus is promising Nathan et al. (2008) demonstrated that LD50 enough for further detailed research and can concentrations of AZA significantly inhibited be incorporated into integrated pest the activity of AChE compared with control. management strategies. The same effect is observed in this study that treated with ethanol leaf extract the amount The phytochemical assays show, the presence of acetyl-choline esterase decreased(Table 3 of alkaloids, phenols, terpenoids. The result and Fig.7). of the phytochemical screening reveals that tanins, alkaloids, flavanoids were present in ethanolic extracts of Asparagus. These phytochemicals may be responsible for their insecticidal properties (Kabaru and Gichia 2001). These compounds could also deter or possibly repel an insect from feeding. It is possible that the insecticidal property present in the selected plant compound may arrest the various metabolic activities. The present observation highlights the potential of the ethanol extract of Asparagus racemosus Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

346 Current status and challenges for conservation and sustainable use of biodiversity against the insect pest of Coconut palm Daniel Blumberg. 2008. Date palm Rhynchophorus ferrugineus. arthropod pests and their management in Israel, 36(5): 411–448. Conclusion De Silva C., Dunphy G.B. & Rau, M.E. The plant extracts had direct changes on red 2000. Dev. Compar. Immunol, 24: 367–379. palm weevil larvae. Ethanol extracts of Franco, O., Rigden, D.J., Melo, F.R. and Grossi-de-Sa,´ M.F. 2002. Plant a-amylase A.racemosus showed significant inhibitors and their interaction with insect a- amylases: structure, function andpotential for morphological changes and cause enzymatic crop protection. Eur. J. Biochem. 269: 397- 412. alterations in treated insects and the effect Lowry, O.H., Rosenbrough, N.J., Farr, A.L. was dose dependent. LD 50 value was and Randall, R.J. 1951. J. Biol.Chem. 193:265-275 pp.. assessed using probity analysis. The effect of Manachini, B., Arizza, V., Parrinello, D. and the plant A.racemosus was found to be most Parrinello, N. 2011. J.Invertebr. Pathol., 106:360−365. significant causing highest mortality Omotoso, O. T. and Adedire, C. O. 2010. compared to other plants. The sublethal Gross Anatomy and Histology of the Alimentary System of the Larva of Palm concentration of this extract elicited Weevil, Rhynchophorus Phoenicis Fabricius (Coleoptera : Curculionidae) Journal of Life significant changes in the enzymatic Sciences, 4(1):1-5. parameters in treated insects compared to Ryan, C.A. 1990. Protease inhibitors in plants: genes for improving defenses against control, sometime it may be lead to several insects and pathogens. Annu. Rev. Phytopathol., 28: 425- 449. physiological disruption in insects body. Senthil Nathan, S., Chunga, P.G. and References Muruganb, K. 2006. Combined effect of biopesticides on the digestive enzymatic Abraham,V. A. and Kurian, C. 1975. profiles of Cnaphalocrocis medinalis Protection and Processing, September 14-25, (Guenee) (the rice leaffolder) (Insecta: Kingston. Lepidoptera: Pyralidae). Ecotoxicology and Environmental Safety. 64, 382–389. Ali, M., Ali Ahmed, Ibrahim, M.A. 2018. Journal of Asia-Pacific Entomology, 21(2), Sivapragasam, A., Arikiah, A. and Ranjit, C. 631-637 A. 1990. Planter, 66: 113 –123. Amaral, I.M.R and Franco, G.B. 2009. Srinivasan, 2006. Structural and functional Micron, 41: 123–129. diversities in lepidopteran serine proteases. Cell. Mol. Biol. Lett., 11: 132-154. Annadurai, R.S. and Rembold, H. 1993. Naturwissenschaften, 80 (3): 127–130. Tiwari, R. K., Pandey, J. P. and Saheli, R. 2002. Indian J. Expt. Biol., 40:1202-1205. Arnason, J.T., Philogene, B.J.R. and Morand, P. 1989. ACS symposium series 387, American Chemical Society, Washington D C. Carl Lowenberger, 2001. Insect Biochemistry and Molecular Biology, 31 219–229. Catherine Regnault Roger, 2014. The potential of botanical essential oils for insect pest control, 34(4):16-27. Child, P. 1974. Coconuts. 2nd Edition. Longman Group Ltd., London. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.339-346 © Principal, Sree Narayana College, Kollam, Kerala, India

Physiological response of freshwater and salinity – acclimated perch (anabas testudineus bloch)… 347 ISBN 978-93-5396-871-7 CHAPTER 50 PHYSIOLOGICAL RESPONSE OF FRESHWATER AND SALINITY – ACCLIMATED PERCH (ANABAS TESTUDINEUS BLOCH) TO WATER - BORNE NITRATE Vijayasree A S* and Oommen V Oommen** *Department of Zoology, Fatima Mata National College, Kollam, Kerala, India **Centre for Venom Informatics, Department of Computational Biology & Bioinformatics, University of Kerala. Correspondence E-mail: [email protected] ABSTRACT Aquatic systems are highly vulnerable due to their tendency to accumulate relatively high concentrations of chemicals entering from surrounding terrestrial systems. The aim of the study was to test the physiological mechanism of stress tolerance in the climbing perch exposed to water-borne nitrate at different concentrations (0.5 and 1.5mM) adapted to either fresh water or seawater for 48 h and 96 hr. The ionoregulatory potential in the test species was quantified by assaying Na+, K+-ATPase activity in the gill, liver, kidney and intestine. Plasma glucose remains unaffected in fresh water groups while it significantly decreased in sea water groups. Lactate and urea significantly decreased in both fresh water and seawater acclimated fishes. Plasma cortisol, the major stress hormone were also measured. Percentage of methaemoglobin increased significantly in nitrate treated FW fish where as it remained unaffected in SA fish. The results suggest that the climbing perch shows more tolerance to nitrate toxicity in salinity condition. The current study explored the utility of the comet assay for in vivo laboratory studies using A. Testudineus species for screening the genotoxic potential of sodium nitrate. Our data also provide evidences that climbing perch can handle nitrate toxicity without relying on urogenic pathway. The present data indicate that water-borne nitrate induces a classic stress response in climbing perch with pronounced metabolic and osmoregulatory disturbances and suggest a protective role of cortisol in nitrate tolerance in these fish. The present investigation illustrates that presence of high concentration of nitrate in water is stressful to fishes. Key words: Ionoregulation, Methaemoglobin, Sodium nitrate, Stress, Perch Introduction Nitrite (NO2-) is a potential contaminant in aquatic environments that receive Aquatic systems are highly vulnerable due to nitrogenous waste and also in recirculated their tendency to accumulate relatively high aquaculture systems (Grosell and Jensen, concentrations of chemicals entering from 1999). surrounding terrestrial systems. They are oftentimes repositories for a large array of The aim of the study was to test the stressor chemicals (Di Giulio and Hinton, physiological mechanism of stress tolerance 2008). Aquatic animals are at higher risk of in the climbing perch exposed to water-borne nitrite intoxication than terrestrial animals. nitrate adapted to either fresh water or seawater. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

348 Current status and challenges for conservation and sustainable use of biodiversity Materials and Methods groups were same as experiment A group of seawater acclimated (SA) fish was held as Experimental animal: Air-breathing fish, control. Anabas testudineus(Bloch) of approximate 50±5g body mass were collected from a local Sampling and analysis: After supplier near Vellayanilake, experimentation, the fish were anaesthetized Thiruvananthapuram. The fish were in 0.1% 2- phenoxyethanol (SRL, Mumbai) acclimated in 50 L glass tanks with tap water and blood was drawn by caudal puncture at 28 ± 1°C (pH 7.2) under natural using heparinised syringe. The blood was photoperiod (12L/12D) for three weeks prior centrifuged at 5000 rpm for 5 min at 4ºC and to experiment. The specimens were given plasma was separated and stored at - 20°C. prophylactic treatment by bathing twice in The fish were sacrificed by spinal 0.05% potassium permanganate solution for transsection and the gills, liver, intestine and two minute to avoid any dermal infections. kidney were excised and kept in ice-cold They were fed with commercial fish feed at a 0.25M SEI buffer (pH 7.1) and stored at - ration of 1.5% of body mass per day. The 80°C until further analysis. Plasma glucose, animal care and the experimentation were lactate and urea concentrations were done according to the regulation of Animal measured in a Systronics Spectrophotometer Ethical Committee of the University and 2202 using commercial test kits. there was no mortality during the Methemoglobin level (%) was determined in experiments. The fish were starved for DDRC laboratory, Trivandrum. Cortisol twenty four hours before sacrifice for getting concentration in plasma samples was optimum experimental conditions. measured by electrochemiluminescence immunoassay, (ECLIA) and the values were Experimental Design: Two sets of expressed as µgdL-1. The ouabain-sensitive experiment were done. In experiment one, Na+, K+ dependent adenosine triphosphatase twenty- four fish were divided into four (Na+, K+-ATPase, E.C. 3.6.3.9) specific groups of six each and placed in separate activity was measured in the gill, liver, glass aquaria. The fish of group one were intestine and kidney tissue homogenates freshwater control, group two and three were according to Flik et al.(1989). The treated with 0.5 and 1.5mM sodium nitrate concentration of nitrate in the plasma (µM/L) (NaNO3) for 48 hr respectively. The group was estimated colorimetrically (Jenkins and four fish were first kept at 1.5mM NaNO3 for Medsken, 1964) using the Brucine method 48h and later kept for recovery in clean and absorbance was measured at 410 nm. freshwater for another 96hr. In the second The alkaline single cell gel experiment, perch acclimated to selected electrophoresis/comet assay was performed dilute seawater (20%) at different intervals. as a three-layer procedure (Singh et al., Artificially-diluted seawater was prepared by 1988). ANOVA followed by Student- dissolving natural crystal salt (20 g L-1) and Newman-Keul’s test was employed to test aerated for 24 h. Fish were transferred to the significant difference between the dilute seawater through transient transfer treatment groups using Instat-3 Software from 0, 5, 10, 15 and finally to 20‰ with a (Graph Pad Software Inc., San Digeo, 12 h interval between two salinities. Fish California). Significant difference between groups exposed to 20‰ salinity were groups was accepted if P<0.05 and the values sampled after 21 days. They were given are in mean ± SEM (n = 6). environmentally-relevant concentrations of sodium nitrate (0.5 -1.5 mM) for 48h and the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

Physiological response of freshwater and salinity – acclimated perch (anabas testudineus bloch)… 349 Results while remained unaffected in fresh water (Fig.7). Renal Na+, K+-ATPase activity was Plasma glucose remains unaffected in fresh significantly decreased (P<0.001) in water groups while it significantly decreased freshwater adapted nitrate recovery fishes (P<0.01) in sea water groups (Fig.1). Lactate (Fig.8). Level of cortisol in the plasma and urea significantly decreased (P<0.001) in significantly increased (*** P<0.001) both in both fresh water and seawater acclimated fresh water and seawater acclimated groups fishes (Fig.2 and 3). Mean methemoglobin (Fig.9). The levels of plasma nitrate concentrations (% of total haemoglobin) significantly increased in freshwater adapted significantly increased (2 fold) compared to fishes (*** P<0.001) but it remained control after 48 hr nitrate treated freshwater unaffected in seawater acclimated fishes (FW) fish, but it remained unchanged in sea (Fig.10).The fish specimens exposed to water acclimated (SA) fishes (Fig. 4). The different concentrations of NaNO3, exhibited activity of gill Na+, K+-ATPase significantly significant DNA damage (P<0.01) in liver increased (P<0.01 and P<0.001) in nitrate- tissues than the control sample. The liver exposed fish of both fresh and sea water. It tissues exhibited significantly (P<0.01) substantially increased (P<0.001) in nitrate- higher DNA damage at all the studied exposed seawater fishes (Fig. 5). Liver Na+, concentrations of the NaNO3.The DNA K+-ATPase activity significantly increased in damage was concentrations-dependent in all fresh water (FW) fish and remained the tissue examined, and the highest DNA unchanged in sea water challenged (SA) damage was at 1.5 mM NaNO3 followed by fishes (Fig. 6). Salinity acclimation (SA) 0.5 mM NaNO3 and recovered fishes (Fig. resulted a significant rise (P<0.01) in the 11). The higher the concentration of the intestinal Na+, K+-ATPase activity after low chemicals, greater the comet tail and the dose nitrate treatment and then a drop in lower the comet head; which means that (P<0.001)the activity after high dose nitrate higher the DNA fragmentation (Fig.12). Fig.1 Plasma glucose levels in freshwater Fig.2 Plasma lactate levels in freshwater (FW) and seawater-acclimated (SA) (20 ppt) (FW) and seawater-acclimated (SA) (20 ppt) climbing perch after 48 h nitrate exposure climbing perch exposed to 48 h nitrate with or without 96 h recovery. Columns are treatment with or without 96 h recovery. means ± SEM for six fish. ** P<0.01 Columns are means ± SEM for six fish. * compared to control. P<0.05, $$ P<0.01 compared to 1.5mM NaNO3 exposed fish. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

350 Current status and challenges for conservation and sustainable use of biodiversity Fig.3 Plasma urea levels in freshwater (FW) Fig.4 Level of methemoglobin (%) in and seawater-acclimated (SA) (20 ppt) freshwater (FW) and seawater acclimated climbing perch exposed to 48 h nitrate (SA) climbing perch after 48 h nitrate treatment with or without 96 h recovery. treatment with or without 96 h recovery. Columns are means ± SEM for six fish. Columns are means ± SEM for six *P<0.05, **P<0.01 when compared to fish.*P<0.05 when compared to control. control, $$ P<0.01 compared to 1.5mM NaNO3 exposed fish. Fig.5 Levels of branchial Na+, K+-ATPase Fig. 6 Levels of liver Na+, K+-ATPase activities in freshwater (FW) and seawater- activities in freshwater (FW) and acclimated (SA) (20 ppt) climbing perch exposed seawater-acclimated (SA) (20 ppt) to 48 h nitrate treatment with or without 96 h climbing perch exposed to 48 h nitrate recovery. Columns are means ± SEM for six fish. treatment with or without 96 h recovery. ** P<0.01when compared to control, $$ P<0.01 Columns are means ± SEM for six fish. and $$$ P<0.001 when compared to 1.5mM *P<0.05 when compared to control, NaNO3 exposed fish. $P<0.05 compared to 1.5mM NaNO3 exposed fish. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

Physiological response of freshwater and salinity – acclimated perch (anabas testudineus bloch)… 351 Fig. 7 Levels of intestine Na+, K+-ATPase Fig. 8 Levels of renal Na+, K+-ATPase activities in freshwater (FW) and seawater- activities in freshwater (FW) and acclimated (SA) (20 ppt) climbing perch exposed seawater-acclimated (SA) (20 ppt) to 48 h nitrate treatment with or without 96 h climbing perch exposed to 48 h nitrate recovery. Columns are means ± SEM for six fish. treatment with or without 96 h recovery. * P<0.05, ** P<0.01 when compared to control, $ Columns are means ± SEM for six fish. P<0.05 compared to 1.5mM NaNO3 exposed fish. $$$ P<0.001 compared to 1.5mM NaNO3 exposed fish. Fig 9. Changes in the plasma cortisol levels in Fig.10 Levels of plasma nitrate in the freshwater (FW) and seawater-acclimated freshwater (FW) and (SA) – acclimated (SA) (20 ppt) climbing perch exposed to 48 h perch exposed to water-borne nitrate for 48h nitrate treatment with or without 96 h recovery. with or without 96h recovery. Columns are Columns are means ± SEM for six fish. *** means ± SEM for six fish.*** P<0.001 P<0.001 when compared to control, $ P<0.05 when compared to control, # # # P<0.001 when 1.5mM NaNO3 exposed fish. compared to 1.5mM NaNO3 exposed fish. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

352 Current status and challenges for conservation and sustainable use of biodiversity Fig. 11 Representative comet images of fish (A. testudineus) liver tissue, depicting the genotoxic effect of nitrate; (A) FW Control; (B) 0.5 mM NaNO3 (C) 1.5 mM NaNO3 (D) 1.5 mM NaNO3 +Rec in FW fish (E) SA fish control (F) 0.5mM NaNO3 (G) 1.5 mM NaNO3 (H) 1.5 mM NaNO3 +Rec in SA fish Fig. 12. Average tail length of A. testudineus treated with sodium nitrate in freshwater and seawater acclimated fishes indicating the DNA damage (Comet assay). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

Physiological response of freshwater and salinity – acclimated perch (anabas testudineus bloch)… 353 Discussion activities distinctive to these organs, with respect to either the activation or The decrease in plasma glucose probably detoxification of pollutants or the repair of rules out the involvement of chromaffin axis different types of strand breaks. The in the nitrate-treated fish. The reduced genotoxic potential of NaNO3 found in this plasma lactate in the nitrate-loaded fish study demands serious concern towards its indicates a high turnover of pyruvate potential dangers to aquatic organisms. oxidation associated with increased mitochondrial respiration. The declined Conclusion plasma glucose, lactate and the elevated urea thus may point to an enhanced metabolic cost The present data indicate that water-borne required to maintain the energy homeostasis sodium nitrate induces an integrated stress in the nitrate-loaded fish. In the present response in the climbing perch with increase study, the reduced plasma urea level in the in the inter renal function and disturbance in perch after nitrate exposure indicates less the metabolic and hydromineral regulations. participation of ureogenic potential in the SA Our data also illustrate that cortisol has a fish. On the contrary, the increased plasma protective role in the regulation of tolerance urea turnover in FW fish indicates an mechanism in the post-stress fish. The increased ureogenic potential in the nitrate- present investigation illustrates that presence treated fish. The unaffected plasma nitrate of high concentration of nitrate in water are even after loading high dose of ambient stressful to fishes. nitrate indicates a tight regulation of nitrate availability in the plasma as a consequence of Acknowledgement tolerating excess nitrate. It is likely that the cortisol is essential for the unique metabolic The authors are grateful to University Grants adaptation which could help the fish to Commission (UGC), New Delhi, Grant No. F tolerate excess nitrate. The Na+, K+-ATPase 3-15/2007 SAP to the Department of activities in these tissues were not affected by Zoology, University of Kerala, nitrate loading, implying that the Thiruvananthapuram and UGC Grant osmoregulatory potential in these tissues is No.F.43/2006 (BSR) RFSMS for the less affected by excess nitrate. Liver cells financial assistance and infrastructural DNA of exposed fishes were observed to facilities. The authors greatly acknowledge migrate rapidly toward the anode than those Dr. Rajesh Ramachandran, Biogenix of control fish which indicates DNA damage Research Centre, Thiruvananthapuram for or fragmentation in the exposed fishes. expertise in comet analysis and Ahmed et al. (2010b) reported higher %Tail interpretations. DNA (38.81 ±0.55%) in liver tissue of A. Testudineus (Bloch) at 2 mg/L concentration References of cadmium exposure. In the present study, the observed tissue specific response to Di Giulio, R. T. and Hinton, D. E. 2008. The nitrate may be due to physiochemical toxicology of Fishes, CRC Press, Taylor & Francis Group, 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 334872742. Flik, G. and Perry, S. F. 1989. Cortisol stimulates whole body calcium uptake and Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

354 Current status and challenges for conservation and sustainable use of biodiversity the branchial calcium pump in freshwater rainbow trout. J. Endocrinol., 120:75-82. Grosell, M. and Jensen, F. B. 1999. NO2 uptake and HCO-3 excretion in the intestine of the European flounder (Platichthysflesus). J. Exp. Biol., 202: 2103 - 2110. Jenkins, D. and Medsken, L. 1964. A Brucine method for the determination of nitrate in ocean, Estuarine, and fresh waters. Anal Chem., 36: 610. Singh, N., McCoy, M., Tice, R. and E. Schneider. 1988. A simple technique or quantification of low levels of DNA damage in individual cells. Exp.Cell Res., 175:184– 191. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.347-354 © Principal, Sree Narayana College, Kollam, Kerala, India

Effects of addition of carbohydrate sources on fish waste fermentation and efficacy 355 ISBN 978-93-5396-871-7 CHAPTER 51 EFFECTS OF ADDITION OF CARBOHYDRATE SOURCES ON FISH WASTE FERMENTATION AND EFFICACY EVALUATION OF FERMENTED PRODUCTS AS BIO-FERTILIZER FOR THE CULTIVATION OF OKRA, ABELMOSCHUS ESCULENTUS (L.) MOENCH Hari B*, Jisha S and Noufiyath N P. G. and Research Department of Zoology Sree Narayana College, Kollam, Kerala *Correspondence E-mail: [email protected] ABSTRACT The fish waste can be used as substrata for microbial fermentation. The present work is aimed to evaluate the suitability of different carbohydrate sources on the fermentation of fish wastes and also to evaluate the efficacy of the fermented products as bio-fertilizer for the cultivation of Ladies Finger (Okra), Abelmoschus esculentus (L.) Moench. Different carbohydrate sources such as cane sugar (Jaggery) (J), rice flour (RF) and wheat flour (WF) were added to the fish waste as fermentable carbohydrate sources at 10% w/w. Propionic Acid at the level of 1% v/w was added to the fish waste as a preservative. A control (C) was maintained without addition of any carbohydrate source. After 30 days of fermentation, the initial pH was increased from 6 to 8 in the control. pH reached to an acidic range of 3 at the end of fermentation in WF and J treatments. However in RF, the pH reached to 3 on 21st day of fermentation and subsequently increased to 8 at the end. Better plant growth in terms of total length in Okra was obtained by the use of bio-fertilizer in potting mixture. The leaf length and breadth was higher in fermented products added treatments when compared to control. Higher number of leaves and node length were recorded in plants which received fermented product as bio-fertilizer compared to control. Properly processed fish waste has great potential in using as bio-fertilizer for the cultivation of vegetables. The results of the present investigation showed that the biotransformation of fish wastes by microbial fermentation was a convenient process for utilizing huge amounts of fish wastes produced and also to solve the problem of solid waste-disposal and subsequent pollution issues. Key words: Fishery waste, Fermentation, Carbohydrate source, Bio-fertilizer, Abelmoschus esculentus (L.) Moench Introduction carbohydrate as a carbon source for microorganisms. Molasses (Brown and The fish waste material can be used as Sumner, 1985; Guerouali et al., 1995; substrata for the fermentation. This also Kompiang et al., 1980) lactose and mixture solves the problem of pollution and waste- of cereal grains and malt (Hassan and Heath, disposal. Fermented fish were made several 1986), dextrose (Lassen, 1994), corn or hundred years ago by the use of lactic acid tapioca flour (Fagbenro and Jauncey, 1993) bacteria previously, the production of fish have been used for such purpose. flour and oil were the old technique adapted for obtaining fish protein (Disney et al., The recycling of nutrients through the land 1997). In the biological silage, the application of fishery waste effluent requires fermentation process requires fermentable the use of crops capable of utilizing these Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

356 Current status and challenges for conservation and sustainable use of biodiversity nutrients. Animal offals are poor in fermentation process. The above mixtures carbohydrates. Fermentation can be effected were mixed thoroughly and stored in four air in offal with the addition of fermentable tight plastic bottles with inner lids, at room sugars. Corn syrup (Levin, 1994) or Molasess temperature (28-32oC). (Durairaj et al., 1976; Urlings et al., 1993; Zuberi et al., 1993; Levin, 1994) have been Aliquots of samples were drawn in specific used as a source of fermentable sugars to sampling period and analysed for pH, ensure successful preservation. The Protein, Free Amino Acids and Total Free utilization of the fermented wastes, a source Sugar. pH of the samples was directly of plant nutrients are finding agricultural use measured using pH indicator paper. Protein will have double benefit such as safe disposal was estimated following Lowry’s method of the fishery waste material and production (Lowry et al., 1955). Free Amino Acids of cost effective organic fertilizer from that were determined by Ninhydrin method for vegetable cultivation at homestead level. (Yemm & Cocking, 1955). Total Free Sugar This approach will definitely be reducing was estimated following Anthrone Method. share of the waste from household as well as Pour Plate Method was employed for fish markets and fish processing industries determining the Total Heterotrophic Bacteria for waste disposal. The aim of this work is to Count (APHA, 2005). Rose Bengal evaluate the suitability of different locally Chloramphenicol Agar of HIMEDIA was available carbohydrate sources as carbon used to assay the Total Mycotic Content. source for ensilaging fish waste materials. Results were expressed in colony-forming Microbial cell growth, metabolite units (cfu) mL-1. productions and substrates uptakes were measured. The efficacy of the fermented An outdoor experiment was conducted at products as bio-fertilizer was assessed in the Post Graduate Department of Zoology, Sree responses of growth of Okara (lady’s finger) Narayana College, Kollam. The experiment plant (Abelmoschus esculentus L. Moench). was designed to study the effects of fermented organic material as bio-fertilizer Materials and Methods source on the growth of Okra (Abelmoschus esculentus L.) which was commonly known Fish wastes obtained in fresh condition after as ‘Ladies fingers’. The experiment was the cleaning and processing of fishes, were conducted in 20 nos. Polythene bags. collected from fish merchants of Polythene bags having a dimension of 8 x 8 x Sakthikulangara Fishing harbor, Kollam 11cm were arranged in the outdoor condition. District. Collected fish waste was kept in Washed sand and coconut pith was used in freezer until use. Fish waste was thawed, cut 1:1 ratio as potting substrate. The fermented in to small pieces and minced in a grinder for product from the treatments WF, RF and J of 5 minutes. Different carbohydrate sources the fermentation trial were added to potting such as Wheat Flour, Rice Flour and Jaggery material at the rate of 2.5% v/w of the potting (Cane Sugar) were added to the fish waste as substrate in treatments P1, P2 and P3. fermentable carbohydrate at 10% (w/w) and Triplicates were maintained for control and the treatments were designated as WF, RF treatments. and J respectively. Control (C) was maintained without addition of fermentable Seeds of the Ladies finger (F1) were carbohydrate. Propionic Acid was added at purchased from FCMC AgroBazar, Kollam 1% (v/w) to all the treatments to control the and sowed in the plastic cover filled with fungal and mould infestation during respective potting mixture. The experiment was conducted for 75 days under outdoor Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

Effects of addition of carbohydrate sources on fish waste fermentation and efficacy 357 conditions with regular irrigation. Total protein content and this observation indicated Plant Length, Leaf Length, Leaf Breadth and that during fermentation the protein was Number of Leaves per Plant were recorded converted to amino acids. Protein content on 7th, 21st, 30th, 45th 60th and 75th days. decreased from the initial range of 220-360 Node Length was measured on 45th, 60th and mg ml-1 in the raw material to 45-70 mg/ml 75th days. All the measurements were in the product after 30 days of fermentation. recorded using a Vernier Caliper and were Similar results were also observed by expressed in cm. One-way ANOVA was Moraler-Ullora and Oetterer (1997) for performed using the SPSS program (IBM biological and enzymatic chemical silages. SPSS Statistics, Release 20.0 © IBM Corporation). If a main effect was significant The effect of varying carbohydrate sources (P<0.05), the ANOVA was followed by on free amino acid content of the fermented Tukey’s test at P < 0.05 level of significance. fish waste was shown in Fig. 3. Amino acid content increased steadily with respect to Results and Discussion time in T3 (23.3 to 44.9 mg ml-1) and WF (24.2 to 46.3 mg ml-1). However, in RF the pH increased from the initial value of 6 in the increase was in a slow pace. In control raw material to 7 in the control and on 21st (J0C1), the free amino acid concentration day it declined to acidic range (pH -6) then showed a declining trend (from 24.2 to 18.7 increased to 8. In RF, the initial pH value of mg ml-1) after 30 days of fermentation. 6 decreased to 5 and maintained that value up Similar result was observed by Morales- to 21 days and then increased to 8 on the Ulloa and Oetterer (1997) and Arason termination of the fermentation experiment (1994). Finally amino acid content increased on 30th day (Fig 1). However, in the case of from the initial level to the final level J, pH was deceased from initial pH of 6 to 4 because all proteins contained in the silage on 7th day and then further decreased to 3 on were degraded to form the amino acids 21th day of fermentation. In WF the pH was during the 30 days of fermentation. remained steady on 6 in 7th, 14th and 21th days and then declined to 3 on 30th day of the The effect of varying carbohydrate sources experiment. Fall in the pH value of fish on carbohydrate levels of the fermented fish waste during fermentation was reported by waste was shown in Fig. 4. In addition to Raa and Gildberg (1982) and Levin (1994). protein, the carbohydrate content was Similar observations were noted when fish gradually decreased from the initial viscera were fermented with different levels carbohydrate content which ranged from 50- of molasses (Ahmed and Mahendrakar, 200 mg/ml to10-70 mg/ml in the final 1995). Urlings et al., (1993) also reported product during the 30 days of fermentation. similar results during fermentation of broiler The Cane Sugar or Jaggery added treatment processing waste. The treatment with Jaggeri (J) was reached the acidic condition early and Wheat Flour were utilized more by the when compared Wheat Flour and Rice Flour microbes as shown by acidic pH of 3 at the added treatments. This may be due to the end of the fermentation. readily available glucose present in the Jaggery. Shaw et al. (1997) reported that The effect of varying carbohydrate sources initial glucose concentration, inoculum level on soluble protein content of the fermented and fermentation time had a significant effect fish waste was shown in Fig.2. The final on acid production in the poultry intestine silage protein content after 30 day of fermentation. fermentation was lower than the initial Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

358 Current status and challenges for conservation and sustainable use of biodiversity The effect of inclusion of various the range of 27 to 31.5cm was recorded in the carbohydrate sources on the Total treatments. While the control plants showed Heterotropic Bacterial (THB) Count of the a Mean Length of only 10.5cm. The Leaf fermented fish waste is presented in Fig. 5. Length and breadth was higher in P3 plants The highest growth (THB) was observed in when compared to other treatments. Lowest RF (45 cfu ml-1) followed by has 43 cfu ml-1. Leaf Length and Breadth were recorded in The THB count was lower (29 cfu ml-1) in control. The data clearly showed that the WF. The control (J0C1) contains 31cfu ml -1. number of leaves was higher in P3, and P2 This revealed that heterotrophic bacteria can compared to control (Table 1). utilize the available carbohydrate in the fish Comparatively higher Node Length was waste even in control where the initial recorded in plants which received fermented carbohydrate content was in the level of 59.9 product as bio-fertilizer. Animal offals are mg ml-1. It was reduced to 12.6mg ml-1 at the poor in carbohydrates. Fermentation can be end of the experiment. Moreover, control effected in offal with the addition of couldn’t reach the acidic environment and fermentable sugars. Corn syrup (Levin, was necessary for the preservation of the 1994) or Molasess (Durairaj et al., 1976; fermented material. Foul smell was reported Urlings et al., 1993; Zuberi et al., 1993; in the control when compared to other Levin, 1994) have been used as a source of treatments. This necessitate addition of fermentable sugars to ensure successful sufficient carbohydrate to the fermented preservation. In the present study, various product is necessary fermentable sugars were added to fish waste and the fermented products were applied to The effect of inclusion of various as the fertilizer for the culture of Ladies carbohydrate sources on the Total Mycotic Finger plants and the higher plant growth Count of the fermented fish waste is depicted was reported in potting mixture in Fig. 6. Presence of large amount of supplemented with fermented product as mycotic growth is observed in C (103cfu/ml). biofertilizer. Low mycotic biota present in the WF and J. 10x 104cfu ml-1 mycotic organisms were Results of the present study indicated that present in each treatment. In RF, the mycotic organic matter is necessary for the plant count was recorded as 34x 104cfu ml-1. In growth for vegetable cultivation. The growth treatment, J and WF have less count of performance of the Lady Finger plants in the micotic organisms, because of the presence treatment (P1 to P3) indicated that the of propionic acid, it inhibited the growth of addition of fermented fish waste augmented the mould. However, in control, large plant growth. These results indicated that number of mycotic growth was present in the addition of fermented product at a level of (103 x104cfu ml-1). While in the study of 2.5% in the potting mixture is necessary to Warth (1989), at pH 3.5, propionic acid was support the plant growth. Rana Ibrahim required 10 times more than benzoic acid to (2014) suggested that dairy effluent, after inhibit the growth of the mould. chemical treatment and proper dilution, can be used as a potential source of water for The Total Plant Length, Leaf Length, Leaf seed germination and plant growth in Breadth and Number of Leaves of the Ladies agricultural practices of Lady’s Fingers Finger (Abelmoschus esculentus L.) plants (Abelmoschus esculentus L.). In the present were presented in Table 1. Fermented study also the fishery waste material added products added treatment showed higher with carbohydrate sources was properly growth compared to control. Plant Length in processed by microbes to produce a stable Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

Effects of addition of carbohydrate sources on fish waste fermentation and efficacy 359 bio-fertilizer which supported plant growth. processing units (fishing harbor, fish markets, fish processing industry, house hold, The results of the present investigation etc.). The process may produce products showed that the biotransformation of wastes with added values which can be applied to by fermentation constitute a very convenient the production of vegetables at homestead process for the recycling of the huge level and/or by self-help groups to make our amounts of wastes produced in the different State of Kerala self reliant in the field of avenues of sea food handling, treating and organically farmed vegetables. Table 1. Effect of various fermented product on the growth of ladies finger ( Abelmoschus esculentus L.) after 75 day of culture Treatments Plant length ( cm) C P1 P2 P3 Leaf length ( cm) 10.5 26.9 32.5 37 Leaf breadth (cm) 6.1 5.3 6.2 10.9 No of leaves 5.1 5.4 12.8 Node length (cm) 6 10 45 7 3.7 2.7 3.2 3.7 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

360 Current status and challenges for conservation and sustainable use of biodiversity Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

Effects of addition of carbohydrate sources on fish waste fermentation and efficacy 361 Acknowledgement APHA. 2005. Standard Methods of Water and Wastewater, 21st Edn., American First author acknowledges the financial Public Health Association, Washington, assistance received from University Grants DC., pp: 2-61, ISBN: 0875530478. Commission through a U.G.C. M.R.P. Arason, S. 1994. Production of Fish Silage. References In: Martin, A.M. (Ed), Fisheries Processing: Biotechnological Applications. Chapman and Ahmed, J. and Mahendrakar, N. S. 1996. Hall, London, pp. 244-272. Autolysis and Rancidity Development in Tropical Freshwater Fish Viscera during Brown, N. and Summer, J. 1985. Fish Silage. Fermentation., Bioresource Technology, 58: FAO-Fisheries Report 313(suppl.), pp. 404- 247-251. 413. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

362 Current status and challenges for conservation and sustainable use of biodiversity Disney, G. J., Tatterson, I. N. and Ollen, J. Morales-Ulloa, D. F. and Oetterer, M. 1997. 1977. Recent Development in Fish Silage. In: Composição em Aminoácidos de Silagens Proceedings of the Conference on the Químicas, Biológicas e Enzimáticas Handling Processing and Marketing of Preparadas com Resíduos de Sardinha. Tropical Fish. London Tropical Products Ciência e Tecnologia de Alimentos., 17 (3): Institute, London, pp 321-340. 252-258. Durairaj, A. S., Santhanaraj, T., Sulthan, K. Raa, J. and Gildberg, J. A. 1982. Fish Silage: M. and Dorairajh, K. A. P. A. 1976. A Review. Critical Reviews in Food Science Utilisation of Trash Fish. Fish Ensilage- and Nutrition, 16: 343-419. Some Aspects of Processing and Storage. In. Proc.Symp. Fish Process. Ind., Central Food Rana Ibrahim Al-Dulaimi, Norli Ismail and Technol. Res. Inst. Mysore, India, pp. 81. Mahamad H. Ibrahim. 2014. Responses of Growth of Lady’s Fingers (Abelmoschus Fagenbro, O. and Jauncey, K. 1993. esculentus L.) to Different Treatments Chemical and Nutritional Quality of Raw, Methods of Dairy Wastewater, Annals of Cooked and Salted Silages. Food Chem., 48: Agricultural and Environmental Medicine., 331-335. 21(1): 42–48. Guerouali, A. M. and Benkerroum, N. 1995. Shaw, D .M., Narasimha, D.R. and Preparation and Utilisation of Fish Silage as Mahendrakar, N.S. 1997. Effect of Different Feed Supplement for Ruminants, Ann. Levels of Molasses, Salt and Antimycotic Zootech., 44(1): 102. Agents on Microbial Profiles During Fermentation of Poultry Intestine, Hassan, T. E. and Heath, J. L. 1986. Bioresource Technology, 63(3): 237-241. Biological Fermentation of Fish Waste for Potential Use in Animal and Poultry Feeds, Urlings, H. A. P., Bijker, P. G. J. and Van Agri. Waste., 15: 1-15. Longtestijn, J. G. 1993. Fermentation of Raw Poultry Byproducts for Animal Nutrition, J. Kompiang, I. P. and Yushadi, C. D. C. 1980. Anim. Sci., 72: 2420-2426. Microbial Fish Silage: Chemical Composition, Fermentation Characterstics Warth, A. D. 1989. Relationships between and Nutritional Value, FAO Fish Rep. No. the Resistance of Yeasts to Acetic, Propionic 230: 38-43. Acids and to Methyl Paraben and pH, Int. J. Food Microbiol., 8: 343-349. Lassen, T. M. 1994. Evaluation of Conditions for Fermentation of Fish Offal, Agri. Sci. Yemm, E.W. and Cocking, E. C. 1955. The Finland, 4: 11-17. Determination of Amino Acids with Ninhydrin, Analyst, 80: 209-213. Levin, R. E. 1994. Lactic Acid and Propionic Acid Fermentation of Fish Zuberi, R., Fathima, R., Shamshad, S. J. and Hydrolysates. In Fisheries Processing – Qadri, R.B. 1993. Preparation of Fish Silage Biotechnological Applications. ed. A.M. by Microbial Fermentation, Trop. Sci., 33: Marlin, pp. 273-310. 171-182. Lowry, O. H., Rosebrough, N. J., Fan, A. L. and Randall, R. J. 1951. Protein Measurement with the Folin - Phenol Reagent, Journal of Biological Chemistry., 270: 27299-27304. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.355-362 © Principal, Sree Narayana College, Kollam, Kerala, India

Differential regulation of na+, k+-atpase by in vitro thyroid hormones in perch… 363 ISBN 978-93-5396-871-7 CHAPTER 52 DIFFERENTIAL REGULATION OF NA+, K+-ATPASE BY IN VITRO THYROID HORMONES IN PERCH (ANABAS TESTUDINEUS) GILLS AND KIDNEYS: EVIDENCE FOR DIRECT AND RAPID ACTIONS Leji J Department of Zoology S. N. College, Sivagiri, Varkala, Thiruvananthapuram, Kerala *Correspondence E-mail: [email protected] ABSTRACT In teleosts, thyroid plays a vital role in the regulation of many biological processes. The Na+-K+-ATPase activity thus provides the driving force for Na+ transport in the osmoregulatory epithelia of gills and kidneys and is widely used as an index of osmoregulation in fishes. The ouabain-sensitive Na+, K+-ATPase activity, a measure of sodium pump activity, was quantified in branchial and renal tissues following in vitro addition of physiological concentration of T3 or T4. Three important responses were observed after supplementing T3 or T4 to branchial or renal explant medium. In vitro supplementation of both T3 and T4 at varied concentrations produced a hyperactivity of Na+, K+-ATPase in branchial tissue, showed a hypoactivity in renal tissue. The addition of T4 produced a similar pattern in Na+, K+-ATPase activity in both branchial and renal tissues. A tissue-specific differential regulation of sodium pump activity by both T3 and T4 was also recorded. In vitro data led us to conclude that both T3 and T4 have specific direct and rapid regulatory action on the sodium pump in the gills and kidneys. Key words: Na+-K+-ATPase activity, Thyroid Hormones, Gills, Kidneys, Anabas testudineus Introduction ions in seawater (McCormick, 2001; Evans et al., 2005; Hwang and Lee, 2007). Na+/K+- In teleosts, thyroid plays a vital role in the ATPase (NKA), a universal membrane-bound regulation of many biological processes enzyme, provides driving force for many Leatherland, 1994; Oommen and Matty, transport systems and help the fish to maintain 1997). Thyroxine (T4) and triiodothyronine Na homeostasis with its presence in a variety (T3), the principal thyroid hormones (TH’s), of osmoregulatory epithelia including gills are known for their metabolic and (Evans et al., 2005). osmoregulatory actions in fish tissues (Peter et al., 2000; Peter, 1996, 2007; Peter and Peter, The Na+-K+-ATPase, the most important 2009). Among the many reported physiologic enzyme associated with Na+ and K+ regulation actions of THs, its role in hydromineral in the fish osmoregulatory epithelia, indirectly regulation seems to be controversial energizes the excretion of NaCl from marine (Leatherland, 1994; Evans et al., 2005). The teleosts (McCormick, 1995; Marshall and transport of Na and Cl across the teleost gill Bryson, 1998). This electrogenic cationic epithelium is facilitated by the chloride cells pump (Skou, 2000) actively transports Na+ out which absorb ion in freshwater while secrete and K+ into the epithelial cells through the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

364 Current status and challenges for conservation and sustainable use of biodiversity plasma membrane and the regulation of this storage tanks with a temperature ranging from enzyme is under hormonal support 27 to 29°C and kept under natural photoperiod (McCormick, 1995; 2002; Evans et al., 2005) (12hL:12hD). Two weeks prior to the and ambient salinity (Nebel et al., 2005; experiment, adult healthy fish were Veillette et al., 2005; Lingwood et al., 2005). transferred to aerated 100L glass tank. Fish The Na+-K+-ATPase activity thus provides the were fed with commercial fish feed at 1.5% of driving force for Na+ transport in the body mass. All experimental fish received no osmoregulatory epithelia of gills and kidneys feed for 24h before sampling. and is widely used as an index of osmoregulation in fishes. Consequently, a In vitro model tight handling of ions has been observed between branchial and renal tissues in A simple in vitro organ explant was developed freshwater tilapia where branchial Na+-K+- as described earlier in tilapia (Bury et al., ATPase activity increases and renal Na+-K+- 1998) with some modifications. Fresh gill ATPase activity decreases following in vivo arch was excised from the gill basket of perch TH treatment (Peter et al., 2000). and washed thrice in Dulbecco’s modified Ca2+/Mg2+ free phosphate buffer. Primary Investigations in tilapia (Peter et al., 2000) filaments were severed approximately 1 cm and perch support the hypothesis that THs just above the septum and were placed in 1ml have a definite role to play in osmoregulation PBS in sterile culture tubes kept at 4°C. as was evident from the activity pattern of Similarly, posterior kidney was also excised Na+, K+-ATPase following in vivo TH and maintained as renal explantation. As we administration. Studies, however, addressing analysed only short-term in vitro effects on a direct action of THs on osmoregulatory branchial and renal explants, PBS alone was markers is not available in fishes (Evans et al., used in this study. The preparation was 2005). Further, such an approach in fish equilibrated for 10·min in PBS at room seems to be inevitable to delineate the temperature (28oC) before in vitro addition of specificity of thyroidal action in fish hormones. Stock solutions of triiodo-L- osmoregulation. thyroxine (T3, Sigma) and L-thyroxine (T4, Sigma) were prepared in alkaline saline. The aim of this study was to identify whether Serial dilutions of hormones were prepared in THs have a direct rapid action on the Na+ PBS, which also used in controls. All pump activity in the gills and kidneys. To this solutions, including control and varied doses, end, we quantified the ouabain-sensitive Na+, contained the same amount of hormone K+-ATPase activity, a measure of sodium solvent. pump activity in branchial and renal tissues following in vitro addition of physiological The tissue explant was brought to room concentrations of triiodo-L-thyronine (T3) or temperature (28±1°C) and incubated for 5 L-thyroxine (T4). minutes in PBS medium (control) or medium containing varied doses of L-T3 (0.651x10- Materials and Methods 9M, 3.255 x 10-9M and 0.651 x 10-8M) or L-T4 (0.889 x 10-9M, 4.44 x 10-9 M and 0.889 x 10- Fish and holding conditions 8M). The tissues were washed thoroughly with SEI buffer (0.3M sucrose, 20 mMNa2 EDTA, Adult freshwater climbing perch, Anabas 0.1M imidazole pH 7.2) several times and testudineus, comprising both sexes were used kept in the same buffer. The branchial or renal in the present study. They were collected tissues were homogenised in ice cold SEI from a local supplier and kept in freshwater Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

Differential regulation of na+, k+-atpase by in vitro thyroid hormones in perch… 365 buffer and after centrifugation (10 min, 500 Results g), supernatants were used for analysis. After measuring the protein concentration (BSA as Dose-responsive effects of THs standard), saponin (20µg/mg protein) was added to the membrane preparations to Tissue explants prepared from branchial and optimise substrate accessibility. renal tissues responded well to in vitro supplementation of both T3 and T4. In vitro Na+-K+-ATPase Specific Activity additions of varied concentrations of T3 viz. 0.651x 10-9M, 3.25 x10-9M and 0.651x10-8M The ouabain-sensitive Na+-K+-ATPase significantly elevated the branchial Na+-K+- specific activity was measured in tissue ATPase activity (Fig. A.1). In contrast, homogenates as described by Flik et al., significant (P<0.05) inhibition in the renal (1989) and modified by Peter et al. (2000). Na+-K+-ATPase activity was observed except All sample preparations were carried out at for the highest dose (0.651x10-9M, Fig. A.2). 4°C. Homogenate samples were incubated for Similarly, the addition of varied doses of T4 15 minutes at 37°C in a medium containing viz. 0.89x 10-9 M, 4.44 x 10-9 M and 0.89x10- 100mmol.L-1 NaCl, 30 mmol.L-1 imidazole, 8 M significantly elevated Na+-K+-ATPase pH 7.4, 0.1 mmol.L-1 EDTA, 5mmol.L-1 activity in a dose-responsive manner in MgCl2 and either 15 mmol.L-1KCl (Medium branchial explants (Fig. A.3). A significant A) or 1mmol. inhibition in the enzyme activity occurred in renal explants (Fig. A .4). L-1 ouabain (Medium E). Na2ATP was added to a final concentration of 3mmol.L-1. The Branchial Na+, K+-ATPase reaction was terminated by adding cold trichloroacetic acid and was transferred to an 25 ice bath. Liberated inorganic phosphate (Pi) was quantified. The specific activity of Na+- µmols Pi/hr/mg protein 20 C K+-ATPase was defined as the difference o… between the release of Pi in medium A and in 15 medium E and was expressed in µmol.L-1Pi.h- 1.mg-1 protein. 10 Statistics 5 Data were collected from at least six tests in 0 234 each subset. Statistical differences among 01 3 .25x10-9 0.651x10-8 subsets were tested by means of one-way analysis of variance (ANOVA) followed by 0.651x10-9 SNK comparison test. Significance between subsets was accepted if P<0.05. Values are in T3 Concentration (M) mean ± SEM of six tests. Fig. A.1: Dose-dependent effect of in vitro T3 addition on Na+, K+-ATPase activity in branchial explant of FW A. testudineus incubated for 5 min. Each column represents mean ± SEM for six fish. Statistical differences between fish were quoted after SNK test. *P<0.05**P<0.01 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

366 Current status and challenges for conservation and sustainable use of biodiversity Renal Na+, K+-ATPase Renal Na+, K+-ATPase µmols Pi/hr/mg protein 16 µmols Pi/hr/mg protein 12 C 14 o… 12 Control 10 10 T3 8 8 * 6 * 6 4 ** 2 4 0 2 ** 0 1 2 3 4 0 0.651x10-9 3 .25x10-9 0.651x10-8 01234 T3 Concentration (M) 0.89x10-9 4.44x10-9 0.89x10-8 Fig. A.2: Dose-dependent effect of in vitro T3 T4 Concentration (M) addition on Na+, K+-ATPase activity in renal Fig. 3A.4: Dose- dependent effect of in vitro explant of FW A. testudineus incubated for 5 T4 addition on Na+, K+-ATPase activity in branchial explant of FW A. testudineus minutes. Each column represents mean ± incubated for 5 min. Each column represents mean ± SEM for six fish. Statistical SEM for six fish. Statistical differences differences between fish were quoted after SNK test. **P<0.01 between fish were quoted after SNK test. *P<0.05 **P<0.01 Branchial Na+, K+-ATPase 20 18 µmols Pi/hr/mg protein 16 ** 14 12 10 Control 8 T4 6 4 2 0 01234 0.89x10-9 4.44x10-9 0.89x10-8 T4 Concentration (M) T4 Concentration (M) Fig. A.3: Dose- dependent effect of in vitro T4 Fig. A.4: Dose- dependent effect of in vitro T4 addition on Na+, K+-ATPase activity in addition on Na+, K+-ATPase activity in renal branchial explant of FW A. testudineus explant of FW A. testudineus incubated for 5 incubated for 5 min. Each column represents min. Each column represents mean ± SEM for mean ± SEM for six fish. Statistical six fish. Statistical differences between fish differences between fish were quoted after were quoted after SNK test. **P<0.01 SNK test. *P<0.05 ***P<0.001 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

Differential regulation of na+, k+-atpase by in vitro thyroid hormones in perch… 367 Discussion monovalent ion transport in the epithelia of renal tissues. The thyroidal regulation of renal Three important responses were recorded in Na+, K+-ATPase is not known. In this study, the branchial and renal explants after supplementing varied doses of both T3 and T4 supplementing T3 or T4 in vitro. (1) In vitro directly on renal tissue media showed a direct additions of both T3 and T4 increased the inhibition on its renal Na+-K+-ATPase activity activity of Na+, K+-ATPase in the branchial (Fig. A.2 & A.4). Inhibition in the activity of explant but showed a reduction in the renal Na+, K+- ATPase in renal tissue may be explant. (2) The pattern of changes observed attributed to the specificity of TH action in in the enzyme activities following T4 addition freshwater renal tubules where Na+ uptake was similar to the T3-induced changes in both appears to be minimal. explants.(3) A tissue-specific differential regulation of sodium pump activity by both T3 The inhibition in the renal Na+, K+-ATPase and T4 could also be observed. activity following TH addition reflects the preferential role of TH in renal sodium Our results are the first to show that both T3 handling in the renal epithelia of freshwater- and T4 in vitro have direct and rapid actions on adapted fish. It is plausible to suggest that the sodium pump activity in the gills and kidneys inhibition in the renal Na+ pump activity could of climbing perch. A significant stimulation in be due to the apparent TH-driven affinity in the branchial Na+, K+ ATPase activity was the intracellular K+, which is a competitive occurred after the addition of both T3 and T4 inhibitor of Na+ activation leading to high Na+, (Fig. A.1 & A. 3). The up-regulation of the K+ ATPase activity. This view agrees with the branchial and down-regulation of the renal notion that modulations in cytoplasmic Na+, K+ ATPase activities upon addition of T3 K+/Na+ antagonises are the potential mode of or T4 clearly indicate a differential regulation pump regulation (Therian and Blostein, 1999). of Na+, K+ ATPase activity by in vitro THs as The production of large volumes of urine by has been observed. freshwater fish must be accompanied by the reabsorption of proportionate amounts of Na The Na+, K+ ATPase is primarily localized and Cl ions by the renal tubules to maintain within the chloride cells of the gill epithelium, positive ion balance. and the concentration of this enzyme is thus a useful measure of the functional state of the The endocrine regulation and its rapid action chloride cells (Li et al., 1997; Dang et al., of the transport of ions in the osmoregulatory 2000). The increased uptake of Na+ followed epithelia of fish is the subject of much by its active efflux occurs in the branchial attention (Foskett et al., 1983; Mayer-Gostan explant by the in vitro T3 or T4 addition. et al., 1987; Marshall, 1995; McCormick, Furthermore, the present data agrees with the 1995, 2001). Evidence of rapid regulation of in vivo data obtained in trout and salmon ion transport comes from in vitro studies (Madsen and Korsgaard, 1989; Trombetti et examining the ion transport in isolated al., 1996; Ventrella et al., 2001). The direct opercular membranes of certain species like action of TH on Na+, K+ ATPase also points tilapia and mummichog. The most difficulty that chloride cells are the major target for TH in this in vitro approach is the lack of an action as has been evident from the reported in appropriate model for ion transport studies vivo studies on the dynamics of chloride cells and the effects of rapid acting hormones on in the gill epithelia of this species . branchial vasculature (McCormick, 2001). Thus, there exists a limited picture on the It appears that addition of THs down regulate hormonal involvement and transport of ions in the active mechanism for absorbing Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

368 Current status and challenges for conservation and sustainable use of biodiversity understanding the rapid regulation of ion appropriate gradients of Na+ and K+ in renal as uptake and secretion. The rapid regulation of well as in gill cells. ion transport in teleosts and its multi hormonal support are therefore complex (McCormick, Overall, these observations led us to conclude 2001). that both T3 and T4 are equally potent to have direct and rapid regulatory action on the The physiological relevance of cation sodium pump in the gills and kidneys. homeostasis and its control by THs in gills and Furthermore, the present data support the kidneys are evident in this study. The hypotheses that both T3 and T4 have tissue- differential regulation of sodium pump specific differential regulation of Na pump activity by THs in these tissues clearly activity recorded in the branchial and renal indicates the thyroidal regulation in the epithelia. branchial and renal Na+ handling in fishes. Similarly, in vitro reports on many other References hormones, such as cortisol (Sunny and Oommen, 2001; Kelly and Wood, 2001, Bury, N. R., Jie, L., Flik, G., Lock, R. A. C. 2002a; Zhou et al., 2003; Veillett and Young, and Wendelaar Bonga, S. E. 1998. Cortisol 2005) and adrenaline (Marshall et al., 1993; protects against copper induced necrosis and Marshall, 1995) are involved in the regulation promotes apoptosis in fish gill chloride cells of Na+, K+-ATPase. Most of the reported in vitro. Aquat. Toxicol., 40: 193–202. regulatory actions of hormones have done in seawater-adapted fish, and there is relatively Dang, Z., Lock, R. A. C., Flik, G. and little information on rapid stimulation of Wendelaar Bonga, S. E 2000. Na+, K+- transport epithelia involved in net ion uptake ATPase immunoreactivity in branchial in fresh water (Marshall, 1995; Kelly and chloride cells of Oreochromis mossambicus Wood, 2002b). exposed to copper. J. Exp. Biol., 203: 379– 387. The present results showed the remarkable osmoregulatory response of gills and kidney Evans, D.H., Piermarini, P.M., and Choe, K.P. to THs. This implies that these tissues are 2005. The multifunctional fish gill: Dominant responsible for maintaining hydromineral site of gas exchange, osmoregulation, acid- balance. The remarkable rapid up regulation base regulation, and excretion of nitrogenous of the Na+ pump activity in freshwater gills waste. Physiol. Revs., 85: 97-177. and a rapid down-regulation of Na+ pump activity in renal tissue by TH reflect the Flik, G. and Perry, S. F. 1989. Cortisol compensatory regulation of Na+ efflux and stimulates whole body calcium uptake and the influx in the gills and kidneys respectively. As branchial calcium pump in freshwater the activity of Na+, K+-ATPase could be used rainbow trout. J. Endocrinol., 120: 75–82. as an indicator of Na+ and K+ transport across osmoregulatory epithelia, the differential Hwang, P.P. and Lee, T.H. 2007. New insights action of TH observed on the sodium pump into fish ion regulation and mitochondrion- activity in branchial and renal epithelia rich cells. Comp Biochem Physiol., 48A: 479- appears to be one of the vital mechanisms of 497. TH regulation on ion pumps. This suggests that TH have rapid regulatory action on the Kelly, S.P. and Wood, C. M. 2001. The Na+, K+ ATPase activity to establish physiological effects of 3, 5’, 3’-triiodo-L- thyronine alone or combined with cortisol on cultured pavement cell epithelia from freshwater rainbow trout gills. Gen. Comp. Endocrinol., 123: 280-294. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

Differential regulation of na+, k+-atpase by in vitro thyroid hormones in perch… 369 Kelly, S.P. and Wood, C. M. 2002a. Cultured multifunctional cell. Comp. Biochem. gill epithelia from freshwater tilapia Physiol., 119A: 97–106. (Oreochromis niloticus): effect of cortisol and homologous serum supplements from stressed McCormick, S. D 1995. Hormonal control of and unstressed fish. J. Membr. Biol., 190: 1- gill Na+, K+-ATPase and chloride cell 14. function. In: Wood CM, Shuttleworth TJ, editors. Cellular and molecular approaches to Kelly, S.P. and Wood, C. M. 2002b. Prolactin fish ionic regulation. San Diego: Academic effects on cultured pavement cell epithelia and Press.14: 285–315. pavement cell plus mitochondria-rich cell epithelia from freshwater rainbow trout gills. McCormick, S. D. (2002). Endocrine control Gen. Comp. Endocrinol., 128: 44-56. of osmoregulation in teleost fish. Am Zoologist, 41:781–794. Leatherland, J. F. 1994. Reflections on the thyroidology of fishes: from molecules to Nebel, C., Romestand, B., Negre-Sadargues, humankind. Guelph. Ichtyol. Rev., 2: 1-64. G., Grousset, E., Aujoulat, F., Bacal, J., Bonhomme, F. and Charmantier, G. 2005. Li, J., Eygensteyn, J., Lock, B. A. C., Differential freshwater adaptation in juvenile Wendelaar Bonga, S. E. and Flik, G. 1997. sea-bass Dicentrarchus labrax: involvement of Na+ and Ca2+ homeostatic mechanisms in gills and urinary system. J. Exp. Biol., 208: isolated chloride cells of the teleost 3859-3871. Oreochromis mossambicus analyzed by confocal laser scanning microscopy. J. Exp. Oommen, O. V. and Matty, A. J. 1997. Biol., 200: 1499-1508. Metabolism in Poikilotherms: Regulation by Thyroid hormones. In: Frontiers in Lingwood, D., Harauz, G. and Ballantyne, J.S. Environmental and Metabolic Endocrinology 2005. Regulation of fish gill Na+, K+- ATPase (Ed. S. K. Maitra) pp. 1-11. The University of by selective sulfatide-enriched raft Burdwan, India. partitioning during seawater adaptation. J. Biol. Chem., 280: 36545-36550. Peter, M.C.S. 1996. Thyroid hormones and intermediary metabolism in fish : Influence of Marshall, W. S. 1995. Transport processes in neem kernel extract. In : Neem and isolated teleost epithelia: opercular epithelium Environment. Ed. Singh, R.P, M.S. Chari, and urinary bladder. In Fish Physiology, A.K. Raheja and W. Kraus. Oxford and IBH Cellular and Molecular Approaches to Fish Publishing Co. New Delhi. 1189-1198 pp. Ionic Regulation (ed. C. M. Wood and T. J. Shuttleworth). Vol. 14, New York: Academic Peter, M.C.S., Lock, R.A.C and Bonga, Press. 1-25. S.E.W. 2000. Evidence for an osmoregulatory Marshall, W.S., Bryson, S.E. and Garg, D. 1993. 2-Adrenergic inhibition of Cl- transport role of thyroid hormones in the freshwater by opercular epithelium is mediated by intracellular Ca2+ Proc. Natl. Acad. Sci., USA, Mozambique tilapia, Oreochromis 90: 5504–5508. mossambicus. Gen Comp Endocrinol., Marshall, W. S. and Bryson, S. E. 1998. Transport mechanisms of seawater teleost 120:157–167. chloride cells: An inclusive model of a Peter, V. S, Joshua E. K, Wendelaar Bonga, S. E. and Peter, M.C.S. 2007. Metabolic and thyroidal response in airbreathing perch (Anabas testudineus) to water-borne kerosene. Gen Comp Endocrinol., 152: 198-205. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

370 Current status and challenges for conservation and sustainable use of biodiversity Peter, M.C.S. and Peter, V.S. 2009. Action of thyroid inhibitor propyl thiouracil on thyroid and interrenal axes in the freshwater tilapia, Oreochromis mossambicus Peters. J Endocrinol Reprod., 13: 37–44. Skou, J.C. (2000). The identification of sodiuim-potassium pump. K. Taniguchi and S. Kaya (eds). Na+, K+-ATPase and related ATPases. Elsevier Science B.V. Sunny, F. and Oommen, O.V. 2001. Rapid action of glucocorticoids on branchial ATPase activity in Oreochromis mossambicus: an in vivo and in vitro study. Comp Biochem Physiol B Biochem., 130: 323–330. Therien, A. G., and Blostein, R. 1999. K+/Na+ antagonism at cytoplasmic cation activation sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation. Am. J. Physiol. Cell Physiol., 277: C891-C898. Trombetti, F., Ventrella, V., Pagliarani, A., Ballestrazzi, R., Galeotti, M., Trigari, G., Pirini, M. and Borgatti, A.R. 1996. Response of rainbow trout gill Na+, K+-ATPase and chloride cells to T3 and NaCl administration. Fish. Physiol. Biochem., 15: 265–274. Veillette, P.A. and Young, G. 2005. Tissue culture of sockeye salmon intestine: functional response of Na+, K+-ATPase to cortisol. Am. J. Physiol. Regul. Integr. Comp. Physiol., 288: R1598-R1605. Ventrella, V., Pagliarani, A., Trombetti, F., Pirini, M., Trigari, G. and Borgatti, A.R. 2001. Response of rainbow trout gill Na+, K+- ATPase to T3 and NaCl administration. Physiol. Biochem. Zool., 74:694–702. Zhou, B., Kelly, S.P., Ianowski, J.P. and Wood, C.M. 2003. Effects of cortisol and prolactin on Na+ and Cl- transport in cultured branchial epithelia from FW rainbow trout. Am. J. Physiol. Integr. Comp. Physiol., 285: R1305-R1316. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.363-370 © Principal, Sree Narayana College, Kollam, Kerala, India

371 Current status and challenges for conservation and sustainable use of biodiversity ISBN 978-93-5396-871-7 CHAPTER 53 LENGTH WEIGHT ANALYSIS OF STOLEPHORUS INDICUS OF KERALA COAST Divya T Dharan* and Sreedevi** *Department of Zoology, Sree Narayana College, Kollam, Kerala **Department of Zoology, All Saints college, Thiruvananthapuram, Kerala *Correspondence Email: [email protected] ABSTRACT Fish has been the most important and the sustaining for the people around the world. Fish forms a major industry for many people in natural areas and supplying the raw material for many industries like manufacture of fish meal for a poultry and the production of oils of various kinds. Length-weight Relationship of organism reflects their pattern of growth, habitat support, feeding behavior, competition and survivability. The present study was conducted to determine the length-weight relationship of Stolephorus indicus. Usually the study on length weight relationship is assist in understanding the general wellbeing and condition of the fishes in aquatic life. The observed b value for the fish is 2.017 indicated an allometric growth of Stolephorus indicus. In the present work the b value is deviating from the ideal b value, less than 3 therefore fishes exhibit a negative allometric length weight relationship it indicates that the fish tend to become thinner as they grow larger. The length-weight relationship in fishes can be affected by a number of factors including season, habitat, gonad maturity, sex, diet, and stomach fullness, health and preservation techniques, and differences in the length ranges of the specimen caught. The exact relationship between length and weight differs among species of fish according to the inherited body shape, and within a species according to the condition (robustness) of individual fish. Key words; Fish, Length-weight, Stolephorus indicus, Habitat, Growth Introduction and animal rearing etc. at present in India, millions of people are suffering from Fish has been the most important and the malnutrition. However, prawns and fishes sustaining for the people around the world. solve the problem of nutrition. Fishes are the Fish forms a major industry for many people cheapest food for the poor people. According in natural areas and supplying the raw to the central health survey fish contain material for many industries like manufacture highest nutrition value followed by eggs and of fish meal for a poultry and the production provides balanced diet at cheaper rates of oils of various kinds. India is the third (Narasaiaah and Rangaig , 2011). largest producer of fish in the world and pisciculture is emerging as an important Length-weight Relationship of organism source of food. Rural areas in developing reflects their pattern of growth, habitat countries depend mostly on fisheries, poultry support, feeding behavior, competition and Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

372 Current status and challenges for conservation and sustainable use of biodiversity survivability. Bio-morphometric and meristic study of fish population dynamics to estimate studies of endemic, threatened or alien the potential yield (Prasad and Ali, 2007). species are more important, enabling to make out strategic stands against their Organisms generally increase in size (length, vulnerability. Invasive alien species (IAS) are weight) during development. The key factors regarded as the second major cause for native that influence the growth of fish are the and endemic species extinction globally quantity of food available, the number of fish (Wilcove et. al., 1998), introduced mainly for utilizing same food source, temperature, improving fishery, ornamental fish trade, bio- oxygen and other water quality factors control of mosquitoes and sport fishing (Biju besides the size, age and sexual maturity of Kumar, 2010). The impact of invasive the fish. Every animal in its life exhibit species on ecosystem is receiving much growth both in length and in weight and the attention due to their detrimental ecological relationship between these two has both instability. The consequences of Aquatic applied and basic importance. The length- Invasive Species are far reaching, including weight relationship is one of the standard degradation of water quality, food-web methods that yield authentic biological disruptions, depletion of native biodiversity, information and is of great importance in as well as secondary economic impacts on fishery assessments. It establishes the fishing, tour-ism, and other related industries. mathematical relationship between the two The Western Ghats is home to some of the variables, length and weight, and helps in world’s most unique fauna, flora and fungi, assessing the variations from the expected as compared to the other biodiversity weight for the known length groups. This is hotspots. particularly useful for computing the biomass of a sample of fish from the length-frequency The length weight relationship is an of that sample. The parameter estimates of important tool in fish biology, physiology, the relationship for a population of fish can ecology and fisheries industry and it has be compared to average parameters for the primary importance in setting yield region, parameter estimates from previous equations. The length weight relationship for years, or parameter estimates among groups the fish stock is generally used to develop the of fish to identify the relative condition or mathematical relationship between two robustness of the population. variables i.e. Length and weight, so if one of the variable is known the other can be Materials and Methods computed. it allows the comparison of species growth between different The Indian anchovy (Stolephorus indicus) for habitat/regions and is required in fishery the present study were collected from the fish management and conservation (Erguden et market of Kattakada during March 2018. A al., 2011) the length weight relationship is total of 50 specimens of the species were used to assess the general well-being of the brought to the laboratory and total length to fish and the type of growth whether it is the nearest cm and total weight to the nearest isometric or allometric. It is helpful in the 0.1g of individual fishes were measured Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

Length weight analysis of stolephorus indicus of Kerala coast 373 using a measuring board and sensitive Log W = log a + b log L weighing balance respectively. When ‘b’ is equal to 3 isometric pattern of The Indian anchovy (Stolephorus indicus), growth occurs, but when ‘b’ is not equal to 3, allometricpattern of growth occurs, which also known as Hardenberg's anchovy, is a may be positive if >3or negative if <3. species of oceanodromous fish in the Results Engraulidae family The total length and weight of Anabas testudineus used in the present study varied Kingdom: Animalia from 7.8 cm to 8.5cm and 18 g to 24.2 g respectively. Table I shows the data on length Phylum: Chordata and weight of collected for the study Stolephorus indicus. The length- weight Class: Actinopterygii relation of Stolephorus indicus is shown in the Fig. 2. Straight line relationship was Order: Clupeiformes found when the log values of the weight were plotted against the log values of length. The Family: Engraulidae correlation coefficient (r) obtained was 0.511. The b value obtained in the length-weight Genus: Stolephorus relation was 2.017.The correlation coefficient (r) obtained from the length weight analysis Species: S. indicus of was 0.5.11 which clearly exhibits the strong positive correlation between length Binomial name and weight of the body of. Stolephorus indicus. The b value obtained was 2.017. It Stolephorus indicus(Van Hasselt,1823) was below the cube value of 3 and hence it implied that Stolephorus indicus showed a Length-weight(log-transformed) relationships negative allometric growth. As the b–value were determined by linear regression analysis less than 3, the growth of Stolephorus indicus using the data analysis package in was not proportionate in all directions. MICROSOFT EXCEL. It was calculated for each 3 cm length category and for the total fishes collected. The length-weight relationship was worked out as per cube law given by Le Cren (1951) W=aLbWhere, W = weight of fish (g), L = the observed total length (cm), ‘a’ is the rate of change of weight with length (regression intercept) and ‘b’is the weight at unit length (regression slope). The logarithmic transformation of the formula is Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

374 Current status and challenges for conservation and sustainable use of biodiversity 1.4 logW= log -0.526 + 2.017 log L R² = 0.511 1.35 Log of weight (log W) 1.3 1.25 1.2 1.15 1.1 1 0.84 0.86 0.88 0.9 0.92 0.94 0.96 0.98 Log of length (log L) Fig.2. Length-weight relationship of Stolephorus indicus Discussion between2.6328 and 3.2012 in the various size categories. However, the body shape of The present study was conducted to Stolephorus indicus tends to change as the determine the length-weight relationship of length increases. This assertion was Stolephorus indicus. Usually the study on supported by Aliakbar and Ali (1978) and length weight relationship is assist in Nwani et al., (2006). According to the view understanding the general wellbeing and of Kunda et al., (2008) that the fluctuations condition of the fishes in aquatic life. The obtained in certain length groups might be observed b value for the fish is 2.017 due to variation in sample size, gonad indicated an allometric growth of Stolephorus conditions, seasonality and fullness of gut indicus. A study on Vulugil scheli by content. Length weights are two indices Moorthy et al. (1999) reported the ‘b’ value determining the age of the fish gonad was 2.62 which showed allometric growth development and estimating the growth of pattern. The results of the present study are fish stock of assessment models and data for correlated with the results of Moorthy et al. sustainable management resources Similarly Babu and Neelakandan(1983) reported the ‘b’ value for male and female In conclusion, the present study found that Lisa parsia as 2.9 and 2.98. All the reasons the species of Stolephorus indicus inhabiting are at par with the present results. in marine water of Kerala had negatively allometric growth pattern (b=2.0214). The The value of ‘b’ remains constant at ‘3’in an sedentary form of habitat may influence the ideal fish (Allen, 1938), but under natural deviation of b-value from isometric value conditions the value of ‘b’ usually ranges (b=3) due to stress factors in the habitat. between 2.5 and 4 (Hile, 1936; Martin, Weatherly and Gill (1987) found that higher 1949). In the present study ‘b’ varied metabolic activity with spawning season Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

Length weight analysis of stolephorus indicus of Kerala coast 375 lowers the ‘b’ value while less metabolic bergii (DeMan) of Dakatia River. activities, accumulation of fat, weight of Bangladesh Journal of Aquaculture., 1(1): gonad etc. during the pre-spawning period 74-78. increases the values. The closeness of ‘b’ values to ‘3’ also suggests a healthy Allen, K.R. 1938. Some observations on the environment for the fishes with respect to biology of the trout (Salmotrutta) in feeding and growth. Windermere, J.Anim.Ecol., 7:333-349 Conclusion Bijukumar, A., Sirajudheen, T.K., Rajesh, L and Radhakrishnan, T. 2010. Marine fish and In the present study, relationship of fisheries of Kerala- Heritage, diversity and Stolephorus indicus not followed the cube conservation paradigms. 32–102pp. law. So the fish has not followed isometric growth pattern. In the present work the b Prasad, G and Anvar Ali, P.H. 2007. Length- value is deviating from the ideal b value, less Weight Relationship Of A Cyprinid Fish than ‘3’ therefore fishes exhibit a negative Puntius Filamentosus From Chalakudy River, allometric length weight relationship it Kerala. Zoos' Print Journal., 22(3): 2637- indicates that the fish tend to become thinner 2638 as they grow larger. Hile, R.1936. Age and growth of the Cisco, The length-weight relationship in fishes can Lieucichthys artedi (Le Sueur) in the lakes of be affected by a number of factors including the North eastern high lands, Wiscorsin. season, habitat, gonad maturity, sex, diet, and Bull.U.S.Bur.Fish., 48: 211-317 stomach fullness, health and preservation techniques, and differences in the length Kurma Rao. R. and Ramesh Babu, K. 2013. ranges of the specimen caught. The exact Studies on length-weight relationship of relationship between length and weight Mugil cephalus (Linnaeus, 1758), east coast differs among species of fish according to of Andhra Pradesh, India. Pelagia Research their inherited body shape, and within a Library Advances in Applied Science species according to the condition Research., 4(3): 172-176 (robustness) of individual fish. Condition sometimes reflects food availability and Kunda, M., Dewan, S., Uddin, M. J., Karim, growth within the weeks prior to sampling. M., Kabir, S. and Uddin, M. S. 2008. Length- But, condition is variable and dynamic. Weight Relationship, Condition Factor and Individual fish within the same sample vary Relative Condition Factor of Macrobrachium considerably, and the average condition of rosenbergii in Rice Fields. Asian Fisheries each population varies seasonally and yearly. Science., 21 (2008):451-456 451. References Moorthy, K. K. and Saha, A. 2000. Aerosol study during INDOEX: Observation of Aliakbar, M. and Ali, M.L. 1978. Length- enhanced aerosol activity over the mid weight relationship of Macrobrachium rosen- Arabian Sea during the northern winter. J. Atmos. Sol. Terr. Phys., 62: 65 – 72. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

376 Current status and challenges for conservation and sustainable use of biodiversity Mnwani, C.D. 2004. Aspects of the biology of mormyrids in Anambra River, Nigeria. Ph.D Thesis University of Nigeriaartin,W.R. 1949, The mechanics of environmental control of body form in fishes. Vié, J. C., Hilton-Taylor, C. Y and Stuart, S. N. 2009. Wildlife in a Changing World - An Analysis of the 2008 IUCN Red List of Threatened Species. IUCN. Gland, Switzerland. 180 pp. Weatherly, A. H. and Gill H. S.1987. The Biology of Fish Growth. Academic Press, London. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.371-376 © Principal, Sree Narayana College, Kollam, Kerala, India

Regulatory role of curcumin in overcoming chemoresistance to 5-fu in breast cancer cells via … 377 ISBN 978-93-5396-871-7 CHAPTER 54 REGULATORY ROLE OF CURCUMIN IN OVERCOMING CHEMORESISTANCE TO 5-FU IN BREAST CANCER CELLS VIA CYCLIN D1 AND BCL2 Vinod B S* Haritha H Nair** and Ruby John Anto** *Department of Biochemistry, Sree Narayana College, Kollam **Division of Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram *Correspondence E-mail: [email protected] ABSTRACT Cancer is a major cause of morbidity and mortality in developing countries including India. According to ICMR, India is likely to have over 17.3 lakh new cases of cancer by 2020, and 8.8 lakh deaths will be due tocancers of breast, lung and cervix. Based on two population based cancer registries in Kerala, it is estimated for the year 2020 that, 147 new cancer cases occur in one day with an annual prevalence of 1, 61,307 cancer patients in Kerala. Studies have shown that, among women, breast cancer rate has increased and cervical cancer incidence has reduced over the past two decades. Breast cancer is a heterogeneous and complex disease, wherein, the therapy response is highly dependent on the expression status of the receptors, ER, PR and HER2. The anti-metabolite agent 5-fluorouracil (5-FU) is widely used in the treatment of severaltypes of cancers including that of breast, but a major problem related to its use is the gradual development of resistance by the cancer cells. Key words: Cytotoxicity, Curcumin, BCL2, CYCLIN D1 Introduction and Bcl-2 in the synergistic cytotoxicity of Curcumin and 5-FU. Our earlier studies have reported that, Materials and Methods Curcumin enhances 5-FU-induced Cell lines: The breast cancer cell line MDA- cytotoxicity in a panel of breast cancer cell MB-231was purchased from National Center for Cell Sciences (Pune, India). Chemicals: lines of diverse receptor status, while Dulbecco’s modified Eagle’s medium were obtained from Life Technologies (Grand beingnon-toxic towards normal/immortalized Island, NY, USA), antibodies against Cyclin D1 and Bcl-2 were purchased from Santa cells(Vinod et al., 2013) Also, we have Cruz Biotechnology (Santa Cruz, CA, USA). 5-FU was procured from Calbiochem (San elucidated the role of various key molecules Diego, CA, USA). All otherchemicals were including NF-κB, (Nuclear factor Kappa B) in regulating the synergistic effect. The objective of the present study is to find out the involvement of two important downstream targets of NF-κB, i.e., Cyclin D1 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.377-380 © Principal, Sree Narayana College, Kollam, Kerala, India

378 Current status and challenges for conservation and sustainable use of biodiversity purchased from Sigma Chemicals (St. Louis, 5-FU-induced a time-dependent activation MO, USA). of Bcl-2 and Cyclin D1 which is significantly down-regulated by curcumin Mode of treatment: In all combination treatments, Curcumin (10 µM) wasadded 6 h Upon treatment of 5-FU (10 µM) in MDA- before 5-FU (10 µM) treatment. MB-231 cells, there was an obvious time- dependent up-regulation of Bcl-2 and Cyclin Western blot analysis: Total protein isolated D1 upto 24 hours(Fig. 1).The expression of from cells after indicated treatments were Bcl-2 begin to increase from 6hrs onwards subjected to western blotting as described and peaked at 12 and 24hrs and began to earlier (Anto, 2002). Briefly, 60 µgof whole decline from 48 hrs; whereas Cyclin D1 cell protein was resolved on a 10–15% expression peaked at 12 hrs and thereafter polyacrylamide gel, transferred to a PVDF begins to decline. Furthermore, we checked membrane, incubated with the corresponding the expression of Bcl-2 and Cyclin D1 when antibody and detected by ECL (Millipore, a combination of 5-FU (10 µM) and Billerica, MA, USA). Curcumin (10 µM) was given in MDA-MB- 231 cells Results (Fig. 2). For the combination treatment, we NF-κB has been shown to regulate the exposed the cancer cells to Curcumin (10 µM) alone for 6hrs and thereafter given a expression of a number of genes whose combination of 5-FU (10 µM) and Curcumin (10 µM) for 24hrs for Bcl-2 and 12hrs for products are involved in tumorigenesis Cyclin D1.There was a clear down-regulation of 5-FU induced Bcl2 and Cyclin D1 (Garg, 2002).These include anti-apoptosis activation by Curcumin in MDA-MB-231 cells which clearly indicate that, Curcumin genes (e.g., cIAP, survivin,TRAF, bcl-2, and treatment is effective in reducing the chemoresistance induced by 5-FU via Bcl-2 bcl-xl); COX-2; MMP-9; genes encoding and Cyclin D1 in this triple negative cell line. adhesion molecules, chemokines, inflammatory cytokines, and iNOS; and cell cycleregulatory genes such asCyclin D1 (Shishodia,2002). NF-κBmediates survival signals that counteract apoptosis by activating the expression of the above said anti-apoptotic genes. The expression status of two proteins which are regulated by NF- κBviz: Bcl-2 and Cyclin D1 wasinvestigated at this point. Fig.1 Kinetics of 5-FU-induced activation of Bcl-2 and Cyclin-D1 in MDA-MB-231 cells after treating them with 5-FU for different time intervals(0-48hrs). The whole cell lysate was immunoblotted using antibody against Bcl-2 and Cyclin-D1 and detected by ECL. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.377-380 © Principal, Sree Narayana College, Kollam, Kerala, India

Regulatory role of curcumin in overcoming chemoresistance to 5-fu in breast cancer cells … 379 Fig.2 Effect of curcumin either alone or in combination with 5-FU in Bcl-2 and Cyclin-D1 expression in MDA-MB-231 cells. Western blot analysis was performed on whole cell lysates after12 hours of drug exposure. Beta actin was used as loading control Discussion Curcumin at this concentration could down- regulate the anti-apoptotic protein Bcl-2 and The development of drug resistance and dose cell cycle regulatory protein Cyclin-D1, both limiting cytotoxicitygreatly impede the use of of which are important candidates of 5-FU, one of the first rationally designed chemoresistance. anti-metabolites used in chemotherapy.One of the major signaling pathway responsible References for chemoresistance induced by various chemotherapeutics including 5-FU is NF-κB. Anto, R.J., Mukhopadhyay, A., Denning, Several studies including ours (2)have shown K. and Aggarwal, B.B. 2002. Curcumin that down-regulation of NF-κB by various (diferuloylmethane) induces apoptosis means could enhance therapeutic efficacy of through activation of caspase-8, BID 5-FU. The ability of curcumin to down- cleavage and cytochrome c release: its regulate constitutive and inducible NF-κB, suppression by ectopic expression of thereby enhancing the activity of various Bcl-2 and Bcl-xl. Carcinogenesis., chemotherapeutics has been well 23(1):143-50. documented. Activation of NF-κB leads to activation of its downstream targets including Garg,A. and Aggarwal, B.B. 2002. anti-apoptotic genes (cIAP, survivin, Bcl2, Nuclear transcription factor-kappaB as a and BCl-xL), cell cycle-regulatory genes target for cancer drug development. (cyclin D1), genes encoding adhesion Leukemia, 16(6):1053-68. molecules, etc. In our earlier studies, we have shown that 5-FU activates NF-κB in a time- Shishodia,S. and Aggarwal, B.B. 2002. dependent manner and Curcumin (10 µM) Nuclear factor-kappaB activation: a could clearly downregulate this activation. question of life or death. J Biochem Mol Furthermore, checking its downstream Biol., 35(1):28-40. targets we could clearly establish that Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.377-380 © Principal, Sree Narayana College, Kollam, Kerala, India

380 Current status and challenges for conservation and sustainable use of biodiversity Vinod, B.S., Antony, J., Nair, H.H., signaling events regulating curcumin- Puliyappadamba, V.T., Saikia, M., mediated chemosensitization of breast Narayanan, S.S., Bevin, A. and Anto, cancer cells to 5-fluorouracil. Cell Death R.J. 2013. Mechanistic evaluation of the Dis., 21;4:e 505pp. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.377-380 © Principal, Sree Narayana College, Kollam, Kerala, India

Current status of molecular phylogeny of wolf spiders 381 ISBN 978-93-5396-871-7 CHAPTER 55 CURRENT STATUS OF MOLECULAR PHYLOGENY OF WOLF SPIDERS Abhijith R S*, Sheeba P** and Sudhikumar A V* *Centre for Animal Taxonomy and Ecology, Department of Zoology, Christ College, Irinjalakuda, Kerala-680125 **Department of Zoology, Vimala College, Thrissur, Kerala *Correspondence E-mail: [email protected] ABSTRACT Taxonomy, the mother of all applied research happening in the field of Biology never loses its importance. Morphological taxonomy of spiders has many challenges because of the presence of sexual dimorphism, polymorphism and lack of taxonomic keys for juveniles. Collaboration of morphological taxonomy and molecular biology already showed its charisma in the taxonomic field. Wolf spiders constitute the fourth largest spider family, with 2443 species described in 124 genera. They pursue an array of different prey capture strategies, from permanently vagrant hunters to permanently burrowing species, and some genera are known to build permanent sheet-webs. Climate change and global warming have influential effects on behaviour and ecology of lycosids. In Barcode of Life Database (BOLD) 9982sequences of Lycosidae are found which encompasses 374 lycosid species. Molecular data from 53 countries deposited in 60 institutions are present here. The pioneering works of molecular phylogeny of lycosids were done by Zehethofer and Sturmbauer in 1998. In 2002 Vink studied the molecular phylogeny of lycosids in New Zealand and Australia. Murphy et al. in 2006 studied lycosid molecular data from Africa, Asia and Neotropics using 28S rRNA, 12S rRNA and ND1 as molecular markers. Pavel et al. in 2018 studied molecular phylogeny of genus Alopecosa from Central Europe to compare reproductive behaviour. Since spider taxonomy has immense potential, the classical and novel applications can be attributed to spider taxonomy. In the case of Indian Lycosidae, Barcode of Life Database (BOLD) systems have 10 entries which constitutes of 5 species. Pardosa paludicola, Hippasagreenalliae, Pardosa birmanica, Hippas amadraspatana and Lycosa bistriata are the lycosid species represented from India in BOLD. Molecular techniques will aid to increase the credibility of the morphological taxonomy. The data derived from the molecular sequences will give the better insight to the evolution of organisms. Key words: DNA barcoding, Molecular taxonomy, Spider, BOLD Introduction (Ellenberg et al., 1986). Not only abundant, but they are one of the most diverse Spiders are one of the main predators in most arthropod orders, with diverse species and of the ecosystems, but have received much exhibit a great variety of foraging strategies less attention than other insect predators (Coddington and Levi, 1991; Foelix, 1996). (Whitehouse and Lawrence, 2001). Spiders The various foraging strategies can be are among the most abundant insectivorous attributed to better predatory control of predators of terrestrial ecosystems (Nyffeler insects and ecological importance. Because and Benz, 1987; Wise, 1993). They have of their high abundance and insectivorous been reported to occur in peak numbers of feeding habits, spiders are expected to playan more than 1,000 individuals per sq. m important role in the balance of nature Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India

382 Current status and challenges for conservation and sustainable use of biodiversity (Whitcomb, 1974; Gertsch, 1979; Luczak, lack of confusions in taxonomic field will aid 1979; Young and Edwards, 1990; Wise, in the betterment of applied research. 1993; Nyffeler et al., 1994a,b). In agro- ecosystems also they play an immense role as Morphological taxonomy or classical natural predators of insect pests. taxonomy is a very well established field and never loses its importance in the field of Wolf spiders (Lycosidae) constitute the biodiversity. Still sole reliability on the fourth largest spider family, with 2443 morphology can led to the misidentification, species described in 124 genera (World results in synonymy. Aid of molecular Spider Catalog, 2020). Their adult body size techniques can somewhat overcome the ranges from 1 to 30 mm. They pursue an problem and help the classical taxonomy to array of different prey capture strategies, reach a newer height of increased credibility. from permanently vagrant hunters to In India the use of molecular taxonomy is permanently burrowing species, and some still coping up the pace. Initially DNA based genera are known to build permanent sheet- taxonomy was much costlier than classical webs (Murphy et al., 2006). Wolf spiders taxonomy. Now a lot of optimisations are may also show very specific microhabitat happening so that costs will reduce, but, still preferences and may be susceptible to it is costlier than traditional taxonomy. But changes in habitat structure (Jogar et al., the benefits of molecular taxonomy outweigh 2004; Marshall and Rypstra, 1999). This the cost. Molecular taxonomy utilizes various ecological diversity may make them suitable molecular markers for the creation of for control of a wide variety of insect pests. molecular barcodes. Cytochrome oxidase They also manifest an immense role in food subunit 1 (COI) is considered to be the best web and maintain the decorum of the and hence widely used molecular marker. ecosystem. Climate change and global Many other molecular markers are in use. As warming has influential effects on behaviour same as a normal barcode uniquely defines a and ecology of lycosids. It is found that some product, a molecular barcode can represent a of the wolf spider species start to increase its whole organism. This concept already altered foraging quantity in order to maintain body the taxonomic view point in a positive way. moisture during increasing ambient Still a lot of species are unknown. It is the temperature. Another important aspect about duty of taxonomists to identify them and spiders is spider silk and its immense report them to the scientific world before applications (Malik and Barhanpurkar, 2018). them extinct from the biosphere. Another promising field of research is spider venom therapeutics. Most spider venoms are Current status of molecular taxonomy of dominated by disulfide-rich peptides that spiders especially lycosids in India is not typically have high affinity and specificity appreciable. Compared to many other for particular subtypes of ion channels and countries the entries from India is much receptors (Saez et al., 2010). Researches on lesser. The data about molecular taxonomy of spider silk and venom along with ecological lycosids are mined from the website of and phylogenetic studies will gain much Barcode of Life Database (BOLD Systems). importance in research scenario due to its Recently most of the taxonomists are direct application to mankind. These may showing interests in addition of molecular make the spider more charismatic to general techniques along with classical approach. So public and led to the avoidance of unwanted it is evident that in the near future the killing and destruction of habitat. In order to contribution from India to BOLD systems make all these possible, initially taxonomy of will increase in alarming rate. the taxa should be done. Developments and Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India

Current status of molecular phylogeny of wolf spiders 383 Results and Discussion that Arctosa and Trochosas hould be separated from Lycosinae and grouped In Barcode of Life Database (BOLD) 9982 together as a new subfamily Arctosinae. They molecular entries with Lycosidae species also recommended establishing a new name are found which encompasses 374 subfamily Hygrolycosinae to accommodate lycosid species. Molecular data of specimens the genus Hygrolycosa. from 53 countries deposited in 60 institutions are present. Pardosa moesta is the most In 2002 Vink studied the molecular represented lycosid in BOLD with 927 phylogeny of lycosids in New Zealand and entries followed by Alopecosa aculeate with Australia which highly enriched the 499 entries, 375 unspecified and Pardosa molecular phylogeny database of lycosids. mackenziana with 332 entries (Fig.1). In the Vink utilized data from nuclear gene NADH case of genera Pardosa is the most Dehydrogenase subunit 1 (ND1) and represented followed by Alopecosa, Piratula mitochondrial genes Cytochrome C oxidase and Trochosa. Canada mostly contributed to subunit 1(CO1) and 12 S rRNA gene. These the data contribution followed by United data were combined with molecular data of States, Germany and Pakistan (Fig.2). Asian, European and North American lycosids to perform phylogeny studies. Various classical taxonomic works on Studied confirmed that Australasian species lycosids can be traced from 18th century form distinct clade from other groups. Vink’s onwards. Works in lycosids using modern studies also revealed the utility of 12 rRNA techniques can be traced back to 1982 when gene data for examining closely related Elliot et.al examined allozymic variation in genera and its limitation to study deeper nine protein producing loci was examined in generic relationships. three species of Pardosa using starch gel electrophoresis. They published study as Murphy et al. in 2006 studied lycosid genetic variation among three species of molecular data from Africa, Asia and Pardosa. The pioneering works of molecular Neotropics using 28 rRNA, 12 S rRNA and phylogeny of lycosids were done by ND1 as molecular markers. Along with Zehethofer and Sturmbauer in 1998. They Parsimony analysis, they also utilized analysed mitochondrial 12 S rRNA of Bayesian analysis for phylogenetic studies. ribosomal small subunit of 27 lycosid species They were skeptical about the usage of 28 from Central European region. Monophyly of rRNA as its results gave implausible results the lycosids were concluded during and omitted it from final analysis. They also phylogenetic works using high bootstarp sequenced paralogous copies of the gene and value. Through this study they also suggested cautionary approach for its further concluded that, tube builders are the most usage in lycosid phylogeny. Dimitrov et al. in ancestral among lycosids. 2017 reinstated the usage of this marker in studies of ecribillate spiders. But, as Murphy He and Song in 1996 studied the questioned the credibility of 28S marker phylogenetic studies of Chinese wolf spiders. specifically in lycosids, it will be better to use This study was based on morphological and other markers in lycosids until further ecological parameters. The result shows that modifications in 28S rRNA analysis. Lycosidae can be divided into five subfamilies: Hippasinae, Hygrolycosinae, Systematics of the new Australian wolf Arctosinae, Lycosinae and Pardosinae. spider genus Tuberculosa was done by Hippasinae found to be the most primitive of Volker W. Framenau and Yoo in 2006. all, then the Hygrolycosa, the Arctosa and Although the study was based on Trochosa, the Lycosinae and Pardosinae morphological data, phylogenetic analysis being the most advanced. They suggested was performed on four Tuberculosa species Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India

384 Current status and challenges for conservation and sustainable use of biodiversity based on 12 morphological characters. studies contradictory findings on Various evolutionary data in the Tuberculosa evolutionary relationships were obtained. So genus were revealed. they have done the molecular phylogeny using five mitochondrial and three nuclear Yan& Yan in 2007 studied phylogenetic genes to reach the conclusion. relationships of Wadicosa in Chinese wolf spiders. Mitochondrial 16S rRNA gene In 2017 Framenau and Hudson studied the sequence was utilized to analyze 26 species morphological and ecological phylogeny of from 6 genera of Lycosidae. Bayesian and halo tolerant wolf spider genus Tetralycosa Maximum parsimony analyses were used for in Australia. A phylogenetic analysis of the phylogenetic analysis.The results indicated genus identified a monophyletic clade of that Wadicosa has the closest phylogenetic eight species that live permanently on the relationship with Pardosa. Pardosaand barren surface of salt lakes suggesting a Wadicosa form a monophyletic group. They single radiation into this extremely suggested the merging of Pardosa and inhospitable habitat. Many of the species in Wadicosa to a subfamily. Polotow et al. in them are highly specialized to inhabit only 2015 studied the total evidence analysis of single salt lake which increases the the phylogenetic relationships of Lycosoidea importance of protecting the ecosystem also. spiders. The study was so extensive, which include molecular and morphological data Pavel et al. in 2018 studied molecular from 7 families in Lycosoidea superfamily. phylogeny of Alopecosa genus from Central The morphological–behavioural matrix Europe to compare it with reproductive comprised 96 characters, and four gene behaviour. They sequenced one fragments were used: 28S (∼737 base pairs), mitochondrial (COI) and two nuclear genes actin (∼371 base pairs), COI (∼630 base (28S, H3) to reconstruct their phylogenetic pairs) and H3 (∼354 base pairs). Monophyly relationships. The results of the phylogenetic of the Lycosidae family and evolution of analyses were largely incompatible with grate shaped tapetum concluded in the study. classical morphology based grouping. The Albo et al. in 2017 conducted molecular monophyly of Alopecosa could not be phylogenetic analyses to show that recovered by the study. Both Bayesian Trechaleidae and Lycosidae are sister groups. inference and Maximum Likelihood were Based on morphological and behavioural conducted. In both the genus showed to split into two different clades. 927 499 375 332 309 294 282 Pardosa Alopecosa Unspecified* Pardosa Pardosa Pardosa Trochosa moesta hyperborea terricola aculeata mackenziana lapponica Fig 1. Bardiagram showing the most represented lycosids in BOLD Systems Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India

Current status of molecular phylogeny of wolf spiders 385 319 239 201 Canada 617 United States Unspecified* 1141 Germany Pakistan 6336 Brazil Fig 2. Pie diagram showing the contribution of various countries to lycosid molecular database in BOLD Systems Conclusion .boldsystems .org /index.php/Public_Search Terms (Accessed on January 11, 2020). The molecular phylogenetics field of organisms is growing at a rapid pace. In Coddington, J. A. and Levi, H. W. 1991. almost all taxa molecular techniques are also Systematics and evolution of spiders being used along with morphological (Araneae). A. Rev. Ecol. Syst., 22: 565–592. characteristics. Many ambiguities in taxonomy, once taxonomists’ nightmare are Dimitrov, D., Benavides, L. R., Arnedo, M. now fading away. In Barcode of Life A., Giribet, G., Griswold, C. E., Scharff, N. Database (BOLD) 9982 molecular entries and Hormiga, G. 2017. Rounding up the with Lycosidae species name are found usual suspects: a standard target‐ gene which encompasses 374 lycosid species. approach for resolving the interfamilial Molecular data of specimens from 53 phylogenetic relationships of ecribellate countries deposited in 60 institutions are orb‐ weaving spiders with a new present. Still contributions from many family‐ rank classification (Araneae, developing countries are very sparse. The Araneoidea). Cladistics, 33(3), 221-250. real aim of taxonomy came into true only when data from entire world is collaborated Ellenberg, H., Mayer, R. and Schauermann, together. J. 1986. Ökosystemforschung-Ergebnisse des Sollingprojekts: 1966–1986. Stuttgart, References Ulmer. Albo, M. J., Bidegaray-Batista, L., Foelix, R. F. 1996. Biology of spiders (2nd Bechsgaard, J., da Silva, E. L. C., Bilde, T. ed.). New York, Oxford University Press and and Pérez-Miles, F. 2017. Molecular Georg ThiemeVerlag. phylogenetic analyses show that Trechaleidae and Lycosidae are sister groups. Framenau, V. W. and Hudson, P. 2017. Arachnology, 17(4):169-177. Taxonomy, systematics and biology of the Boldsystems.org. 2019. Bold Systems v4. Australian halotolerant wolf spider genus [online] Available at: http: // www Tetralycosa (Araneae: Lycosidae: Artoriinae). European Journal of Taxonomy, (335). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India