Ankola, K.

Arun Prakash, .

Puttaraju, H.P.

Rajas Rao, .

Supriya, V.K.

Vaishnavi, N.

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors,nonprofit publishers, academic institutions, research libraries, and research funders in the common goal ofmaximizing access to critical research.An Efficient Innate Immune Response of Danausgenutia (Lepidoptera: Nymphalidae) Larvae isIndependent of Hemocyte Concentration Against E.coliAuthor(s): Kunal Ankola, N. Vaishnavi, V. K. Supriya, ArunPrakash, Rajas Rao, and H. P. PuttarajuSource: Entomological News, 125(2):147-150.Published By: The American Entomological SocietyDOI: http://dx.doi.org/10.3157/021.125.0208URL: http://www.bioone.org/doi/full/10.3157/021.125.0208BioOne (www.bioone.org) is a nonprofit, online aggregation of coreresearch in the biological, ecological, and environmental sciences. BioOneprovides a sustainable online platform for over 170 journals and bookspublished by nonprofit societies, associations, museums, institutions, andpresses.Your use of this PDF, the BioOne Web site, and all posted and associatedcontent indicates your acceptance of BioOne’s Terms of Use, available atwww.bioone.org/page/terms_of_use.Usage of BioOne content is strictly limited to personal, educational, andnon-commercial use. Commercial inquiries or rights and permissionsrequests should be directed to the individual publisher as copyright holder.AN EFFICIENT INNATE IMMUNE RESPONSE OFDANAUS GENUTIA (LEPIDOPTERA: NYMPHALIDAE)LARVAE IS INDEPENDENT OF HEMOCYTE CONCENTRATION AGAINST E. COLI1Kunal Ankola,2,3 N. Vaishnavi,2 V. K. Supriya,2 Arun Prakash,2Rajas Rao,2 and H. P. Puttaraju2,3,4The immunobiology of insects is different from that of vertebrates by havingonly nonspecific innate immune systems (Freitak et al., 2003). Many parasitesand pathogens of bacteria, virus, fungi, protozoa and metazoa are known to infectinsects during different developmental periods (Tunaz and Stanley, 2009). How -ever, insects have their own strategies to combat the infection by cellular andhumoral defense mechanisms of their nonspecific immune response (Gil les pie,et al., 1997). The immune system of insects exhibits different com pon ents in -clud ing hemocytes and an enzyme, phenoloxidase (Satterfield, et al., 2013). Thehemocytes are the specific blood cells that play a central role in the immuneresponse involving recognition, phagocytosis, and encapsulation of invadingpathogens (Rolff and Reynolds, 2009; Satterfield et al., 2013).Danaus genutia (Lepidoptera: Nymphalidae) is one of the common milkweedbutterflies found in India. The butterfly is known as the milkweed butterfly sincethe larvae feed on milkweeds (Apocynaceae) which possess a high alkaloid con-tent. Feeding on alkaloid-rich foliage makes the larvae unpalatable to their pred-ators. Due to their abundance and attractive coloration, these butterflies arereared in both open and closed butterfly conservatories. However, it is estimatedthat the population of D. genutia has drastically declined in India during the pastfew decades (Atluri et al., 2010) due to various reasons.The survival of any insect in its natural habitat is directly influenced by itsimmune response (Satterfield et al., 2013). In migratory butterflies, the energyallocation for movement has direct influence on the energy spent on immunity(Rolff and Siva-Jothy, 2003; Satterfield et al., 2013). However, the larval stagesin butterflies are more susceptible than the adults to various microbial diseases.Infection decreases the lifespan, especially in migratory insects (Satterfield et al.,2013) like butterflies. There are several types of hemocytes present in the hemo -lymph which aid in resisting the physiological abnormalities induced by variouspathogens. The present study was carried out to gather preliminary informationVolume 125, Number 2, August 2015 147______________________________1 Received on September 7, 2014.  Accepted on February 25, 2015.2 Department of Biological Sciences, School of Natural Science, JB Campus, Bangalore University,Bengaluru, 560056, Karnataka, India.3 Department of Biological Sciences, School of Natural Science, JB Campus, Bangalore University,Bengaluru, 560056, Karnataka, India.4 Corresponding author Email: puttarajuhp@hotmail.comMailed on September 3, 2015regarding the hemocyte efficiency of Danaus genutia larvae against the commongram negative bacteria E. coli. Eggs and larvae of Danaus genutia were collected from their host plants(Asclepias curassavica and Cynanchum dalhousiee) from Jnana Bharathi cam-pus of Bangalore University and transported to the Insectary, Division of Bio -logi cal Science. Larvae were fed with the leaves of both Cynanchum dalhousieeand Asclepias curassavica. Only third instar (and above) larvae were used for theexperiments. E. coli cultures were obtained from the Department of Micro bi -o logy, Bangalore University and were maintained on nutrient agar. The cultureswere kept in an incubator shaker (overnight) at 37°C. Subculturing was donewith sufficient plates and broth after 24 hours of incubation. The hemolymphwas collected from the larvae by severing the third proleg and then diluted withan anticoagulant solution (0.01 M ethylenediamine tetraacetic acid, 0.1 M glu-cose, 0.062 M NaCl, and 0.026 M citric acid, pH 4.6) and phosphate buffer (pH7, 10 mM).The hemolymph was then subjected to tenfold serial dilution in 10 vials con-taining 45µl distilled water. For each vial, 0.3 µl of E. coli was added and incu-bated overnight at 37°C in an incubator shaker. For each tube, optical densitywas measured at 600 nm through an Eppendorf biophotometer. The antibacteri-al activity of hemolymph was analyzed by the disc diffusion method. Three Agarplates were made up of 1ml E. coli suspension, each with four divisions whichwere a, b, c, d. A Watmann paper disc of 0.55mm dia. was used for the experi-ment with different volumes of hemolymph in each division. The zone of inhibi-tion was recorded and analyzed in each division of all three plates.The hemolymph was subjected to different dilutions to obtain different con-148 ENTOMOLOGICAL NEWSVolume 125, Number 2, August 2015 149centrations against a constant bacterial load. The minimum concentration ofhemo lymph required for the inhibition of bacterial growth was determined byusing the spectroscopic method and the resistance capacity was analyzed by thedisc diffusion method. The higher concentration of hemolymph obviously couldreduce bacterial growth but also the lowest concentration was good enough toinhibit the proliferation of bacteria (Fig. 1). Further, there was a steady variationin the bacterial proliferation which was not much influenced by hemolymph con-centration (Fig. 2). The zone of inhibition was similar in all the divisions of thethree plates. Both these outcomes signify high efficiency of the larval immune systemagainst E. coli. The hemocytes consist of several components which are essentialfor defense against various pathogens (Clark et al., 1997; Fang et al., 2010). Thehumoral immunity of the insect is equipped with antimicrobial peptides and theenzyme phenoloxidase, which critically eliminates the microbial infection fromthe insect system. Moreover, phagocytosis and encapsulation aids the cellularimmune response along with the plasmatocytes (Clark et al., 1997). However, thepresent study did not specify the role of individual hemocytes against bacterialinfection but instead focused on the overall performance of innate immunity.This preliminary information could be a good platform for further investigationin the immunobiology of this butterfly.LITERATURE CITEDAtluri, J. B., M. Bhupathi Rayalus, and D. Sandhya Deepika. 2010. Life history and larval per-formance of the common tiger butterflies, Danaus genutia Cramer (Lepidoptera; Rhopalocera;Danaidae). The Bioscan 5(3): 511-515.Clark, K. D., L. L. Pech, and M. R. Strand. 1997. Isolation and identification of a plasmatocytespreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens. Journalof Biological Chemistry 272(37): 23440-23447. Fang, Q., L. Wang, J. Zhu, Y. Li, Q. Song, D. W. Stanley, Z. Akhtar, and G. Ye. 2010.Expression of immune response genes in lepidopteran host is suppressed by venom from an endo -parasitoid, Pteromalus puparum. http://www.biomedcentral.com/1471-2164/11/484. Freitak, D., I. Ots, A. Vanatoa, and P. Horak. 2003. Immune response is energetically costly inwhite cabbage butterfly pupae. Biology Letters DOI 10.1098/rsbl.2003.0069. Gillespie, J. P., M. R. Kanost, and T. Trenczek. 1997. Biological mediators of insect immunity.Annual Review of Entomology 42: 611-643. Rolff, J. and M. T. Siva-Jothy. 2003. Invertebrate ecological immunology. Science 301(5632):472-475.Rolff, J. and S. E. Reynolds. 2009. Insect Infection and Immunity: Evolution, Ecology and Me cha -nisms. New York: Oxford University Press.Satterfield, D. A., A. E. Wright, and S. Altizer. 2013. Lipid reserves and immune defense inhealthy and diseased migrating monarchs Danaus plexippus. Current Zoology 59 (3): 393-402.Tunaz, H. and D. Stanley. 2009. An immunological axis of biocontrol: infections in field trappedinsects. Naturwissenschaften 96: 115-119.150 ENTOMOLOGICAL NEWS

An Efficient Innate Immune Response of Danaus Genutia (Lepidoptera: Nymphalidae) Larvae is Independent of Hemocyte Concentration against E. Coli

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An Efficient Innate Immune Response of Danaus Genutia (Lepidoptera: Nymphalidae) Larvae is Independent of Hemocyte Concentration against E. Coli

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