International Journal of Entomology Research 16 International Journal of Entomology Research ISSN: 2455-4758; Impact Factor: RJIF 5.24 www.entomologyjournals.com Volume 2; Issue 4; July 2017; Page No. 16-20 Expression of regucalcin gene in cold acclimated diapause eggs of red cotton bug Dysdercus cingulatus (Fabricius, 1775) Shahjahan AS, * Encily R Martin Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Abstract The expression of regucalcin gene in cold acclimated diapause eggs of Dysdercus cingulatus was studied. The DNA and RNA were isolated from diapause induced eggs to low temperature and were used for the amplification of regucalcin gene. The PCR gel electrophoresis image shows that RNA and DNA obtained from the eggs had no traces of regucalcin gene. Thus present study shows that regucalcin gene was not expressed in diapause eggs and it may not play any role in cold tolerance diapause eggs. Keywords: regucalcin gene, cold acclimation, diapause and Dysdercus cingulatus Introduction In nature, organisms exposed to changing temperature generally inflict stress which may result in the evolution of adaptive genetic mechanisms to cope up with extreme temperatures (Dillon, et al., 2009; Ayrinhac, et al., 2004 and Rako and Hoffmann, 2006 [7, 2, 17] there are limited studies that support the involvement of genes for survival at low temperature in insects, (Clark and Worland, 2008) [5] .Goto (2000) [9] identified gene for cold tolerance in Drosophila by using substractive hybridization and reported that the Dca gene, is involved in cold acclimation, and it is upregulated at the transcription level, in D. melanogaster flies at 15ºC for 1 day. Similarly, differential gene expression of Dca in D. subobscura was found in flies reared at either 13ºC or 22ºCby microarray studies (Laayouni, et al., 2007) [14] . The microarrays and real time-polymerase chain reaction (RT- PCR) techniques have showed that Dca is downregulated after a cold shock at 0ºC for 1–3 h (Sinclair, et al., 2007) [20] . Studies have shown that Dca is most likely involved in cold adaptation and not in response after a cold shock. Arboleda- Bustos, et al., (2011) [1] reported that regucalcin gene is present in all genes of Drosophila Gene expression in diapausing organism is observed in various tissues and brain. The brain is the main regulatory part that serves as the repository of the diapause programme (Giebultowicz and Denlinger, 1986) [8] . Diapause specific proteins are expressed in brain during the pupal diapause inflesh fly, Sarcophaga crassipalpis which plays a significant role in silencing many genes, but a few genes are uniquely expressed during diapause (Joplin, et al., 1990) [12] . Many proteins are synthesized in brain of non-diapausing and diapausing fly pupae (Joplin, et al., 1990) [12] . In S. crassipalpis the genes are identified prior to expression, during and after diapause in the brain of non-diapausing pupae, and it encodes for heat shock 70 cognate protein, and 28S ribosomal protein (Rinehart et al., 2000) [19] , and the cell cycle regulators cyclin E, p21 and p53 (Tammariello and Denlinger, 1998) [22] . Though the cell cycle arrest is presumably common to all diapauses, the stage of the arrest does not appears to be the same in all species (Tammariello, 2001) [21] . The cell of the optic lobe enlarges in the tobacco hornworm M.sexta in a G2 arrest during pupal diapause (Champlin and Truman, 1998) [3] , similarly, in the cells of B. mori during embryonic diapause (Nakagaki, et al., 1991) [16] . Numerous genes are known to regulate the G2/M transition, including two cdc2-related Ser/Thr kinases in B. mori (Iwasaki, et al., 1997) [11] and regucalcin gene with unknown regulation (Goto and Denlinger, 2002) [10] . The relation between the Dca gene expression and cold stress was studied by Goto (2000) [9] . The Dca gene is up-regulated after acclimation to 15ºC and is single copy in genome, located at position 88D on chromosome 3R, and has some introns with molecular weight of 33.3 KDa (Clowers, et al., 2010) [6] . Nevertheless, in D. melanogaster, regucalcin and smp30, gene for thermal adaptation are the names of two distinct genes that code for proteins with 71.9% 5 amino acid identities (Reis, et al., 2011) [18] . The sequence variation in Dca is associated with variation in chill coma recovery time, which indicates its adaptive role in cold tolerance (Clowers, et al., 2010) [6] . Itappears that Dca arose by a duplication event from the ancestral regucalcin like gene after the spilt of Sophophora and Drosophila subgenera. This gene is present in only a single copy, which bears more sequence to regucalcin than Dca (Arboleda-Bustos and Segarra, 2011) [1] . The function of Dca or regucalcin in insect is not known but in mammals, it regulates regucalcin that is expressed in the cells as a Ca²+ binding protein that hold EF-hand motif of the Ca²+ binding domain, (Yamaguchi, 2013) [25] . The regucalcin gene is highly conserved in mammals and invertebrates, over 15 species consisting of regucalcin family in vertebrate and invertebrate species have been identified with different function (Yamaguchi, 2011) [24] . In the present study, the regucalcin gene expression in cold acclimated diapause eggs of red cotton bug, Dysdercus cingulatus was studied as this gene is reported in various species of Dorsophila sp. Material and Method The red cotton bug, Dysdercus cingulatus, was reared in the laboratory for 2 generation and fed on cotton soaked seeds. After 2 generation of rearing, eggs were taken for experiment. For artificial induction of eggs into diapause, eggs were