School Of Basic And Applied Sciences
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Item Modulation of midgut peritrophins' expression during plasmodium infection in anopheles stephensi (Diptera: Culicidae)(Indian Academy of Sciences, 2017) Venkat Rao, V.; Kolli, S.K.; Bargava, S.; Chaitanya, R.K.The peritrophic matrix (PM) serves as a barrier to pathogens in many disease vectors including mosquitoes. The Plasmodium ookinete has to cross the PM barrier for its successful establishment in the mosquito midgut and subsequent transmission. It is conceived that alterations to PM may lead to a block in infection. Peritrophins which are the major constituents of PM are yet to be elucidated at molecular level. The present study demonstrates Anopheles stephensi midgut peritrophins' expression during Plasmodium berghei infection. Eight peritrophin genes (Per 10, Per 16, Per 22, Per 25, Per 26, Per 28, Per 30 & Per 43) of A. stephensi were identified from vectorbase, isolated from the adult midgut, and expression pattern monitored in real-time, in normal and infected blood meal conditions. Temporal expression of peritrophins in the midgut was monitored every 6 h till 24 h post blood meal. Results showed that the Per 10, Per 16, Per 22, Per 25 and Per 26 expression was significantly downregulated during Plasmodium infection whereas Per 30 and Per 43 expression was markedly up-regulated. The Per 28 expression was low initially but elevated later. This data clearly indicates that peritrophins are differentially modulated in infected midgut. The significance of differential expression of peritrophins' in parasite transmission is discussed further.Item Cloning and characterization of a riboflavin-binding hexamerin from the larval fat body of a lepidopteran stored grain pest, Corcyra cephalonica(Elsevier Inc., 2016) Rao, V. Venkat; Ningshen, Thuirei Jacob; Chaitanya, R. K.; Senthilkumaran, B.; Dutta-Gupta, Aparna; Rao, V.V.; Ningshen, T.J.; Chaitanya, R.K.; Senthilkumaran, B.; Dutta-Gupta, A.In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat body of Corcyra cephalonica. The complete cDNA (2121 bp) encodes a 706-amino acid protein with a molecular mass ~ 82 kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is prominently expressed during the last instar larval development. Homology modeling and docking studies predicted riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect. However, JH in the presence of 20HE maintains the RbHex 82 expression which indicates the JH's role as a status quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran pest. Further, the possibility of RbHex 82 as a pest control target is discussed. ? 2016 Elsevier Inc.Item Cloning, characterization and transmission blocking potential of midgut carboxypeptidase A in Anopheles stephensi(Elsevier B.V., 2017) VenkatRao, V.; Kumar, S.K.; Sridevi, P.; Muley, V.Y.; Chaitanya, R.K.Transmission-blocking vaccines (TBV) interrupt malaria parasite transmission and hence form an important component for malaria eradication. Mosquito midgut exopeptidases such as aminopeptidase N & carboxypeptidase B have demonstrated TBV potential. In the present study, we cloned and characterized carboxypeptidase A (CPA) from the midgut of an important malarial vector, Anopheles stephensi. ClustalW amino acid alignment and in silico 3-dimensional structure analysis of CPA predicted the presence of active sites involved in zinc and substrate binding that are conserved among all the known mosquito species. Real-time PCR analysis demonstrated that CPA is predominantly expressed in the midgut throughout the mosquito life cycle and that this gene is significantly elevated in P. berghei-infected mosquitoes compared to uninfected blood-fed controls. The high midgut CPA activity correlated with the prominent mRNA levels observed. Peptide-based anti-CPA antibodies were raised that cross-reacted specifically to ?48?kDa and ?37?kDa bands, which correspond to zymogen and active forms of CPA. Further, the addition of CPA-directed antibodies to P. berghei-containing blood meal significantly reduced the mosquito infection rate in the test group compared to control and blocked the parasite development in the midgut. These results support further development of A. stephensi CPA as a candidate TBV. ? 2016 Elsevier B.V.Item Larval mid-gut responses to sub-lethal dose of cry toxin in lepidopteran pest Achaea janata(Frontiers Media S.A., 2017) Chauhan, Vinod K.; Dhania, Narendra K.; Chaitanya, R. K.; Senthilkumaran, Balasubramanian; Dutta-Gupta, Aparna; Chauhan, V.K.; Dhania, N.K.; Chaitanya, R.K.; Senthilkumaran, B.; Dutta-Gupta, A.The lack of homogeneity in field application of Bacillus thuringiensis formulation often results in ingestion of sub-lethal doses of the biopesticide by a fraction of pest population and there by promotes the toxin tolerance and resistance in long term. Gut regeneration seems to be one of the possible mechanism by which this is accomplished. However, the existing information is primarily derived from in vitro studies using mid-gut cell cultures. Present study illustrates cellular and molecular changes in mid-gut epithelium of a Bt-susceptible polyphagous insect pest castor semilooper, Achaea janata in response to a Cry toxin formulation. The present report showed that prolonged exposure to sub-lethal doses of Cry toxin formulation has deleterious effect on larval growth and development. Histological analysis of mid-gut tissue exhibits epithelial cell degeneration, which is due to necrotic form of cell death followed by regeneration through enhanced proliferation of mid-gut stem cells. Cell death is demonstrated by confocal microscopy, flow-cytometry, and DNA fragmentation analysis. Cell proliferation in control vs. toxin-exposed larvae is evaluated by bromodeoxyuridine (BrdU) labeling and toluidine blue staining. Intriguingly, in situ mRNA analysis detected the presence of arylphorin transcripts in larval mid-gut epithelial cells. Quantitative PCR analysis further demonstrates altered expression of arylphorin gene in toxin-exposed larvae when compared with the control. The coincidence of enhanced mid-gut cell proliferation coincides with the elevated arylphorin expression upon Cry intoxication suggests that it might play a role in the regeneration of mid-gut epithelial cells. ? 2017 Chauhan, Dhania, Chaitanya, Senthilkumaran and Dutta-Gupta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).