Browsing by Author "Chaitanya, R.K."

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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.
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.
Gut-specific arylphorin mediates midgut regenerative response against Cry-induced damage in Achaea janata
(Elsevier Inc., 2021-04-13T00:00:00) Dhania, Narender K.; Chauhan, Vinod K.; Abhilash, Dasari; Thakur, Vivek; Chaitanya, R.K.; Dutta-Gupta, Shourya; Dutta-Gupta, Aparna
Development of insect resistance to biopesticides is a current and pertinent global issue. Earlier, it was established that lepidopteran larvae can recover from Bt intoxication via a midgut regenerative response and subsequently generate resistance. Molecular aspects of restoration of the midgut integrity following toxin exposure are emerging recently. In the present study, we bring out the pivotal role of gut arylphorin in mediating the midgut regenerative response following sublethal Bt exposure in Achaea janata. Bt-induced midgut damage was characterized by microscopic analysis using differential interference contrast (DIC) and immunofluorescence (IF). Extensive disruption of brush-border membrane, associated with the underlying cytoskeletal alterations including F-actin, ?-actin and ?-tubulin was observed. Single-photon fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) established the metabolic state associated with enhanced stem cell proliferation and migration from the basal side towards the luminal side following the damage. In-silico analysis revealed the phylogenetic relationship of gut arylphorin with closely related insect species and indicated the presence of two different subunits. Transient RNAi knockdown of the arylphorin resulted in diminished expression of mitotic Cyclin B mRNA levels. Human monoclonal Cyclin B antibody cross-reactivity with the Cyclin B located in the stem cells further validate the role of arylphorin as the mitogenic factor responsible for stem cell proliferation and epithelial regeneration. An in-depth understanding of resistance mechanisms will aid in the design of new strategies for the long-term usage and efficacy of Bt technology against pest control. � 2021
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).
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.
Plasmodium's journey through the Anopheles mosquito: A comprehensive review
(Elsevier B.V., 2020-12-17T00:00:00) Singh, Meghna; Suryanshu; Kanika; Singh, Gurmeet; Dubey, Alok; Chaitanya, R.K.
The malaria parasite has an extraordinary ability to evade the immune system due to which the development of a malaria vaccine is a challenging task. Extensive research on malarial infection in the human host particularly during the liver stage has resulted in the discovery of potential candidate vaccines including RTS,S/AS01 and R21. However, complete elimination of malaria would require a holistic multi-component approach. In line with this, under the World Health Organization's PATH Malaria Vaccine Initiative (MVI), the research focus has shifted towards the sexual stages of malaria in the mosquito host. Last two decades of scientific research obtained seminal information regarding the sexual/mosquito stages of the malaria. This updated and comprehensive review would provide the basis for consolidated understanding of cellular, biochemical, molecular and immunological aspects of parasite transmission right from the sexual stage commitment in the human host to the sporozoite delivery back into subsequent vertebrate host by the female Anopheles mosquito. � 2020 Elsevier B.V. and Soci�t� Fran�aise de Biochimie et Biologie Mol�culaire (SFBBM)
Role of midgut digestive proteases upon sub-lethal exposure of Cry toxin in the larvae of castor semilooper, Achaea janata
(Central University of Punjab, 2018) Sureshgopi, D; Chaitanya, R.K.
Development of synthetic insecticides to reduce the level of infestation led to deleterious effects on the environment and human health. This lead to the development of eco-friendly pest management alternatives including Bacillus thuringensis (Bt). Bt produces Crystal (Cry), Cytotoxic (Cyt) and Vegetative (Vip) proteins with insecticidal activity against different orders of Lepidoptera. Of late, pest resistance against Bt is being reported in many parts of the country. The foliar spray may lead to the reduced toxicity of Bt formulation from degradation by UV light, wash-off by rain, drying, temperature, and soil acidity as well as its chemistry. Further, insects sense pesticides through odorant receptors and move away quickly. Under these conditions, there is always a possibility of a population of larvae to get exposed to sub-lethal doses of toxin which might exhibit variable effects and escape mortality and eventually generate resistance. The role played by digestive proteases in the activation of the Cry toxin and digestion when administered in sub-lethal concentration could help in the elucidation of key phenomena like adaptations for survival and resistance development. Elucidation of these digestive proteases would enhance our understanding of resistance development. The current study is an attempt to monitor the transcript levels of digestive proteases upon sub-lethal exposure of Cry toxin in the larvae of an economically important polyphagous pest castor semilooper, Achaea janata prevalent in the Indian subcontinent.