Gut-specific arylphorin mediates midgut regenerative response against Cry-induced damage in Achaea janata.

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, α-actin and ß-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.

Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure.

The midgut of lepidopteran larvae is a multifunctional tissue that performs roles in digestion, absorption, immunity, transmission of pathogens and interaction with ingested various molecules. The proteins localized at the inner apical brush border membrane are primarily digestive proteases, but some of them, like aminopeptidase N, alkaline phosphatase, cadherins, ABC transporter C2, etc., interact with Crystal (Cry) toxins produced by Bacillus thuringiensis (Bt). In the present study, aminopeptidase N (APN) was characterized as Cry-toxin-interacting protein in the larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which have less than 40% sequence similarity but show the presence of characteristic 'GAMENEG' and zinc-binding motifs. Feeding a sublethal dose of Cry toxin caused differential expression of various APN isoform. Further, 6thgeneration Cry-toxin-exposed larvae showed reduced expression of APN2. This report suggests that A. janata larvae exploit altered expression of APNs to overcome the deleterious effects of Cry toxicity, which might facilitate toxin tolerance in the long run.

RNA-Seq analysis and de novo transcriptome assembly of Cry toxin susceptible and tolerant Achaea janata larvae.

Larvae of most lepidopteran insect species are known to be voracious feeders and important agricultural pests throughout the world. Achaea janata larvae cause serious damage to Ricinus communis (Castor) in India resulting in significant economic losses. Microbial insecticides based on crystalline (Cry) toxins of Bacillus thuringiensis (Bt) have been effective against the pest. Excessive and indiscriminate use of Bt-based biopesticides could be counter-productive and allow susceptible larvae to eventually develop resistance. Further, lack of adequate genome and transcriptome information for the pest limit our ability to determine the molecular mechanisms of altered physiological responses in Bt-exposed susceptible and tolerant insect strains. In order to facilitate biological, biochemical and molecular research of the pest species that would enable more efficient biocontrol, we report the midgut de novo transcriptome assembly and clustering of susceptible Cry toxin-exposed and Cry toxin tolerant Achaea janata larvae with appropriate age-matched and starvation controls.

Midgut de novo transcriptome analysis and gene expression profiling of Achaea janata larvae exposed with Bacillus thuringiensis (Bt)-based biopesticide formulation.

India is the major producer and exporter of castor oil in the world. Castor semilooper, Achaea janata is one of the main castor crop pests, which causes serious economic loss of crop, hence management and control of the pest are important. Currently, Bacillus thuringiensis (Bt) based biopesticides are being used for their control. However, the insects are known to develop resistance not only against chemical pesticides but also to Bt based biopesticides. In the present study, de novo transcriptome analysis was conducted to monitor the expression pattern of larval midgut genes in Achaea janata exposed to sublethal dose of Bt formulation. A total of 34,612 and 41,109 transcripts were identified in control and toxin-exposed larval midgut samples out of which 18,836 in control and 21,046 in toxin-exposed samples are annotated. Microarray data analysis employed to monitor the gene expression upon Cry toxin exposure revealed that 375 genes were upregulated and 579 genes were downregulated during all the time points (12-60 h) of toxin exposure. The differentially expressed transcripts include i.e. Cry toxin receptors, gut proteases, arylphorin, REPATs, detoxification enzymes and aquaporins. Validation of microarray data was performed by real-time quantitative PCR using few randomly selected genes and the results obtained were in corroboration. This is the first study on transcriptome data from the castor semilooper and the results would provide valuable resources for the characterization of Bt toxin response in the pest.

Larval Mid-Gut Responses to Sub-Lethal Dose of Cry Toxin in Lepidopteran Pest Achaea janata.

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.

Insecticidal Effects of Hemocoelic Delivery of Bacillus thuringiensis Cry Toxins in Achaea janata Larvae.

Insecticidal effects of Bacillus thuringiensis Cry toxins in hemocoel of larvae have not been properly evaluated. In the present study, hemocoelic injection of four representative Cry toxins i.e., Cry1Aa, Cry1Ab, Cry1Ac, and DOR5 to an economically important lepidopteran insect pest Achaea janata, induced larval mortality, reduced larval growth rate and gave rise to smaller pupae, all in a dose-dependent manner. We observed extensive degeneration as well as the disintegration of larval tissues, most notably, fat body, and the possible involvement of lysosomal enzymes in tissue histolysis. The resultant "hypoproteinemia" and most relevantly, the drastic reduction of 80-85 kDa hexamerin proteins levels of hemolymph could be attributed to the pathological state of the fat body induced by Cry toxin injection. Formation of non-viable larval-pupal intermediates and emergence of defective adults also indicate toxicity effects of Cry toxins during metamorphosis. Thus, findings from our study suggest Cry toxins in larval hemocoel are also toxic to A. janata larval survival and subsequent development.

Differential oxidative stress responses in castor semilooper, Achaea janata.

Balance between reactive oxygen species (ROS) and the antioxidant (AO) defense mechanisms is vital for organism survival. Insects serve as an ideal model to elucidate oxidative stress responses as they are prone to different kinds of stress during their life cycle. The present study demonstrates the modulation of AO enzyme gene expression in the insect pest, Achaea janata (castor semilooper), when subjected to different oxidative stress stimuli. Antioxidant enzymes' (catalase (Cat), superoxide dismutase (Sod), glutathione-S-transferase (GST) and glutathione peroxidase (Gpx)) partial coding sequences were cloned and characterized from larval whole body. Tissue expression studies reveal a unique pattern of AO genes in the larval tissues with maximum expression in the gut and fat body. Ontogeny profile depicts differential expression pattern through the larval developmental stages for each AO gene studied. Using quantitative RT-PCR, the expression pattern of these genes was monitored during sugar-induced (d-galactose feeding), infection-induced (Gram positive, Gram negative and non-pathogenic bacteria) and pesticide-induced oxidative stress (Bt Cry toxin). d-Galactose feeding differentially modulates the expression of AO genes in the larval gut and fat body. Immune challenge with Escherichia coli induces robust upregulation of AO genes when compared to Bacillus coagulans and Bacillus cereus in the larval fat body and gut. Cry toxin feeding predominantly induced GST upregulation in the gut. The current study suggests that though there are multiple ways of generation of oxidative stress in the insect, the organism tailors its response by insult- and tissue-specific recruitment of the antioxidant players and their differential regulation for each inducer.