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

ABSTRACT: 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.

SUBMITTER: Ningshen TJ

PROVIDER: S-EPMC5423935 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

Project description:Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.

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A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase.

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Gene expression analysis during time dependent Cry toxin exposure in Achaea janata larva.

Project description: Bacillus thuringiensis Cry toxin is used for insect pest management. When susceptible insect larvae ingest the Bt toxin it shows various physiological and cellular changes. We have employed Agilent One Color matrix based microarray to check the mid-gut gene expression upon Cry toxin exposure in Achaea janata (Castor semilooper). Microarray analysis shows significant variation in gene expression upon Cry toxin exposure.

2018-05-26 | GSE114934 | GEO

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

Project description: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.

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Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins.

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Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins.

Project description:Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.

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Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins.

Project description:Bacillus thuringiensis (Bt) constitutes the active ingredient of many successful bioinsecticides used in agriculture. In the present study, the genetic diversity and toxicity of Bt isolates was investigated by characterization of native isolates originating from soil, fig leaves and fruits from a Turkish collection. Among a total of 80 Bt isolates, 18 of them were found carrying a vip3 gene (in 23% of total), which were further selected. Insecticidal activity of spore/crystal mixtures and their supernatants showed that some of the Bt isolates had significantly more toxicity against some lepidopteran species than the HD1 reference strain. Five isolates were analyzed by LC-MS/MS to determine the Cry protein composition of their crystals. The results identified the Cry1Ac protein and a Cry2A-type protein in all isolates, Cry1Ea in 3 of them and Cry1Aa in one. The sequence analysis of the new vip3 genes showed that they had a high similarity to either vip3Aa, vip3Af or vip3Ag (94-100%). The vip3Aa gene of the 6A Bt isolate was cloned and sequenced. The protein was named Vip3Aa65 by the Bacillus thuringiensis Nomenclature Committee. The expressed and purified Vip3Aa65 protein was tested against five lepidopteran species and its toxicity compared to that of a reference protein (Vip3Aa16). Both proteins had similar toxicity against Grapholita molesta and Helicoverpa armigera, whereas Vip3Aa65 was less active than Vip3Aa16 against three species from the Spodoptera genus. A tetrameric structure of the Vip3Aa65 protein was detected by gel filtration chromatography. The study revealed some isolates with high insecticidal activity which can be considered promising candidates to be used in pest control.

| S-EPMC6211755 | biostudies-literature

Molecular and insecticidal characterization of a novel Cry-related protein from Bacillus thuringiensis toxic against Myzus persicae.

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