Identification of salivary proteins of the cowpea aphid Aphis craccivora by transcriptome and LC-MS/MS analyses.

Aphid salivary proteins mediate the interaction between aphids and their host plants. Moreover, these proteins facilitate digestion, detoxification of secondary metabolites, as well as activation and suppression of plant defenses. The cowpea aphid, Aphis craccivora, is an important sucking pest of leguminous crops worldwide. Although aphid saliva plays an important role in aphid plant interactions, knowledge of the cowpea aphid salivary proteins is limited. In this study, we performed transcriptomic and LC-MS/MS analyses to identify the proteins present in the salivary glands and saliva of A. craccivora. A total of 1,08,275 assembled transcripts were identified in the salivary glands of aphids. Of all these assembled transcripts, 53,714 (49.11%) and 53,577 (49.48%) transcripts showed high similarity to known proteins in the Nr and UniProt databases, respectively. A total of 2159 proteins were predicted as secretory proteins from the salivary gland transcriptome dataset, which contain digestive enzymes, detoxification enzymes, previously known effectors and elicitors, and potential proteins whose functions have yet to be determined. The proteomic analysis of aphid saliva resulted in the identification of 171 proteins. Tissue-specific expression of selected genes using RT-PCR showed that three genes were expressed only in the salivary glands. Overall, our results provide a comprehensive repertoire of cowpea aphid salivary proteins from the salivary gland and saliva, which will be a good resource for future effector functional studies and might also be useful for sustainable aphid management.

CRISPR/Cas9 mediated editing of pheromone biosynthesis activating neuropeptide (PBAN) gene disrupts mating in the Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

The Fall armyworm, Spodoptera frugiperda, is a globally important invasive pest, primarily on corn, causing severe yield loss. Overuse of synthetic chemicals has caused significant ecological harm, and in many instances control has failed. Therefore, developing efficient, environmentally friendly substitutes for sustainable management of this pest is of high priority. CRISPR/Cas9-mediated gene editing causes site-specific mutations that typically result in loss-of-function of the target gene. In this regard, identifying key genes that govern the reproduction of S. frugiperda and finding ways to introduce mutations in the key genes is very important for successfully managing this pest. In this study, the pheromone biosynthesis activator neuropeptide (PBAN) gene of S. frugiperda was cloned and tested for its function via a loss-of-function approach using CRISPR/Cas9. Ribonucleoprotein (RNP) complex (single guide RNA (sgRNA) targeting the PBAN gene + Cas9 protein) was validated through in vitro restriction assay followed by embryonic microinjection into the G0 stage for in vivo editing of the target gene. Specific suppression of PBAN by CRISPR/Cas9 in females significantly affected mating. Mating studies between wild males and mutant females resulted in no fecundity. This was in contrast to when mutant males were crossed with wild females, which resulted in reduced fecundity. These results suggest that mating disruption is more robust where PBAN is edited in females. The behavioural bioassay using an olfactometer revealed that mutant females were less attractive to wild males compared to wild females. This study is the first of its kind, supporting CRISPR/Cas9 mediating editing of the PBAN gene disrupting mating in S. frugiperda. Understanding the potential use of these molecular techniques may help develop novel management strategies that target other key functional genes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03798-3.

Genome profiling of an indigenous Bacillus thuringiensis isolate, T405 toxic against the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

In this study, we present the molecular and insecticidal characteristics of an indigenous Bt isolate T405 toxic against the maize fall armyworm (FAW), Spodoptera frugiperda. The presence of cry1, cry2 (cry2Aa & cry2Ab) and vip3A1 genes in T405 was confirmed. The SDS-PAGE gel analysis confirmed the occurrence of Cry and Vip proteins with molecular masses of 130, ∼88 and 65 kDa in T405. LC50 estimates of T405 and HD1 were 161.37 and 910.73 µg ml-1 for neonates whereas, 412.29 and 1014.95 µg ml-1 correspondingly for 2nd instar FAW larvae. Scanning Electron Microscopy depicted the existence of bipyramidal, spherical and cubic crystals in T405 spore suspension. The whole genome sequencing and assembly of T405 produced a total of 563 scaffolds with a genome size of 6,673,691 bp. The BLAST similarity search showed that 12 plasmids were distributed in this genome. Genome annotation revealed the presence of 6174 protein coding genes, 13 rRNA and 98 tRNA, in which 6126 genes were completely annotated for their functions through sequence similarity search, domains/motifs identification and gene ontology studies. Further analysis of these genes identified the presence of many insecticidal toxin protein coding genes viz., cry1Ac32, cry1Ab9, cry1Aa6, cry1Ac5, cry1Aa18, cry1Ab8, cry1Ab11, cry2Aa9, cry1Ia40, cry2Aa9, cry1Ia40, cry2Ab35, cyt, vip3Aa7 and tpp80Aa and several additional virulence assisted factors viz., immune inhibitor A, phospholipase C, sphingomyelinase, cell wall hydrolases, chitinase, hemolysin XhlA and seven urease subunit coding genes (ureA, ureB, ureC, ureD, ureE, ureF, ureG) in the annotated genome.

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.

Genetic characterization of resistance to deltamethrin in Plutella xylostella (Lepidoptera: Plutellidae) from India.

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is considered as one of the most difficult pests to control. It has developed resistance not only to synthetic insecticides but also to Bacillus thuringiensis-based pesticides. We tested the hypothesis that selection in a P. xylostella population, from Hosur, India, with deltamethrin would give a broad spectrum of resistance to several insecticides. We also were interested in genetically classifying resistance to deltamethrin in the selected population and in evaluating whether resistance can be suppressed using synergists. Bioassays (at generation 1, G1) using deltamethrin indicated a resistance ratio of 161-fold compared with a laboratory-susceptible population of P. xylostella (Lab-UK). At G2, the field-derived population was divided into two subpopulations; one population was selected (G1 to G8) with deltamethrin (Delta-SEL), and the second population was left unselected (UNSEL). Bioassays at G9 indicated that selection with deltamethrin gave a resistance ratio of 15-fold compared with UNSEL and 1,647-fold compared with Lab-UK. The resistance to deltamethrin in the UNSEL population was stable. The Delta-SEL population maintained resistance to lambda-cyhalothrin, but there was no cross-resistance to indoxacarb, DDT, or Cry1Ac. Crossing experiments indicated that resistance to deltamethrin in Delta-SEL was multigenic and inherited in an incompletely dominant fashion. Piperonyl butoxide (PBO) and S.S.S-tri-n-butyl phosphorotrithioate with potent inhibitory activity against esterases and/or monooxygenases significantly increased the toxicity of deltamethrin against both UNSEL and Delta-SEL, but they showed no such synergism with Lab-UK. Thus, it can be predicted that development of resistance to deltamethrin would be delayed under appropriate control strategies that favor the dilution of resistance alleles by enhanced flow of susceptible alleles. Further analysis suggested that mixing PBO and deltamethrin could eliminate the substantial resistance to deltamethrin in this population.