Insect Innate Immunity Research Articles

The Molecular Toll Pathway Repertoire in Anopheline Mosquitoes.

Innate immunity in mosquitoes has received much attention due to its potential impact on vector competence for vector-borne disease pathogens, including malaria parasites. The nuclear factor (NF)-κB-dependent Toll pathway is a major regulator of innate immunity in insects. In mosquitoes, this pathway controls transcription of the majority of the known canonical humoral immune effectors, mediates anti-bacterial, anti-fungal and anti-viral immune responses, and contributes to malaria parasite killing. However, besides initial gene annotation of putative Toll pathway members and genetic analysis of the contribution of few key components to immunity, the molecular make-up and function of the Toll pathway in mosquitoes is largely unexplored. To facilitate functional analyses of the Toll pathway in mosquitoes, we report here manually annotated and refined gene models of Toll-like receptors and all putative components of the intracellular signal transduction cascade across 19 anopheline genomes, and in two culicine genomes. In addition, based on phylogenetic analyses, we identified differing levels of evolutionary constraint across the intracellular Toll pathway members, and identified a recent radiation of TOLL1/5 within the An. gambiae complex. Together, this study provides insight into the evolution of TLRs and the putative members of the intracellular signal transduction cascade within the genus Anopheles.

BmToll9-1 Is a Positive Regulator of the Immune Response in the Silkworm Bombyx mori.

Toll receptors are involved in the development and innate immunity of insects. BmToll9-1 is an important immune receptor in the Toll pathway. Previous studies have focused on its role as a receptor in immune response. In this study, we aimed to investigate the role of BmToll9-1 as a regulator in the immune response. The expression profiles demonstrated that BmToll9-1 was predominantly expressed in the midgut. RNA interference (RNAi) of BmToll9-1 was found to be effective in the midgut via the injection of dsRNA, which resulted in smaller and lighter larvae and cocoons. Most signaling genes in the Toll pathway and downstream effector genes were downregulated after the RNAi of BmToll9-1. The hemolymph from BmToll9-1-silenced larvae showed decreased antibacterial activity against Escherichia coli, either in growth curve or inhibition zone experiments. The above results indicate that BmToll9-1 might be positively involved in the immune pathway of silkworm. As a positive regulator, BmToll9-1 might function mainly in the gut to maintain microbial homeostasis to regulate the growth of silkworms. Silencing of BmToll9-1 downregulates the signaling genes in the Toll pathway and antimicrobial peptide (AMP) production, resulting in decreased antibacterial activity in the hemolymph.

Current status of innate immune responses in Malpighian tubules of insects: A review focusing on the Toll signaling pathway

AbstractInnate immunity is the immediate and exclusive defense mechanism in invertebrates. It acts against foreign agents and pathogens. In insects, this defense mechanism involves a combination of humoral and cellular responses, orchestrated by specific recognition receptors and the activation of various signaling pathways. Remarkably, Toll and Toll‐related proteins play a pivotal role in insect immunity by inducing the production of antimicrobial peptides (AMPs) in response to pathogen attacks. Toll receptors, which are type I transmembrane proteins, significantly affect insect development and the innate immune response, primarily via the nuclear factor kappa‐B signaling pathway. The Malpighian tubules of insects play a crucial role in waste elimination, hemostasis maintenance, and immune defense. The Toll signaling pathway in Malpighian tubules is mainly involved in the production of AMPs. This review provides valuable insights into the mechanisms of the Toll signaling pathway and the morphological and physiological attributes of Malpighian tubules. Moreover, it discusses the classification and functionalities of AMPs synthesized in Malpighian tubules in response to pathogens, thereby contributing to our understanding of innate immunity in insects.

Insights into midgut cell types and their crucial role in antiviral immunity in the lepidopteran model Bombyx mori.

The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect's physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect's innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect's immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms.

Immunomodulatory Peptides as Vaccine Adjuvants and Antimicrobial Agents.

The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides.

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus

BackgroundMosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment.ResultsWe found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid.ConclusionsNovel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

It has been reported that C-type lectins (CTLs), which are pattern recognition receptors of the insect innate immunity response, may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in insects. However, to date, which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not clear. In this study, 33 CTL genes were identified from S. exigua. Based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures, they were classified into three groups: (1) nineteen CTL-S (single-CRD), (2) eight immulectin (dual-CRD) and (3) six CTL-X (CRD with other domains). RT-qPCR analysis revealed that expression levels of SeCTL-S15, IML-4 and CTL-X6 were upregulated after challenge with Bt and Cry1Ab. Tissue and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva, respectively. Knockdown of SeCTL-S15 significantly increased Bt susceptibility, as indicated by reduced survival and larval weight. These results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in S. exigua. Our results provide new insights into CTL function in insects.

Discovery and Characterization of MaK: A Novel Knottin Antimicrobial Peptide from Monochamus alternatus.

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease.

Molecular characterization and functional analysis of a beta-1,3-glucan recognition protein from oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

The beta-1,3-glucan recognition protein (BGRP) is an important pattern recognition protein (PRP), which plays an important role in immune recognition and signaling pathway of insect innate immunity. Herein, a BGRP gene was obtained from the transcriptome of Grapholita molesta and its expression was verified by PCR. The full cDNA of the GmBGRP gene was 1691 bp encoding 486 amino acid residues. The calculated molecular mass of the mature protein was 54.83 kDa with an estimated pI of 6.14. The amino acid sequence of GmBGRP was highly homologous to BGRPs of other lepidopterans including Leguminivora glycinivorella BGRP-3. Expression profile of GmBGRP at different developmental stages and different tissues of 5th instar larvae showed that the expression level of this gene tends to slightly increase and then decrease at the adult stage, with the highest at the pupa stage; and mainly expressed in the epidermis, fat body and hemocytes compared with other tissues. In addition, we investigated the transcription levels of other immune-related genes, such as Serine-1, Serine-2, Serine-3, Serpin, SRCB (scavenger receptor gene), Toll, PPO (prophenoloxidase) upon GmBGRP gene silencing, indicating that GmBGRP expression is associated with immune responses of G. molesta. This was further supported by the upregulation of the mRNA level of GmBGRP following fungal infection. Taken together, these results provide experimental evidence for the role of GmBGRP gene in immune defense in G. molesta larvae.

Trade-Offs among Immune Mechanisms: Bacterial-Challenged Spodoptera frugiperda Larvae Reduce Nodulation Reactions during Behavioral Fever.

Insect innate immunity is composed of cellular and humoral reactions, the former acting via circulating hemocytes and the latter via immune signaling that lead to the production of antimicrobial peptides and phenol oxidase-driven melanization. Cellular immunity involves direct interactions between circulating hemocytes and invaders; it includes internalization and killing microbes (phagocytosis) and formation of bacterial-laden microaggregates which coalesce into nodules that are melanized and attached to body walls or organs. Nodulation can entail investing millions of hemocytes which must be replaced. We hypothesized that biologically costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae of fall armyworms, Spodoptera frugiperda, that were allowed to fever. We tested our hypothesis by infecting larvae with the Gram-negative bacterium, Serratia marcescens, placing them in thermal gradients (TGs) and recording their selected body temperatures. While control larvae selected about 30 °C, the experimental larvae selected up 41 °C. We found that 4 h fevers, but not 2, 6 or 24 h fevers, led to increased larval survival. Co-injections of S. marcescens with the prostaglandin (PG) biosynthesis inhibitor indomethacin (INDO) blocked the fevers, which was reversed after co-injections of SM+INDO+Arachidonic acid, a precursor to PG biosynthesis, confirming that PGs mediate fever reactions. These and other experimental outcomes support our hypothesis that costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae under appropriate conditions.

Serpin-1a and serpin-6 regulate the Toll pathway immune homeostasis by synergistically inhibiting the Spätzle-processing enzyme CLIP2 in silkworm, Bombyx mori.

The Toll receptor signaling pathway is an important innate immune response of insects to pathogen infection; its extracellular signal transduction involves serine protease cascade activation. However, excessive or constitutive activation of the Toll pathway can be detrimental. Hence, the balance between activation and inhibition of the extracellular protease cascade must be tightly regulated to achieve favorable outcomes. Previous studies have shown that serpins-serine protease inhibitors-negatively regulate insect innate immunity by inhibiting extracellular protease cascade signaling. Although the roles of serpins in insect innate immunity are well described, the physiological mechanisms underlying their synergistic effects remain poorly understand. Here, we characterize the molecular mechanism by which serpin-1a and serpin-6 synergistically maintain immune homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Through in vitro biochemical assays and in vivo bioassays, we demonstrate that clip-domain serine protease 2 (CLIP2), as the Toll cascade-activating terminal protease, is responsible for processing proSpätzle1 to induce the expression of antimicrobial peptides. Further biochemical and genetic analyses indicate that constitutively expressed serpin-1a and inducible serpin-6 synergistically target CLIP2 to maintain homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Taken together, this study provides new insights into the precise regulation of Toll cascade activation signals in insect innate immune responses and highlights the importance and complexity of insect immune homeostasis regulation.

Effect of aminergic signaling on the humoral innate immunity response of Drosophila.

Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. In this work, we analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. We found that the neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, we showed that either blocking the neurotransmission in their specific aminergic neurons by expressing shibirets (Shits) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, our results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation.

Immune functions of pattern recognition receptors in Lepidoptera.

Pattern recognition receptors (PRRs), as the "sensors" in the immune response, play a prominent role in recognizing pathogen-associated molecular patterns (PAMPs) and initiating an effective defense response to pathogens in Lepidoptera. It is becoming increasingly clear that damage-associated molecular patterns (DAMPs) normally play a physiological role within cells; however, when exposed to extracellular, they may become "part-time" critical signals of the immune response. Based on research in recent years, we review herein typical PRRs of Lepidoptera, including peptidoglycan recognition protein (PGRP), gram-negative binding protein (GNBP), β-1,3-glucan recognition protein (βGRP), C-type lectin (CTL), and scavenger receptor (SR). We also outline the ways in which DAMPs participate in the immune response and the correlation between PRRs and immune escape. Taken together, these findings suggest that the role of PRRs in insect innate immunity may be much greater than expected and that it is possible to recognize a broader range of signaling molecules.

Double-strand RNAs targeting MaltRelish and MaltSpz reveals potential targets for pest management of Monochamus alternatus

Overcoming the innate immunity of insects is a key process to improve the efficiency of biological control. Antimicrobial peptides (AMPs) are important effectors in insect innate immunity, usually mediating resistance to pathogenic microorganisms through Toll and IMD signaling pathways. This study investigated the effect of key genes on upstream immune recognition receptor (GNBP3) and downstream effectors (AMPs) by RNAi technology. The transcriptome KEGG enrichment analysis and differential gene annotation results showed that the immune response genes MaltSpz and MaltRelish are important regulators of Toll and IMD signaling pathways, respectively. Both dsSpz and dsRelish could affect AMP gene expression and increase the expression of the immune recognition receptor MaltGNBP3. Moreover, they significantly reduce the survival rate of Monochamus alternatus and promote hyphal growth after Beauveria bassiana infection. This helps to improve the biological control effect of B. bassiana, control the population of vector insects and cut off the transmission route of pine wood nematode. The combined MaltSpz and MaltRelish knockdown increased the infection rate of M. alternatus larvae from 20.69% to 83.93%, achieving the best efficiency in synergistic B. bassiana infection. Our results showed important roles of MaltRelish- and MaltSpz-mediated regulation of AMP genes function in insect entomopathogenic fungi tolerance and induced significant mortality in larvae. Based on this study, MaltSpz and MaltRelish could represent candidate gene targets for the biological control of M. alternatus by RNAi.

TGF-β Type II Receptor Punt Suppresses Antimicrobial Peptide Expression and Influences Development in Tribolium castaneum.

The transforming growth factor-β (TGF-β) superfamily in insects regulated various physiological events, including immune response, growth and development, and metamorphosis. This complex network of signaling pathways involves conserved cell-surface receptors and signaling co-receptors that allow for precisely coordinated cellular events. However, the roles of TGF-β receptors, particularly the type II receptor Punt, in mediating the innate immunity in insects remains unclear. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of TGF-β type II receptor Punt in mediating antimicrobial peptide (AMP) expression. Developmental and tissue-specific transcript profiles revealed Punt was constitutively expressed throughout development, with the highest transcript level in 1-day female pupae and the lowest transcript level in 18-day larvae. Tissue specific expression profiles showed the highest transcript level of Punt was observed in the Malpighian tubule and ovary in 18-day larvae and 1-day female adults, respectively, suggesting Punt might have distinct functions in larvae and adults. Further results indicated that Punt RNAi in the 18-day larvae led to increased transcript level of AMP genes through transcription factor Relish, leading to inhibition of Escherichia coli proliferation. Knockdown of Punt in larvae also led to splitting of adult elytra and abnormal compound eyes. Furthermore, knockdown of Punt during the female pupal stage resulted in increased transcript levels of AMP genes, as well as abnormal ovary, reduced fecundity, and failure of eggs to hatch. This study deepens our understanding of the biological significance of Punt in insect TGF-β signaling and lays the groundwork for further research of its role in insect immune response, development, and reproduction.

Evolution of insect innate immunity through domestication of bacterial toxins

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Treatment of Rapamycin and Evaluation of an Autophagic Response in the Gut of Bactericera cockerelli (Sulč)

Autophagy is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic content, abnormal protein aggregates, and excess of/or damaged organelles to promote cell survival. Autophagy is also a component of innate immunity in insects and is involved in the clearance of pathogens, including bacteria. The potato psyllid, Bactericera cockerelli, transmits the plant bacterial pathogen 'Candidatus Liberibacter solanacearum' (Lso) in the Americas and causes serious damage to solanaceous crops. Our previous studies showed that autophagy could be involved in the psyllid response to Lso and could affect pathogen acquisition. However, the tools to evaluate this response have not been validated in psyllids. To this end, the effect of rapamycin, a commonly used autophagy inducer, on potato psyllid survival and the expression of autophagy-related genes was evaluated. Further, the autophagic activity was assessed via microscopy and by measuring the autophagic flux. Artificial diet-feeding assays using rapamycin resulted in significant psyllid mortality, an increase in the autophagic flux, as well as an increase in the amount of autolysosomes. This study represents a stepping stone in determining the role of autophagy in psyllid immunity.

Insect egg-induced innate immunity: Who benefits?

Plants perceive the presence of insect eggs deposited on leaves as a cue of imminent herbivore attack. Consequential plant signaling events include the accumulation of salicylic acid and reactive oxygen species, transcriptional reprogramming, and cell death. Interestingly, egg-induced innate immunity shows similarities with immune responses triggered upon recognition of microbial pathogens, and in recent years, it became apparent that egg perception affects plant-microbe interactions. Here, we highlight recent findings on insect egg-induced innate immunity and how egg-mediated signaling impacts plant-microbe interactions. Ecological considerations beg the question: Who benefits from egg perception in these complex interactions?

Identification and analysis of C-type lectins from Helicoverpa armigera in response to the entomopathogenic fungus Metarhizium rileyi infection

C-type lectins (CTLs) function as pattern recognition receptors (PRRs) and play an important role in the innate immunity of insects. To investigate the role of CTLs in the antifungal responses, we analyzed expression profiles of 36 CTLs of Helicoverpa armigera in the tissues (hemocytes, fat bodies, and midgut) after the infection by entomopathogenic fungus Metarhizium rileyi. The expression levels of many HaCTLs were found to be up-regulated after the infection. Four recombinant HaCTLs (rHaCTL11, rHaCTL12, rHaCTL27, and rHaCTL45) were expressed and purified. Analysis of the purified rHaCTLs revealed that rHaCTLs were able to bind to conidia and hyphal bodies of M. rileyi, and the affinity of rHaCTL11 and rHaCTL27 for hyphal bodies was weaker than for conidia. All these rHaCTLs agglutinate conidia and hyphal bodies in a calcium (Ca2+) dependent manner. Sugar specificity assays showed that d-trehalose, mannan, β-1,3-glucan, d-galactose, glucose, d-raffinose, lipopolysaccharide, and d-xylose can inhibit the binding of HaCTLs to M. rileyi. Additionally, survival assays showed that pretreatment of fungal conidia with rHaCTL11 significantly reduced the rate of host death, and knockdown of HaCTL11 significantly increased H. armigera sensitivity to fungal infection. These results suggest that HaCTLs play significant role as PRRs in the defense of H. armigera against M. rileyi infection.

"Metabolomic diversity of local strains of Beauveria bassiana (Balsamo) Vuillemin and their efficacy against the cassava mite, Tetranychus truncatus Ehara (Acari: Tetranychidae)".

A desirable substitute for chemical pesticides is mycopesticides. In the current investigation, rDNA-ITS (Internal transcribed spacer) and TEF (Transcriptional Elongation Factor) sequencing were used for molecular identification of six Beauveria bassiana strains. Both, leaf discs and potted plant bioassaye were carried out to study their pathogenicity against the cassava mite, Tetranychus truncatus. LC50 and LC90 values of potential B. bassiana strains were estimated. We also discovered a correlation between intraspecific B. bassiana strains pathogenicity and comprehensive metabolome profiles. Bb5, Bb6, Bb8, Bb12, Bb15, and Bb21 strains were identified as B. bassiana by sequencing of rDNA-ITS and TEF segments and sequence comparison to NCBI (National Center for Biotechnology Information) GenBank. Out of the six strains tested for pathogenicity, Bb6, Bb12, and Bb15 strains outperformed against T. truncatus with LC50 values 1.4×106, 1.7×106, and 1.4×106 and with a LC90 values 7.3×107, 1.4×108, and 4.2×108 conidia/ml, respectively, at 3 days after inoculation and were considered as potential strains for effective mite control. Later, Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the above six B. bassiana strains was done on secondary metabolites extracted with ethyl acetate revealed that the potential B. bassiana strains (Bb6, Bb12, and Bb15) have higher levels of acaricidal such as Bis(dimethylethyl)-phenol: Bb6 (5.79%), Bb12 (6.15%), and Bb15 (4.69%). Besides, insecticidal (n-Hexadecanoic acid), and insect innate immunity overcoming compound (Nonadecene) were also identified; therefore, the synergistic effect of these compounds might lead toa higher pathogenicity of B. bassiana against T. truncatus. Further, these compounds also exhibited two clusters, which separate the potential and non-potential strains in the dendrogram of Thin Layer Chromatography. These results clearly demonstrated the potentiality of the B. bassiana strains against T. truncatus due to the occurrence of their bioactive volatile metabolome.