Antimicrobial Peptide Genes Research Articles

Biocontrol Potential and Mechanism of Bacillus SPP. against Phytopathogens: A Review

The biocontrol potential of Bacillus spp. clearly indicated by several authors are presented crop wise. The understanding of mechanism of action of Bacillus spp. against phytopathogens is essential to support their biocontrol potential. In this regard the various mechanism of action of Bacillus is elaborately discussed with suitable subheadings along with their plant growth promoting ability. The available literature presented below as review strongly emphasized their distinct biocontrol potential coupled with plant growth promoting effect through their various behavioral, biological, biochemical, induction of resistance, role of antimicrobial peptide genes etc.

Cadmium exposure through the food chain reduces the parasitic fitness of Chouioia cunea to Hyphantria cunea pupae: An ecotoxicological risk to pest control

The toxicity of heavy metals on various trophic levels along the food chain has been extensively investigated, but no studies have focused on parasitic natural enemy insects. Herein, we constructed a food chain consisting of soil–Fraxinus mandshurica seedlings–Hyphantria cunea pupae–Chouioia cunea to analyze the effects of Cd exposure through food chain on the fitness of parasitic natural enemy insects and its corresponding mechanism. The results showed that the transfer of Cd between F. mandshurica leaves and H. cunea pupae and between H. cunea pupae and C. cunea was a bio-minimization effect. After parasitizing Cd-accumulated pupa, the number of offspring larvae, and the number, individual size (body weight, body length, abdomen length) and life span of offspring adults decreased significantly, while the duration of embryo development extended significantly. The contents of malondialdehyde and H2O2 in Cd-exposed offspring wasps increased significantly, accompanied by a significantly decrease in antioxidant capacity. The cellular immunity parameters significantly decreased in Cd-accumulated pupae, including the number of hemocytes, melanization activity and the expression level of cellular immunity genes (e.g. Hemolin-1 and PPO1). The humoral immunity disorder was found in the Cd-accumulated pupae, as evidenced by that the expression level of immune recognition gene (PGRP-SA), signal transduction genes (IMD, Dorsal, and Tube), as well as all antimicrobial peptide genes (e.g. Lysozym and Attacin) decreased significantly. Cd exposure decreased the content of glucose, trehalose, amino acid, and free fatty acid in H. cunea pupae. The expression of Hk2 in glycolysis pathway and the expression of Idh2, Idh3, Cs, and OGDH in TCA cycle pathway were significantly down-regulated in Cd-accumulated pupae. Taken together, exposure to Cd through the food chain causes oxidative damage on the offspring wasps and disrupts energy metabolism of the host insect, ultimately reducing the parasitic fitness of C. cunea to H. cunea pupae.

Proteomics and Immune Response Differences in Apis mellifera and Apis cerana Inoculated with Three Nosema ceranae Isolates.

Nosema ceranae infects midgut epithelial cells of the Apis species and has jumped from its original host A. cerana to A. mellifera worldwide, raising questions about the response of the new host. We compared the responses of these two species to N. ceranae isolates from A. cerana, A. mellifera from Thailand and A. mellifera from France. Proteomics and transcriptomics results were combined to better understand the impact on the immunity of the two species. This is the first combination of omics analyses to evaluate the impact of N. ceranae spores from different origins and provides new insights into the differential immune responses in honeybees inoculated with N. ceranae from original A. cerana. No difference in the antimicrobial peptides (AMPs) was observed in A. mellifera, whereas these peptides were altered in A. cerana compared to controls. Inoculation of A. mellifera or A. cerana with N. ceranae upregulated AMP genes and cellular-mediated immune genes but did not significantly alter apoptosis-related gene expression. A. cerana showed a stronger immune response than A. mellifera after inoculation with different N. ceranae isolates. N. ceranae from A. cerana had a strong negative impact on the health of A. mellifera and A. cerana compared to other Nosema isolates.

Intake of caffeine containing sugar diet remodels gut microbiota and perturbs Drosophila melanogaster immunity and lifespan

The diet-microbiome-immunity axis is one among the many arms that draw up the “we are what we intake” proclamation. As such, studies on the effect of food and beverage intake on the gut environment and microbiome and on modulating immunological responses and the host's susceptibility to pathogens are on the rise. A typical accompaniment in different sustenance we consume on daily basis is the trimethylxanthine alkaloid caffeine. Being a chief component in our regular aliment, a better understanding of the effect of caffeine containing food and beverages on our gut-microbiome-immunity axis and henceforth on our health is much needed. In this study, we shed more light on the effect of oral consumption of caffeine supplemented sugar diet on the gut environment, specifically on the gut microbiota, innate immunity and host susceptibility to pathogens using the Drosophila melanogaster model organism. Our findings reveal that the oral intake of a dose-specific caffeine containing sucrose/agarose sugar diet causes a significant alteration within the fly gut milieu demarcated by microbial dysbiosis and an elevation in the production of reactive oxygen species and expression of immune-deficiency (Imd) pathway-dependent antimicrobial peptide genes. The oral intake of caffeine containing sucrose/agarose sugar diet also renders the flies more susceptible to bacterial infection and shortens their lifespan in both infection and non-infection settings. Our findings set forth additional insight into the potentiality of diet to alter the gut milieu and highlight the importance of dietary control on health.

Specific Expression of Antimicrobial Peptides from the Black Soldier Fly in the Midgut of Silkworms (Bombyx mori) Regulates Silkworm Immunity.

Antimicrobial peptides are molecules with strong antimicrobial activity and are of substantial interest for the immunization of insects. As a type of dipteran insect that can turn organic waste into animal feed, the black soldier fly (BSF) can "turn waste into treasure". In this study, we investigated the antimicrobial activity of the antimicrobial peptide genes, HiCG13551 and Hidiptericin-1, of BSF in silkworms, by overexpressing the genes specifically in the midgut. Changes in the mRNA levels of the transgenic silkworms after infection with Staphylococcus aureus were evaluated using transcriptome sequencing. The results showed that Hidiptericin-1 had stronger antimicrobial activity than HiCG13551. KEGG enrichment analysis showed that the differentially expressed genes in the transgenic overexpressed Hidiptericin-1 silkworm lines from the D9L strain were mainly enriched in the starch and sucrose metabolism, pantothenate and CoA biosynthesis, drug metabolism (other enzymes), biotin metabolism, platinum drug resistance, galactose metabolism, and pancreatic secretion pathways. In addition, immune-related genes were up-regulated in this transgenic silkworm strain. Our study may provide new insights for future immune studies on insects.

Disruption of peritrophic matrix chitin metabolism and gut immune by chlorantraniliprole results in pathogenic bacterial infection in Bombyx mori

Chlorantraniliprole (CAP) is widely used in pest control, and its environmental residues affect the disease resistance of non-target insect silkworms. Studies have demonstrated that changes in gut microbial communities of insects are associated with susceptibility to pathogens. In the present study, we examined the effects of CAP exposure on the immune system and gut microbial community structure of silkworms. The results showed that after 96 h of exposure to low-concentration CAP, the peritrophic matrix (PM) of silkworm larvae was disrupted, and pathogenic bacteria invaded hemolymph. The trehalase activity in the midgut was significantly decreased, while the activities of chitinase, β-N-acetylglucosaminidase, and chitin deacetylase were increased considerably, resulting in decreased chitin content in PM. In addition, exposure to CAP reduced the expressions of key genes in the Toll, IMD, and JAK/STAT pathways, ultimately leading to the downregulation of antimicrobial peptides (AMPs) genes and alterations in the structure of the gut microbial community. Therefore, after infection with the conditional pathogen Enterobacter cloacae (E. cloacae), CAP-exposed individuals exhibited significantly lower body weight and higher mortality. These findings showed that exposure to low-concentration CAP impacted the biological defense system of silkworms, changed the gut microbial community structure, and increased silkworms' susceptibility to bacterial diseases. Collectively, these findings provided a new perspective for the safety evaluation of low-concentration CAP exposure in sericulture.

Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

RNAi-mediated CrebA silencing inhibits reproduction and immunity in Locusta migratoria manilensis

Locusta migratoria manilensis is a major agricultural pest that causes severe direct and indirect damage to several crops. Thus, to provide a theoretical foundation for pest control, the role of CrebA in the reproduction and immune regulation of L. migratoria was investigated. CrebA is a bZIP transcription factor that critically regulates intracellular protein secretion. In this study, CrebA was widely expressed in the brain, fat body, integument, midgut, and reproductive tissues of different maturity stages of adult locusts, especially in the female fat body. RNA interfering (RNAi)-mediated silencing of CrebA inhibited locusts ovarian development, and key reproduction gene expressions, Vgs, VgRs, Chico, and JHAMT were downregulated. After the locusts were injected with Micrococcus luteus or Escherichia coli, M. luteus activated lysozyme expression, while the E. coli activated both phenol oxidase cascade and lysozyme expression. Furthermore, both bacteria stimulated the upregulation of the antimicrobial peptide genes DEF3 and DEF4. However, CrebA silencing is fatal to locusts infection with E. coli, with a mortality rate of up to 96.3%, and resulted in a significant decrease in the expression of DEF3 and DEF4 and changes in the activities of phenol oxidase and lysozyme of locusts infected by bacteria. Collectively, CrebA may be involved in diverse biological processes, including reproduction and immunity. CrebA inhibited locusts reproduction by regulating JH signaling pathway and inhibits the expression of immune genes TLR6, IMD, and AMPs. These results demonstrate CrebA seems to play a crucial role in reproduction and innate immunity.

Molecular identification of a novel antimicrobial peptide in giant Triton snail Charonia tritonis: mRNA profiles for tissues and its potential antibacterial activity

Antimicrobial peptides (AMPs) play an important role in innate immunity against microorganisms. AMPs is an effective antibacterial agent, and the chances of causing pathogens to develop is very low. However, there is little information about AMPs in the giant Triton snail Charonia tritonis. In this research, an antimicrobial peptide gene (termed Ct-20534) was identified in C. tritonis. The open reading frame of Ct-20534 is 381 bp in size and it encodes a basic peptide precursor containing 126 amino acids. Ct-20534 gene was found to be expressed in all five tissues examined by real-time fluorescence quantitative PCR (qPCR), but the highest expression was found in the proboscis. This is the first report that antibacterial peptides have been found in C. tritonis, and it has been proved that Ct-20534 has antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, among which the activity of Staphylococcus aureus is most significantly inhibited, this suggests that the newly discovered antimicrobial peptides in C. tritonis may play an important role in the immune system and bacterial resistance of C. tritonis. This study presents the discovery of a newly identified antibacterial peptide from C. tritonis, with its structural properties fully characterized and potent antibacterial activity confirmed. The results provide essential fundamental data for the development of preventive and therapeutic measures against aquatic animal diseases, which in turn can promote the sustainable and stable growth of the aquaculture industry and create economic benefits. Additionally, this research lays the foundation for future development of novel anti-infective drugs.

Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor.

Although Toll-like receptors have been widely identified and functionally characterized in mammalian models and Drosophila, the immunological function of these receptors in other insects remains unclear. Here, we explored the relevant innate immune response of Tenebrio molitor (T. molitor) Toll-3 against Gram-negative bacteria, Gram-positive bacteria, and fungal infections. Our findings indicated that TmToll-3 expression was mainly induced by Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of young T. molitor larvae. Surprisingly, Escherichia coli systemic infection caused mortality after TmToll-3 knockdown via RNA interference (RNAi) injection, which was not observed in the control group. Further analyses indicated that in the absence of TmToll-3, the final effector of the Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated after E. coli challenge. Our results indicated that the expression of almost all AMP genes was suppressed in silenced individuals, whereas the expression of relevant genes was positively regulated after fungal injection. Therefore, this study revealed the immunological involvement of TmToll-3 in T. molitor in response to systematic infections.

HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection

Tomato spotted wilt virus (TSWV) causes a serious plant disease and is transmitted by specific thrips including the western flower thrips, Frankliniella occidentalis. The persistent and circulative virus transmission suggests an induction of immune defenses in the thrips. We investigated the immune responses of F. occidentalis to TSWV infection. Immunofluorescence assay demonstrated viral infection in the larval midguts at early stage and subsequent propagation to the salivary gland in adults. In the larval midgut, TSWV infection led to the release of DSP1, a damage-associated molecular pattern, from the gut epithelium into the hemolymph. DSP1 up-regulated PLA2 activity, which would lead to biosynthesis of eicosanoids that activate cellular and humoral immune responses. Phenoloxidase (PO) activity was enhanced following induction of PO and its activating protease gene expressions. Antimicrobial peptide genes and dual oxidase, which produces reactive oxygen species, were induced by the viral infection. Expression of four caspase genes increased and TUNEL assay confirmed apoptosis in the larval midgut after the virus infection. These immune responses to viral infection were significantly suppressed by the inhibition of DSP1 release. We infer that TSWV infection induces F. occidentalis immune responses, which are activated by the release of DSP1 from the infection foci within midguts.

Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes.

Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus. Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased, with the available genomes of varying accuracy and completeness. We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species; Citrus australis (round lime) using highly accurate PacBio HiFi long reads, complemented with Hi-C scaffolding. Hifiasm with Hi-C integrated assembly resulted in a 331Mb genome of C. australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3Mb and 98.8% genome assembly completeness (BUSCO). Repeat analysis showed that more than 50% of the genome contained interspersed repeats. Among them, LTR elements were the predominant type (21.0%), of which LTR Gypsy (9.8%) and LTR copia (7.7%) elements were the most abundant repeats. A total of 29 464 genes and 32 009 transcripts were identified in the genome. Of these, 28 222 CDS (25 753 genes) had BLAST hits and 21 401 CDS (75.8%) were annotated with at least one GO term. Citrus specific genes for antimicrobial peptides, defense, volatile compounds and acidity regulation were identified. The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2, 4, 7 and 8. This chromosome scale, and haplotype resolved C. australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.

A peptidoglycan recognition protein regulates the immune response of Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) during exposure to pathogenic Gram-positive bacteria and fungi

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a tremendously destructive insect pest of palm trees worldwide. Although some biological agents have been used to fight against RPW larvae, the control efficiency is still dissatisfactory. This study aimed to determine the role of a peptidoglycan recognition protein (PGRP), RfPGRP-S3, in RPW immunity. RfPGRP-S3 is a secreted protein with a DF (Asp85-Phe86) motif, implying that it can discriminate Gram-positive bacteria. The abundance of RfPGRP-S3 transcripts in the hemolymph was significantly higher than that in other tissues. The expression of RfPGRP-S3 can be markedly induced by challenge with Staphylococcus aureus and Beauveria bassiana. After RfPGRP-S3 was silenced, the ability of individuals to clear the pathogenic bacteria in the body cavity and gut was significantly compromised. Furthermore, silencing RfPGRP-S3 dramatically impaired the survival rate of RPW larvae upon challenge with S. aureus. RT‒qPCR revealed that the expression levels of RfDefensin in the fat body and gut were decreased by RfPGRP-S3 silencing. Taken together, these results demonstrated that RfPGRP-S3 acts as a circulating receptor to promote the expression of the antimicrobial peptide gene upon the discrimination of pathogenic microbes.

Transcriptome Analysis Reveals the Immunoregulation of Replacing Fishmeal with Cottonseed Protein Concentrates on Litopenaeus vannamei

Cottonseed protein concentrate (CPC) is a new non-food protein source with high crude protein, low price, and abundant resources, making it an ideal substitute for fishmeal. In this study, we investigated the effects of CPC re placing fishmeal on the immune response of Litopenaeus vannamei using transcriptome sequencing. L. vannamei (initial body weight: 0.42 ± 0.01 g) were fed four isonitrogenous and isolipid feeds for eight weeks, with CPC replacing fishmeal at 0% (control, FM), 15% (CPC15), 30% (CPC30), and 45% (CPC45), respectively. At the end of the feeding trial, the changes of the activities and expression of immune-related enzymes were consistent in L. vannamei in the CPC-containing group when compared with the FM group. Among them, the activities of ACP, PO, and LZM in the group whose diet was CPC30 were significantly higher than those in the FM group. Moreover, the activities of AKP, SOD, and CAT were significantly higher in the group containing CPC than in the FM group. Furthermore, all CPC groups had considerably lower MDA levels than the FM group. This suggests that the substitution of fishmeal with CPC leads to a significant immune response in L. vannamei. Compared with the FM group, transcriptome analysis identified 805 differentially expressed genes (DEGs) (484 down and 321 up), 694 (266 down and 383 up), and 902 (434 down and 468 up) in CPC15, CPC30, and CPC45, respectively. Among all DEGs, 121 DEGs were shared among different CPC-containing groups compared with the FM group. Most of these differential genes are involved in immune-related signaling pathways. The top 20 signaling pathways enriched for differential genes contained toxoplasmosis, pathogenic Escherichia coli infection, insulin resistance, and Toll and immune deficiency (IMD) pathways, in which NF-kappa-B inhibitor Cactus were involved. In addition, trend analysis comparison of the DEGs shared by the group with CPC in the diet and the FM group showed that Cactus genes were significantly down-regulated in the group with CPC in the diet and were lowest in the CPC30 group. Consistently, the expression of antimicrobial peptide genes was significantly higher in both diet-containing CPC groups than in the FM group. In conclusion, the moderate amount of CPC substituted for fishmeal may improve the immunity of L. vannamei by suppressing the expression of Cactus genes, thereby increasing the expression of antimicrobial peptide (AMP) genes.

The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor.

Upon infection, insect hosts simultaneously express a cocktail of antimicrobial peptides (AMPs) which can impede pathogen colonization and increase host fitness. It has been proposed that such a cocktail might be adaptive if the effects of co-expressed AMPs are greater than the sum of individual activities. This could potentially prevent the evolution of bacterial resistance. However, in vivo studies on AMPs in combination are scarce. Attacins are one of the relatively large AMP families, which show anti-Gram-negative activity in vitro. Here, we used RNA interference (RNAi) to silence three members of the Attacin family genes in the mealworm beetle, Tenebrio molitor: (TmAttacin1a (TmAtt1a), TmAttacin1b (TmAtt1b), and TmAttacin2 (TmAtt2) both individually and in combination. We then infected T. molitor with the Gram negative entomopathogen Pseudomonas entomophila. We found that survival of the beetles was only affected by the knockdown of TmAttacin1b, TmAttacin2 and the knockdown of all three Attacins together. Triple knockdown, rather than individual or double knockdowns of AMPs, changes the temporal dynamics of their efficiency in controlling the colonization of P. entomophila in the insect body. More precisely, AMP gene expression influences P. entomophila load early in the infection process, resulting in differences in host survival. Our results highlight the importance of studying AMP-interactions in vivo.

Tachinid parasitoid Exorista japonica affects the utilization of diet by changing gut microbial composition in the silkworm, Bombyx mori

Changes in both intake and digestion of feed have been demonstrated in the host following parasitization. However, its regulatory mechanism has not been clarified. In this study, silkworms and Exorista japonica were used as research objects to analyze the effect of parasitism on the midgut immune system of the silkworm. After being parasitized, the expressions of antimicrobial peptide (AMP) genes of silkworms showed a fluctuating trend of first upregulation and then downregulation, while phenoloxidase and lysozyme activities were inhibited. To study the possible impact of the downregulation of AMP genes on intestinal microorganisms, the characteristics of the intestinal microbial population of silkworms on the third day of parasitism were analyzed. The relative abundance of Firmicutes, Proteobacteria, and Bacteroidota decreased, while that of Actinobacteriota increased. The increased abundance of conditionally pathogenic bacteria Serratia and Staphylococcus might lead to a decrease in the amount of silkworm ingestion. Meanwhile, the abundance of Acinetobacter, Bacillus, Pseudomonas, and Enterobacter promotes an increase in the digestion of nutrients. This study indicated that the imbalance of intestinal microbial homeostasis caused by parasitism may affect the absorption and digestion of nutrients by the host. Collectively, our findings provided a new clue for further exploring the mechanism of nutrient transport among the host, parasitoid, and intestinal microorganisms.

Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, we found that several antimicrobial peptide genes, such as metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

Heterologous Expression and Bioactivity Determination of Monochamus alternatus Antibacterial Peptide Gene in Komagataella phaffii (Pichia pastoris)

Insects have evolved to form a variety of complex natural compounds to prevent pathogen infection in the process of a long-term attack and defense game with various pathogens in nature. Antimicrobial Peptides (AMPs) are important effector molecules of the insect immune response to the pathogen invasion involved in bacteria, fungi, viruses and nematodes. The discovery and creation of new nematicides from these natural compounds is a key path to pest control. A total of 11 AMPs from Monochamus alternatus were classified into 3 categories, including Attacin, Cecropin and Defensin. Four AMP genes were successfully expressed by Komagataella phaffii KM71. The bioassay results showed that the exogenous expressed AMPs represented antimicrobial activity against Serratia (G-), Bacillus thuringiensis (G+) and Beauveria bassiana and high nematicide activity against Bursaphelenchus xylophilus. All four purified AMPs' protein against B. xylophilus reached LC50 at 3 h (LC50 = 0.19 mg·mL-1 of MaltAtt-1, LC50 = 0.20 mg·mL-1 of MaltAtt-2 and MaltCec-2, LC50 = 0.25 mg·mL-1 of MaltDef-1). Furthermore, the AMPs could cause significant reduction of the thrashing frequency and egg hatching rate, and the deformation or fracture of the body wall of B. xylophilus. Therefore, this study is a foundation for further study of insect biological control and provides a theoretical basis for the research and development of new insecticidal pesticides.

The susceptibility of Hyphantria cunea larvae to microbial pesticides Bacillus thuringiensis and Mamestra brassicae nuclear polyhedrosis virus under Cd stress

Heavy metal pollution is an abiotic factor that can affect the efficiency of pest control. In this study, two microbial pesticides, Bacillus thuringiensis and Mamestra brassicae nuclear polyhedrosis virus (MbNPV), were used to treat Hyphantria cunea larvae with Cd pre-exposure, and the humoral and cellular immunity of H. cunea larvae with Cd exposure were evaluated. The results showed that Cd exposure increased the susceptibility of H. cunea larvae to microbial pesticides B. thuringiensis and MbNPV, and the lethal effect of Cd exposure and microbial pesticides on H. cunea larvae was synergistic. Cd exposure significantly decreased the expression of pathogen recognition genes (GNBP1 and GNBP3), signal transduction genes (Relish, Myd88, Tube, and Imd), and antimicrobial peptide gene (Lebocin) in the humoral immunity of H. cunea larvae compared with the untreated larvae. Parameters of cellular immunity, including the number of hemocytes, phagocytic activity, melanization activity, encapsulation activity, and the expression of three phagocytic regulatory genes (HEM1, GALE1, GALE2), were also found to decrease significantly in Cd-treated larvae. TOPSIS analysis showed that humoral immunity, cellular immunity, and total immunity levels of H. cunea larvae with Cd exposure were weaker than those in untreated larvae. Correlation analysis showed that the mortality of two microbial pesticides investigated in H. cunea larvae was negatively correlated with the humoral and cellular immunity of larvae. Taken togther, Cd exposure results in immunotoxic effects on H. cunea larvae and the use of microbial pesticides are an effective strategy for pest control in heavy metal-polluted areas.