Gut bacterium Burkholderia cepacia (BsNLG8) and immune gene Defensin A contribute to the resistance against Nicotine-induced stress in Nilaparvata lugens (Stål).

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts.

MicroRNA Targets PAP1 to Mediate Melanization in Plutella xylostella (Linnaeus) Infected by Metarhizium anisopliae.

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3' untranslated region (3' UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest.

Nicotine perturbs the microbiota of brown planthopper (Nilaparvata lugens stål Hemiptera: Delphinidae).

Bacterial symbionts exhibiting co-evolutionary patterns with insect hosts play a vital role in the nutrient synthesis, metabolism, development, reproduction, and immunity of insects. The brown planthopper (BPH) has a strong ability to adapt to various environmental stresses and can develop resistance to broad-spectrum insecticides. We aimed to investigate whether gut symbionts of BPH play a major role in the detoxification of insecticides and host fitness in unfavorable environments. Nicotine-treated rice plants were exposed to BPH (early stage) and the gut microbiome of the emerging female adults were analyzed using high throughput sequencing (HTS). Nicotine administration altered the diversity and community structure of BPH symbionts with significant increases in bacterial members such as Microbacteriaceae, Comamondaceae, Enterobacteriaceae, and these changes may be associated with host survival strategies in adverse environments. Furthermore, the in-vitro study showed that four intestinal bacterial strains of BPH (Enterobacter NLB1, Bacillus cereus NL1, Ralstonia NLG26, and Delftia NLG11) could degrade nicotine when grown in a nicotine-containing medium, with the highest degradation (71%) observed in Delftia NLG11. RT-qPCR and ELISA analysis revealed an increased expression level of CYP6AY1 and P450 enzyme activities in Delftia NLG11, respectively. CYP6AY1 increased by 20% under the action of Delftia and nicotine, while P450 enzyme activity increased by 18.1%. After CYP6AY1 interference, nicotine tolerance decreased, and the mortality rate reached 76.65% on the first day and 100% on the third day. Moreover, Delftia NLG11 helped axenic BPHs to increase their survival rate when fed nicotine in the liquid-diet sac (LDS) feeding system. Compared with axenic BPHs, the survival rate improved by 25.11% on day 2% and 6.67% on day 3. These results revealed an altered gut microbiota and a cooperative relationship between Delftia NLG11 and CYP6AY1 in nicotine-treated BPH, suggesting that insects can adapt to a hostile environment by interacting with their symbionts and providing a new idea for integrated pest management strategies.

Combined transcriptomic and proteomic analysis of developmental features in the immune system of Plutella xylostella during larva-to-adult metamorphosis.

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is considered one of the most destructive worldwide agricultural pests and has developed various defence mechanisms to fight against the available pesticides. Understanding the host-defence system of P. xylostella is vital for developing biocontrol-based pest management strategies. Although there are several studies on P. xylostella, little is known about the changes in the immune system during the larva-to-adult metamorphosis. RNA-seq and iTRAQ investigations of P. xylostella from 2-day-old fourth instar larvae (L4D2), pupa (P0), and adult (A0) were done to understand these alterations at a molecular level. A total of 412/ 584 up-regulated and 1430/ 757 down-regulated genes/proteins between larva and pupa, 813/ 589 up-regulated and 1206/ 846 down-regulated genes/proteins between pupa and adult were identified. It was shown that the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) expression were up-regulated during the pupation and emergence of metamorphosis. The pathway enrichment analysis demonstrated that DEGs and DEPs were mainly associated with the energy generation and metabolism and innate immunity of the insect. The expression of immune-related and developmental-related genes were significantly different during the developmental process of P. xylostella. Moreover, the expression of four focused genes, i.e., serine proteinase inhibitor (Serpin-15), prophenoloxidase activating proteinase 1 (PAP-1) and 3a (PAP-3a), Gram-negative bacteria-binding protein (GNBP-6), was different in developmental stages and after Bacillus thuringiensis HD73 and Metarhizium anisopliae infection. The phenoloxidase (PO) activity in plasma was also significantly up-regulated during the pathogen infection. Recombinant proteins PAP-1, PAP-3a, GNBP-6 could significantly trigger the PO activity in vitro, Serpin-15 could suppress the PO activity. Taken together, these results indicate that Serpin-15, PAP-1, PAP-3a, and GNBP-6 might have the potential for co-regulation of immunity and development in P. xylostella. In conclusion, this study provided the immune system dynamics in the developmental process of P. xylostella and identified four candidate genes that can serve as potential targets for pest control strategies.

Recent Advances and Future Potential of Long Non-Coding RNAs in Insects.

Over the last decade, long non-coding RNAs (lncRNAs) have witnessed a steep rise in interest amongst the scientific community. Because of their functional significance in several biological processes, i.e., alternative splicing, epigenetics, cell cycle, dosage compensation, and gene expression regulation, lncRNAs have transformed our understanding of RNA's regulatory potential. However, most knowledge concerning lncRNAs comes from mammals, and our understanding of the potential role of lncRNAs amongst insects remains unclear. Technological advances such as RNA-seq have enabled entomologists to profile several hundred lncRNAs in insect species, although few are functionally studied. This article will review experimentally validated lncRNAs from different insects and the lncRNAs identified via bioinformatic tools. Lastly, we will discuss the existing research challenges and the future of lncRNAs in insects.

Transcriptomic Analysis of Metarhizium anisopliae-Induced Immune-Related Long Non-Coding RNAs in Polymorphic Worker Castes of Solenopsis invicta.

Long non-coding RNAs (lncRNAs) represent a class of RNA molecules that do not encode proteins. Generally studied for their regulatory potential in model insects, relatively little is known about their immunoregulatory functions in different castes of eusocial insects, including Solenopsis invicta, a notoriously invasive insect pest. In the current study, we used Metarhizium anisopliae, an entomopathogenic fungus, to infect the polymorphic worker castes (Major and Minor Workers) and subjected them to RNA sequencing at different intervals (6, 24, and 48 h post-infection (hpi)). Comprehensive bioinformatic analysis identified 5719 (1869 known and 3850 novel) lncRNAs in all libraries. Genomic characteristics analysis showed that S. invicta lncRNAs exhibited structural similarities with lncRNAs from other eusocial insects, including lower exon numbers, shorter intron and exon lengths, and a lower expression profile. A comparison of lncRNAs in major and minor worker ants revealed that several lncRNAs were exclusively expressed in one worker caste and remained absent in the other. LncRNAs such as MSTRG.12029.1, XR_005575440.1 (6 h), MSTRG.16728.1, XR_005575440.1 (24 h), MSTRG.20263.41, and MSTRG.11994.5 (48 h) were only present in major worker ants, while lncRNAs such as MSTRG.8896.1, XR_005574239.1 (6 h), MSTRG.20289.8, XR_005575051.1 (24 h), MSTRG.20289.8, and MSTRG.6682.1 (48 h) were only detected in minor workers. Additionally, we performed real-time quantitative PCR and experimentally validated these findings. Functional annotation of cis-acting lncRNAs in major worker ants showed that lncRNAs targeted genes such as serine protease, trypsin, melanization protease-1, spaetzle-3, etc. In contrast, apoptosis and autophagy-related genes were identified as targets of lncRNAs in minor ants. Lastly, we identified several lncRNAs as precursors of microRNAs (miRNAs), such as miR-8, miR-14, miR-210, miR-6038, etc., indicating a regulatory relationship between lncRNAs, miRNAs, and mRNAs in antifungal immunity. These findings will serve as a genetic resource for lncRNAs in polymorphic eusocial ants and provide a theoretical basis for exploring the function of lncRNAs from a unique and novel perspective.

Analysis of synonymous codon usage patterns of HPRT1 gene across twelve mammalian species.

Genetic changes in Hypoxanthine guanine phosphoribosyltransferace (HPRT1) gene can alter the expression of the dopamine neurotransmitter leads to abnormal neuron function, a disease called Lesch-Nyhan syndrome (LNS). Although different studies were conducted on LNS, information on codon usage bias (CUB) of HPRT1 gene is limited. The present study examines the genetic determinants of CUB in HPRT1 gene using twelve mammalian species. In the coding sequence of HPRT1 genes, A/T ending codons was most frequently used. A higher ENC value was observed indicating lower HPRT1 gene expression in the selected mammalian species. Correlation analysis indicates that compositional constraints under mutation pressure can involve in CUB of HPRT1 genes among the selected mammalian species. Relative synonymous codon usage (RSCU) value revealed that the codons such as ACT, AGG, ATT and AGC were over-represented in each of the mammalian species. Result from the analysis of the RSCU indicates that compositional constraint is a key driver for the variation in codon usage. Ratio of nonsynonymous (dN) and synonymous (dS) substitution further suggested that purifying selection occurs among the HPRT1 gene of studied mammals to maintain its protein function under the process of evolution. Our findings report an insight into the codon usage patterns of HPRT1 gene and will be useful for LNS management.

Role of serine protease inhibitors in insect-host-pathogen interactions.

Serine protease inhibitors (serpins), evolutionary old, structurally conserved molecules, are a superfamily of proteins found in almost all living organisms. Serpins are relatively large, typically 350-500 amino acids in length, with three ß-sheets and seven to nine α-helices folding into a conserved tertiary structure with a reactive center loop. Serpins perform various physiological functions in insects, including development, digestion, host-pathogen interactions, and innate immune response. In insects, the innate immune system is characterized as the first and major defense system against the invasion of microorganisms. Serine protease cascades play a critical role in the initiation of innate immune responses, such as melanization and the production of antimicrobial peptides, and are strictly and precisely regulated by serpins. Herein, we provide a microreview on the role of serpins in the insect-host-pathogen interactions, emphasizing their role in immune responses, particularly in diamondback moth (Plutella xylostella), highlighting the important discoveries and also the gaps that remain to be explored in future studies.

Alternative splicing and insect ryanodine receptor.

Phylogenetic tree of the ryanodine receptor (RyR) family based on maximum likelihood estimation.

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella.

Expression of dsRNA in recombinant Isaria fumosorosea strain targets the TLR7 gene in Bemisia tabaci.

BACKGROUND: RNA interference (RNAi) technology shows a great potential in controlling agricultural pests, despite the difficulty of introducing exogenous dsRNA/siRNA into target pests. Isaria fumosorosea is a common fungal pathogen of the B-biotype Bemisia tabaci (whitefly), which is a widespread pest. Entomopathogenic fungi directly penetrate the cuticle and invade insect hemocoel. Application of I. fumosorosea expressing dsRNA of whitefly immunity-related gene may aid in developing RNAi technology to effectively control whiteflies. METHODS: A dsRNA expression plasmid, psTLR7, was constructed by introducing the Toll-like receptor 7 (TLR7) gene of B-biotype whitefly to the silent vector, pSilent-1. The plasmid psTLR7 was transferred into the protoplast of the I. fumosorosea strain IfB01. Then, the recombinant strain was screened out based on the biological stability and bioactivity against whitefly. RESULTS: A genetically stable recombinant strain IfB01-TRL7 was screened out. The impact of IfB01-TRL7 against whitefly TRL7 gene was validated by qPCR. Lower expression levels of the TLR7 gene was observed in the whiteflies infected by the recombinant strain. The bioassay results indicated that compared to IfB01 strain, IfB01-TRL7 increased the mortality of whitefly nymphs, and decreased and shortened the values of LC50 and LT50, thus indicating higher virulence of IfB01-TRL7. CONCLUSION: The expression of the dsRNA of whitefly TLR7 gene in recombinant I. fumosorosea strain successfully knocked down the host target gene by infecting the nymphs and enhanced the whiteflies mortality. The present study will give insight to new application of RNAi technology for more effective biocontrol of this pests.

Molecular characterization of a short peptidoglycan recognition protein (PGRP-S) from Asian corn borer (Ostrinia furnacalis) and its role in triggering proPO activity.

Peptidoglycan recognition proteins (PGRPs) are non-specific immune molecules of insects, and vertebrates etc., but are not present in plants and nematodes. In the current experiment, a PGRP DNA sequence (2,910 bp containing four exons) was identified from genomic DNA library of Asian corn borer, Ostrinia furnacalis, and a full-length cDNA programming PGRP was cloned (designed as OfPGRP-S) with an open reading frame of 579 bp, having 192 amino acid. This inferred amino acid sequence showed maximum similarity to known lepidopteran PGRPs. Quantitative real-time PCR investigation disclosed the level of mRNA of OfPGRP-S to be constitutively expressed in the whole developmental stages and with higher expression in the mature larvae. Even more the OfPGRP-S was mainly expressed in immune capable organs i.e., fat body and midgut, and was strongly induced by injecting gram-positive bacteria i.e., Staphylococus aureus. Recombinant protein OfPGRP-S could bind to S. aureus and Bacillus thuringiensis which enhance proPO activation in the presence of these microbes. The results indicated that OfPGRP-S is an inducible protein acting as a receptor-type PGRP for enhancing the proPO activation on exposure to bacteria.

Two-Sex Life Table Analysis for Optimizing Beauveria bassiana Application against Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae).

Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is a highly dispersive, polyphagous insect pest that severely defoliates crops. Excessive reliance on synthetic insecticides leads to ecological pollution and resistance development, urging scientists to probe eco-friendly biopesticides. Here, we explore the virulence of an entomopathogenic fungus, Beauveria bassiana, against S. exigua, resulting in 88% larval mortality. Using an age-stage, two-sex life table, we evaluated the lethal and sublethal effects of B. bassiana on the demographic parameters of S. exigua, including survival, development, and reproduction. Sublethal (LC20) and lethal concentrations (LC50) of B. bassiana impacted the parental generation (F0), with these effects further influencing the demographic parameters of the first filial generation (F1). The infected F1 offsprings showed a reduced intrinsic rate of increase (r), mean generation time (T), and net reproduction rate (R0). Larval developmental duration varied significantly between the control (10.98 d) and treated groups (LC20: 10.42; LC50: 9.37 d). Adults in the treated groups had significantly reduced lifespans (M: 8.22; F: 7.32 d) than the control (M: 10.00; F: 8.22 d). Reduced fecundity was observed in the B. bassiana-infected groups (LC20: 313.45; LC50: 223.92 eggs/female) compared to the control (359.55 eggs/female). A biochemical assay revealed elevated levels of detoxification enzymes (esterases, glutathione S-transferases, and acetylcholinesterase) in the F0 generation after B. bassiana infection. However, the enzymatic activity remained non-significant in the F1 generation likely due to the lack of direct fungal exposure. Our findings highlight the enduring effects of B. bassiana on the biological parameters and population dynamics of S. exigua, stressing its use in eco-friendly management programs.

Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival.

Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Chromosome-level genome assembly of the two-spotted spider mite Tetranychus urticae.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a notorious pest in agriculture that has developed resistance to almost all chemical types used for its control. Here, we assembled a chromosome-level genome for the TSSM using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled contigs had a total length of 103.94 Mb with an N50 of 3.46 Mb, with 87.7 Mb of 34 contigs anchored to three chromosomes. The chromosome-level genome assembly had a BUSCO completeness of 94.8%. We identified 15,604 protein-coding genes, with 11,435 genes that could be functionally annotated. The high-quality genome provides invaluable resources for the genetic and evolutionary study of TSSM.

Transcriptomic Analysis Reveals the Impact of the Biopesticide Metarhizium anisopliae on the Immune System of Major Workers in Solenopsis invicta.

The red imported fire ant (Solenopsis invicta Buren, 1972) is a globally significant invasive species, causing extensive agricultural, human health, and biodiversity damage amounting to billions of dollars worldwide. The pathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (1883), widely distributed in natural environments, has been used to control S. invicta populations. However, the interaction between M. anisopliae and the immune system of the social insect S. invicta remains poorly understood. In this study, we employed RNA-seq to investigate the effects of M. anisopliae on the immune systems of S. invicta at different time points (0, 6, 24, and 48 h). A total of 1313 differentially expressed genes (DEGs) were identified and classified into 12 expression profiles using short time-series expression miner (STEM) for analysis. Weighted gene co-expression network analysis (WGCNA) was employed to partition all genes into 21 gene modules. Upon analyzing the statistically significant WGCNA model and conducting Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the modules, we identified key immune pathways, including the Toll and Imd signaling pathways, lysosomes, autophagy, and phagosomes, which may collectively contribute to S. invicta defense against M. anisopliae infection. Subsequently, we conducted a comprehensive scan of all differentially expressed genes and identified 33 immune-related genes, encompassing various aspects such as recognition, signal transduction, and effector gene expression. Furthermore, by integrating the significant gene modules derived from the WGCNA analysis, we constructed illustrative pathway diagrams depicting the Toll and Imd signaling pathways. Overall, our research findings demonstrated that M. anisopliae suppressed the immune response of S. invicta during the early stages while stimulating its immune response at later stages, making it a potential biopesticide for controlling S. invicta populations. These discoveries lay the foundation for further understanding the immune mechanisms of S. invicta and the molecular mechanisms underlying its response to M. anisopliae.

Eurycomanone (EN) Activates Transcription Factor FoxO by Inhibiting the Insulin Signaling Pathway to Suppress the Development of Spodoptera frugiperda.

The insulin-like signaling (IIS) pathway is essential for insect growth and development. In this study, we showed that eurycomanone (EN) is an active compound with growth inhibitory activity against Spodoptera frugiperda larvae. Experiments in cells and RNA-seq analysis in the midgut showed that EN targeted the IIS pathway in S. frugiperda to activate the transcription factor SfFoxO (S. frugiperda forkhead boxO) to regulate mRNA levels associated with nutrient catabolism. Additionally, mass spectrometry imaging revealed that EN was distributed in the larval gut and enriched in the inner membrane of the gut. Immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EN induced program cell death (PCD) in the larvae midgut. Thus, EN targeted the insulin receptor to inhibit the IIS signaling pathway, exerting inhibitory activity on the growth and development of S. frugiperda larvae. Our results suggest that EN has great potential as a botanical pesticide, and the IIS signaling pathway may be an effective target for botanical pesticides.

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis.

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.