In this review, we show that predatory ants have a wide range of foraging behavior, something expected given their phylogenetic distance and the great variation in their colony size, life histories, and nesting habitats as well as prey diversity. Most ants are central-place foragers that detect prey using vision and olfaction. Ground-dwelling species can forage solitarily, the ancestral form, but generally recruit nestmates to retrieve large prey or a group of prey. Typically, ants are omnivorous, but some species are strict predators preying on detritivorous invertebrates or arthropod eggs, while those specialized on termites or other ants often have scouts that localize their target and then trigger a raid. They can use compounds that ease this task, including chemical insignificance, mimicry, and venoms triggering submissive behavior. Army ants include 8 Dorylinae and some species from other subfamilies, all having wingless queens and forming raids. Dorylinae from the Old World migrate irregularly to new nesting sites. The foraging of most New World species that prey on the brood of other ants is regulated by their biological cycle that alternates between a “nomadic phase” when the colony relocates between different places and a “stationary phase” when the colony stays in a bivouac constituting a central place. Among arboreal ants, dominant species forage in groups, detecting prey visually, but can use vibrations, particularly when associated with myrmecophytes. Some species of the genera Allomerus and Azteca use fungi to build a gallery-shaped trap with small holes under which they hide to ambush prey.

Hemiptera is one of the most significant orders of insect pests, including whiteflies, true bugs, aphids, planthoppers, psyllids, and so forth, which have led to substantial economic losses in agricultural industries and have significantly affected food yields through their ability to suck the phloem sap of crops and transmit numerous bacterial and viral pathogens. Therefore, explorations of pest-specific, eco-friendly and easy-to-adopt technologies for hemipteran pest control are urgently needed. To the best of our knowledge, microRNAs (miRNAs), which are endogenous non-coding small RNAs approximately 22 nucleotides in length, are involved in regulating gene expression via the direct recognition and binding of the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) or by acting as a center of a competitive endogenous RNA (ceRNA) network at the post-transcriptional level. This review systematically outlines the characterization and functional investigation of the miRNA biogenesis pathway in hemipteran pests, such as whiteflies, true bugs, aphids and planthoppers. In addition, we explored the results of small RNA sequencing and functional observations of miRNAs in these pests, and the results suggest that the numerous miRNAs obtained and annotated via high-throughput sequencing technology and bioinformatic analyses contribute to molting development, fitness, wing polyphenism, symbiont interactions and insecticide resistance in hemipteran pests. Finally, we summarize current advances and propose a framework for future research to extend the current data and address potential limitations in the investigation and application of hemipteran miRNAs.

The gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect-specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single-nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV-infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single-nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single-cell level.

Insect herbivores adapt and develop strategies to counteract plant chemical defenses. The aphid Uroleucon formosanum is a serious sap-sucking pest that infests lettuces containing toxic sesquiterpene lactones (STLs). Herein, we employed a combination of genome sequencing and RNA-seq transcriptome profiling to understand the mechanisms underlying phytotoxin tolerance in U. formosanum. We generated the first chromosome-level genome assembly for U. formosanum, with a total size of 453.26 Mb and a scaffold N50 of 33.22 Mb. Comparative genomic analyses revealed an enrichment of signals for positive selection and gene family expansion in immune-related pathways. Specifically, the expanded set of heat shock protein 70 (HSP70) genes showed upregulation after treatment with lactucin, suggesting that they may play a role in the immune response against STLs. The expression of takeout-like genes and cuticle-associated genes was also significantly increased in the lactucin-treated samples. Additionally, 53 cytochrome P450 monooxygenase, 30 carboxylesterase, 19 glutathione S-transferase, 32 uridine diphosphate glycosyltransferase and 63 ATP-binding cassette (ABC) transporter genes were identified in the U. formosanum genome. CYP4C1, CYP6A13 and 7 ABC genes were strongly upregulated in response to lactucin treatment, indicating the involvement of detoxifying enzymes in the tolerance of U. formosanum to STLs. Our findings suggest that the cuticle barrier, immune response and enzyme-mediated metabolic detoxification jointly enhance the tolerance of U. formosanum to phytotoxins and promote its adaptation to host plants. This study presents a valuable genomic resource and provides insights into insect adaptation to plant chemical challenges and future technological developments for pest management.

The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for “NGG” or “CCN” target sequences in the BmNPV genome, excluding non-recurring and potential targets in the B. mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome—one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild-type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains.

The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles’ progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed “direct parental” CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.

No mechanoreceptor potential C (NompC) is a major mechanotransduction channel with an important role in sensing of external mechanical stimuli by insects, which help these organisms to avoid injury and adapt to environmental changes. To explore the biological functions of NompC in Bactrocera dorsalis, a notorious agricultural pest, we successfully generated NompC knockout strains using clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) technology. BdorNompC knockout led to an adult lethal phenotype, with approximately 100% mortality at 3 d after eclosion. Morphological observation revealed that the legs and wings of BdorNompC knockout insects were deformed, while behavioral assays showed that the locomotion was impaired in both adults and larvae, relative to that of the wild-type strain. Moreover, BdorNompC knockout reduced gentle-touch response in larvae. These results suggest that BdorNompC is critical for B. dorsalis survival, and that this mechanosensation channel represents a potential new target for pest control agents. Our findings also represent novel evidence indicating that insect NompC is involved in modulating adult wing and leg morphology.

In the organisms with XX/XY sex chromosomes, Y chromosome is unique to males and plays an important role in male reproductive development. The study of Y chromosome genes will contribute to the development of pest genetic prevention and control technology. In this study, we identified 9 Y chromosome genes in Zeugodacus tau (Diptera: Tephritidae), including gene 16222. Protein structure analysis showed that 16222 was highly similar to odorant binding protein, and thus gene 16222 was named obp-Y. Obp-Y knockout (KO) significantly reduced hatching rate of offspring. Sperm detection results showed that obp-Y KO did not affect sperm number in the testes or sperm transfer during mating. We further examined the storage of sperms in females, and found that sperms in females mating with wild-type males began to transfer from spermathecal ducts to the spermathecae at hour 0 after the end of mating (AEM), and at 0–24 h AEM, the sperm count in the spermathecae gradually increased. However, no sperms were observed in spermathecae of females mating with mutant males at hours 0, 4, 8, 24 and 48 AEM. In summary, this study revealed that Y chromosome gene obp-Y was necessary for the storage of sperms in females. Our findings not only provide theoretical basis for elucidating the function of the Y chromosome, but also offer a molecular target for the genetic control over Z. tau.

In addition to preventing precocious larval metamorphosis, juvenile hormone (JH), synthesized in corpora allata (CA), is known to stimulate female reproduction of insects. JH titer is extremely low or absent during metamorphosis, but thereafter rapidly increases in the previtellogenic stage and rises to a peak in the vitellogenic phase. However, the mechanisms underlying the biosynthesis of high levels of JH in adults remain unclear. We found in this study that 12 genes involved in JH synthesis pathway were highly expressed in the CA of adult locusts. By transcriptome analysis and quantitative real-time – polymerase chain reaction validation, a total of 106 evolutionary conserved micro RNAs (miRNAs) and 163 species-specific miRNAs were identified in locust CA. Dual-luciferase assay revealed that 17 miRNAs bound to 10 JH synthesis genes (JHSGs) and downregulated their expression. These miRNAs were expressed in low levels during vitellogenic stage, which was oppositive from that of targeting JHSGs. Six miRNAs including miR-971-3p, miR-31a, miR-9-5p, miR-1-3p, miR-315, and miR-282 were selected for function study. Co-application of agomiRs resulted in significantly decreased levels of targeting JHSGs, accompanied by significantly reduced vitellogenin expression as well as arrested ovarian development. The data suggest that multiple miRNAs expressed synchronously at low levels in the vitellogenic phase, thereby ensuring the high levels of JHSG expression to facilitate JH biosynthesis required for JH-dependent female reproduction. The findings provide important information for deciphering miRNA—messenger RNA modules for JH biosynthesis as well as JH regulation of insect metamorphosis and reproduction.

Insect lytic polysaccharide monooxygenases (LPMO15s) are newly discovered copper-dependent enzymes that promote chitin degradation in insect through oxidative cleavage of glycosidic bonds. They are potential pesticide targets due to their critical role for chitin turnover in the integument, trachea, and peritrophic matrix of the midgut during insect molting. However, the knowledge about whether and how LPMO15s participate in chitin turnover in other tissues is still insufficient. Here, using the orthopteran pest Locusta migratoria as a model, a novel alternative splicing site of LmLPMO15-1 was discovered and it produces 2 variants, LmLPMO15-1a and LmLPMO15-1b. The transcripts of LmLPMO15-1a and LmLPMO15-1b were specifically expressed in the trachea and foregut, respectively. RNA interference targeting LmLPMO15-1 (a common fragment shared by both LmLPMO15-1a and LmLPMO15-1b), a specific region of LmLPMO15-1a or LmLPMO15-1b all significantly reduced survival rate of nymphs and induced lethal phenotypes with developmental stasis or molt failure. Ultrastructure analysis demonstrated that LmLPMO15-1b was specifically involved in foregut old cuticle degradation, while LmLPMO15-1a was exclusively responsible for the degradation of the tracheal old cuticle. This study revealed LmLPMO15-1 achieved tissue-specific functional differentiation through alternative splicing, and proved the significance of the spliced variants during insect growth and development. It provides new strategies for pest control targeting LPMO15-1.

Bombyx mori ELAV-like-1 (BmEL-1) and B. mori ELAV-like-2 (BmEL-2) are 2 members of the ELAV-like family of RNA-binding proteins. Mutations in Bmel-1 and Bmel-2 resulted in 5.8% and 28.5% decreases in larval weight on the 3rd day of the 5th instar larva (L5D3), respectively. Triglycerides (TG) are the most important energy resource and are the main component of neutral fat (NF) in animals. To investigate the role of Bmelav-like genes in the synthesis and decomposition of TG, transcriptomic, and metabolic analyses were performed on the whole bodies on the 1st day of the 2nd instar larvae (L2D1) and on fat bodies on L5D3 of Bmel-1⁻ and Bmel-2⁻ mutants, respectively. As compared with the control silkworm, differentially expressed genes generated in both mutants were mainly enriched in lysine degradation, fatty acid (FA) metabolism, and unsaturated FAs biosynthesis. The diglyceride and phosphatide contents were significantly lower in Bmel-1⁻ and Bmel-2⁻ fat bodies than those of the control group. Consistently, the NF content of both mutants’ fat bodies were reduced by 50% and 60%, respectively. BmEL-2 positively regulates BmAGPATγ (B. mori 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma, LOC101741736) and BmFaF2 (B. mori fatty acid synthetase-associated factor 2, LOC101739090) expression by binding to the specific regions of their 3′ untranslated regions in BmN cells. This study suggests that BmEL-2 may be an important regulator of BmAGPATγ and BmFAF2 expression and thereby participates in TG metabolism in the silkworm fat body.

Respiration is a vital process essential for organism survival, with most terrestrial insects relying on a sophisticated tubular tracheal network. In the current study, a gene with repetitive sequence was identified within the silkworm genome. Designated as BmMuc91C, it contains a dozen repeated motifs “PSSSYGAPX” and “GGYSSGGX” in its sequence. BmMuc91C exhibits specific expression in the tracheal system of silkworm larvae, with significantly higher expression levels during the molting stage. Overexpression of BmMuc91C in individual silkworms resulted in a marked increase in tracheal diameter, particularly during the molting stage. Immunofluorescence staining using a BmMuc91C antibody revealed a noticeable thickening of the apical extracellular matrix in the trachea. Tensile testing confirmed a considerable enhancement in tracheal elasticity. Additionally, a BmMuc91C mutation strain of silkworms was generated using the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 system. Although no significant differences were observed in the growth, development, and molting of BmMuc91C mutant silkworms, mechanical tests demonstrated a decrease in tracheal elasticity. Transcriptomic techniques revealed that a significant number of cuticular and chitin-binding proteins were among the differentially expressed genes between mutant and wild-type silkworms. Furthermore, the recombined BmMuc91C protein was successfully expressed using the Escherichia coli system. Cross-linking experiments with horseradish peroxidase demonstrated the formation of macromolecular complexes of BmMuc91C, which exhibited spontaneous luminescent properties under ultraviolet light. This research sheds light on the role of elastic proteins in insect tracheae and provides valuable insights for the development of elastic biomaterials.

Diapause is an adaptive strategy employed by insects to endure adverse environmental conditions and is characterized by reduced metabolic activity, primarily due to a decreased respiratory rate. AMP-activated protein kinase (AMPK) serves as an intracellular energy regulator, modulating energy metabolism in response to metabolic fluctuations. However, its role in pupal diapause of the cotton bollworm, Helicoverpa armigera, remains unclear. In this study, we found that AMPK and its active form, P-AMPK, are highly expressed in diapause-destined pupae. Furthermore, activation of AMPK delayed the development of nondiapause-destined pupae, suggesting a critical role for AMPK in the regulation of pupal diapause in H. armigera. Manipulating AMPK activity in H. armigera epidermal (HaEpi) cells and pupae significantly influenced the expression of hypoxia-inducible factor-1α (HIF-1α), which our laboratory previously reported as a key inducer of pupal diapause through the reduction of mitochondrial activity in H. armigera. Histone deacetylase 4 (HDAC4), a shuttle protein phosphorylated by AMPK which translocates between the cytoplasm and the nucleus, was found to exhibit significantly higher expression in diapause-destined pupal brains compared to their nondiapause counterparts. AMPK in both HaEpi cells and pupae positively regulated the protein levels of P-HDAC4 by binding to the HDAC4 promoter. Additionally, HDAC4 was shown to enhance HIF-1α expression in diapause-destined individuals. HDAC4 binds to and deacetylates heat shock protein 70 (HSP70), and reduced acetylation of HSP70 was found to significantly elevate HIF-1α protein levels. The AMPK-HIF-1α signaling pathway appears to play a pivotal role in reducing mitochondrial activity and facilitating diapause induction in H. armigera pupae.

Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (ΔMrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with ΔMrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

Juvenile hormones (JHs) play a crucial role in regulating development and reproduction in insects. Most insects predominantly synthesize JH III, which typically involves esterification followed by epoxidation, lepidopteran insects use a pathway of epoxidation followed by esterification. Although hemipteran insects have JH III and JH skipped bisepoxide III (JH SB3), the synthesis pathway and key epoxidases remain unclear. This study was conducted on Aphis craccivora, and demonstrated that corpora allata, microsomes, Ac-CYP15C1, and Ac-JHAMT catalyze JH III production in vitro, establishing the pathway of epoxidation followed by esterification. These findings were further confirmed through RNA interference and molecular docking. The presence of JH III and JH SB3 in A. craccivora was identified, and their synthesis pathway was elucidated as follows: Ac-CYP15C1 oxidizes farnesic acid to JH A, followed by methylation to JH III by Ac-JHAMT, possibly providing an epoxidation site on the second carbon for JH SB3. This alteration may significantly contribute to the differentiation and functional diversification of JH types in insects.

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye-staining properties were used to characterize hemocyte types from T. castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2 h), then increased over time (at 4 and 6 h) and eventually returned to the naive state by 24 h post-injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T. castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph in vivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes in vitro, forming capsules to encapsulate and melanize nickel–nitrilotriacetic acid (Ni–NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T. castaneum larvae, offering valuable insights into cell-mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T. castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T. castaneum hemocytes in combating pathogens.

Insulin-like peptides (ILPs) act as crucial reproductive neuropeptides in insects, regulating insect reproduction through the insulin signaling pathway (ISP). Our previous studies have found that the sublethal concentrations (LC₁ and LC₁₀) of lambda-cyhalothrin (λCy) could induce severe reproductive toxicity in the lacewing, Chrysoperla sinica (Tjeder), but the toxicological mechanism remains unclear. This study discovered that λCy could inhibit CsILP transcription, leading to a decrease in insulin content and downregulation of C. sinica insulin receptor (CsInR) and C. sinica forkhead box O (CsFOXO) expression in ISP. Interference with CsILP expression resulted in downregulation of C. sinica vitellogenin (CsVg) and decreasing fecundity, while exogenous injection of bovine insulin promoted upregulation of CsVg expression and facilitated reproduction in female adults of C. sinica. Meanwhile, interference with FOXO of ILP downstream transcription factor could lead to downregulation of CsVg, hindering ovarian development and resulting in a decrease in egg production. However, exogenous injection of bovine insulin could remedy the effects caused by FOXO interference. In addition, ILP mediates juvenile hormone and 20-hydroxyecdysone biosynthesis by acting on their synthetic regulatory enzymes and influences the signal transduction of the 2 reproductive endocrine hormones, thereby regulating the reproductive endocrine environment in C. sinica. In conclusion, λCy inhibits CsILP expression, leading to disorder of ISP, leading to the reduced fecundity of C. sinica.

In insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E) pathways jointly regulate fecundity, but only methyl farnesoate (MF) and ponasterone A exist in mites. Comparative transcriptomic analysis in Panonychus citri showed that E75B was significantly downregulated when exposed to lufenuron. Knockdown of E75B significantly affects the expression of vitellogenin (Vg), Fushi tarazu factor 1 (Ftz-f1) and juvenile hormone acid O-methyltransferase (JHAMT), reducing fecundity in mites. The knockdown of Ftz-f1 produced a more significant effect than the knockdown of E75B, indicating that the ponasterone A pathway positively regulates fecundity in P. citri. After the knockdown of JHAMT, the expression levels of both Vg and Ftz-f1 and fecundity were significantly increased, along with the inhibition of Kr-h1, suggesting that JHAMT was negatively correlated with fecundity in the regulatory network. Knockdown of Kr-h1 inhibited the expression of Vg and Ftz-f1 and fecundity, and whether the drop in fecundity is caused by Kr-h1 or Ftz-f1 is unclear. Subsequent feeding with MF induced Kr-h1 and Vg expression, whereas no significant effects were observed for JHAMT and Ftz-f1. Therefore, the MF pathway stimulates fecundity independently. RNA interference (RNAi) showed that JHAMT and Ftz-f1 inhibited each other, resulting in opposite effects of MF and ponasterone A pathways on steady-state fecundity when either factor changed. Meanwhile, JHAMT knockdown led to increased fecundity, indicating that the stimulating effect of the ponasterone A pathway was greater than the inhibiting effect of the MF pathway, and demonstrating the dominant role of the ponasterone A pathway. Therefore, the interaction between JHAMT and Ftz-f1 may be closely associated with the maintenance of MF–ponasterone A regulatory network homeostasis and is involved in the reduction of fecundity in P. citri induced by exposure to lufenuron.

American foulbrood (AFB) disease, caused by the bacterium Paenibacillus larvae, is a devastating disease affecting honeybee (Apis mellifera L.) populations worldwide. Commonly treated with antibiotics, which have negative impacts on both honeybees and the environment, there is an urgent need for alternatives in AFB control. This study aimed to investigate the effects of gallic acid (GA) on honeybee larvae challenged with P. larvae spores and explore its modulation of larval microbiota. Our results demonstrated that in the presence of P. larvae spores, coadministration of 125 µg/mL GA significantly increased the survival rate and body weight of honeybee larvae. Molecular docking analyses revealed that GA competitively binds to spore germination proteins YndE and GerM, with affinities comparable to L-tyrosine and stronger than uric acid, respectively, suggesting interference with P. larvae spore germination. 16S rRNA gene amplicon sequencing revealed that GA treatment augmented bacterial diversity and enriched lactic acid bacteria (LAB) in honeybee larvae. Whole-genome sequencing of 2 LAB strains, Apilactobacillus kunkeei GL-2 and Enterococcus faecium GL-6, isolated from GA-treated larvae, unveiled their potential to produce antimicrobial secondary metabolites and bacteriocins, which may contribute to their competitive advantages against P. larvae. Notably, the E. faecium GL-6 strain possessed genes encoding gallate decarboxylase, enabling GA utilization, and 2 putative bacteriocinogenic genetic clusters for enterolysin A and enterocin L50 a/b. These findings suggest that GA and the GL-6 strain hold potential as preventive measures against AFB disease in honeybees through modulation of gut microbiota and competitive inhibition of P. larvae.

Previous studies have shown oviposition deterring properties of 8 coconut free fatty acid (CFFA) compounds on fruit flies with different key deterrent components for different species. Here we evaluated oviposition deterrence of CFFA using laboratory 2-choice bioassays against Zeugodacus cucurbitae, determined key-bioactive deterrent compounds, and evaluated their behavioral mode. Unlike other reported fruit fly species, CFFA mixture increased Z. cucurbitae oviposition when directly applied on an oviposition substrate. When tested individually in subsequent tests, 4 compounds (caprylic, capric, oleic, and linoleic acids) significantly reduced the oviposition (“negative-compounds”), 1 compound (stearic acid) had no effect (“neutral-compound”), and 3 compounds (lauric, myristic, and palmitic acids) stimulated the oviposition (“positive-compounds”). The 4-component negative-compound blend was effective at reducing oviposition. However, adding stearic acid to the 4-component blend (5-component blend, 5c) further reduced oviposition. Adding any of the positive-compounds to the 5c resulted in loss of oviposition deterrence, suggesting the 5c as the key deterrent component blend. The blend was also effective in no-choice assays and when applied on cucumbers, a preferred host of Z. cucurbitae. When given a choice, Z. cucurbitae made 48.5% fewer visits, spent 39% less time, and oviposited 88.2% fewer eggs per min on 5c treated pumpkin agar than on control agar, suggesting that the 5c blend has both spatial repellency and contact deterrence. Given that all compounds are registered food additives and generally regarded as safe, this blend has potential application in behavioral control strategies, such as push–pull, to protect host fruit against Z. cucurbitae.

Oviposition preferences of plant-feeding predators remain a complex topic, as such omnivores choose oviposition sites by assessing both plant characteristics and the quality and quantity of nearby animal food sources. Orius predators are omnivores that oviposit endophytically, thus plant characteristics play an important role in their oviposition choices. In this study, we assessed the oviposition and foraging preferences of O. laevigatus and O. majusculus on vegetative and flowering chrysanthemum plants, and assessed the survival of their offspring on differently aged tissues. Our results show a preference of O. laevigatus for young and tender chrysanthemum tissues, where the survival of the nymphs was longer on a plant diet. In contrast, O. majusculus selected older plant parts when laying its eggs, and nymphs did not survive long on any of the plant tissues offered. The foraging activity of Orius females for animal prey (Ephestia kuehniella eggs) did not reveal any specific pattern for either of the two predators. Furthermore, we tested the plasticity of the within-plant oviposition preferences of O. laevigatus, by offering sentinel prey (E. kuehniella eggs) on distinct plant parts. We found that more eggs were laid in older plant tissue when animal prey was offered lower on the plant. Overall, our findings show that oviposition choices of Orius predators are based on a dynamic interplay between plant characteristics, presence of animal and/or floral food sources among other factors, and that differences may well occur between closely related species based on the importance of plant resources in their diet.

Spiders are an abundant group of natural enemies preying on insect pests in agroecosystem. But their potential in biological control has not been fully realized due to difficult mass production. One hindrance is the intense intraspecific aggression in spiders. Neurotransmitters such as serotonin play important roles in modulating aggression. Here, we investigated the regulatory function of serotonin (5-hydroxytryptamine [5-HT]) signaling in the intraspecific aggression in a wandering spider Pardosa pseudoannulata (Araneae, Lycosidae). The aggression was quantified with 5 escalated aggression behaviors as approach, chasing, lunging, boxing, and biting. Virgin (VG) females exhibited higher aggression levels but less 5-HT content than post-reproductive (PR) females. Systemic increase of 5-HT via 5-HT injection decreased aggression, while decrease of 5-HT via RNA interference (RNAi) of the tryptophan hydroxylase gene, increased aggression. The involvement of the four 5-HT receptors were determined via individual or combined RNAi. Co-RNAi of the three 5-HT1 genes increased overall aggression with decreased incidents of approach, chasing, lunging, and increased biting. RNAi of 5-HT1B decreased approach and increased biting, whereas RNAi of 5-HT1A or 5-HT1C did not affect aggression. RNAi of 5-HT7 decreased approach only. Therefore, different 5-HT receptor types contribute to different aspects of the inhibitory effects of 5-HT on aggression and provide several pharmacological targets for manipulating spider aggression. 5-HT injection did not affect spiders’ predation on their insect prey, the brown planthopper Nilaparvata lugens. The findings reveal 1 neuronal mechanism regulating intraspecific aggression in spiders and provide an insight in developing aggression suppression strategies for spider mass rearing.

In many animals, drastic changes are observed during sexual maturation characterized by the reproductive system development concomitantly to the sexual behavior ontogenesis. These modifications are under the control of internal and external factors such as food. Sexual maturation requires considerable energetic investment, and diet has been shown to affect reproductive activities in many taxonomic groups, especially in insects and vertebrates. By contrast, diet effects on sexual behavior development remain largely unexplored. To elucidate this aspect, we used the male moth Agrotis ipsilon which undergoes sexual maturation occurring between the third and the fifth day postemergence. During this period, males are sensitive to female sex pheromones and a stereotypical sexual behavior characterized by female-oriented flight takes place. In our study, we compared (1) sex pheromone detection by electroantennography recordings and (2) behavioral response in wind tunnel assays between males fed with different diets found in nature. Compared to standard sucrose diet, males fed with sucrose, fructose, and glucose supplemented with sodium (a mineral element necessary for the locomotor activity in several moths) did not respond better to female sex pheromones but clearly exhibited an earlier behavioral response. Thus, such a diet accelerates the development of sex pheromone-mediated oriented flight, probably by facilitating the central processing of sex pheromone information in male A. ipsilon moths. Our results provide new information on the influence of nutritional intake on the ontogenesis of male sexual behavior in animals.

Species distributed across wide elevational gradients are likely to experience local thermal adaptation and exhibit high thermal plasticity, as these gradients are characterised by steep environmental changes over short geographic distances (i.e., strong selection differentials). The prevalence of adaptive intraspecific variation in thermal tolerance with elevation remains unclear, however, particularly in freshwater taxa. We explored variation in upper and lower thermal limits and acclimation capacity among Iberian populations of adults of the widespread water beetle Agabus bipustulatus (Dytiscidae) across a 2000 m elevational gradient, from lowland to alpine areas. Since mean and extreme temperatures decline with elevation, we predicted that populations at higher elevations will show lower heat tolerance and higher cold tolerance than lowland ones. We also explored whether acclimation capacity is positively related with climatic variability across elevations. We found significant variation in thermal limits between populations of A. bipustulatus, but no evidence of local adaptation to different thermal conditions across the altitudinal gradient, as relationships between thermal limits and elevation or climatic variables were largely nonsignificant. Furthermore, plasticities of both upper and lower thermal limits were consistently low in all populations. These results suggest thermal niche conservatism in this species, likely due to gene flow counteracting the effects of divergent selection, or adaptations in other traits that buffer exposure to climate extremes. The limited adaptive potential and plasticity of thermal tolerance observed in A. bipustulatus suggest that even generalist species, distributed across wide environmental gradients, may have limited resilience to global warming.