Termites are not only social insects but also significant global insect pests. Investigating the molecular mechanisms regulating immune defense response in termites is beneficial for developing novel approaches to termite management. Currently, research mainly focuses on coding RNAs in termite immunity, with limited exploration of non-coding RNAs. Here, we identified miR-701, a markedly downregulated microRNA (miRNA) in the globally significant termite pest Coptotermes formosanus after Metarhizium anisopliae infection, which targets the immune gene Toll4. Transcriptome analysis of termites injected with miR-701 agomir revealed that miR-701 affects the immune-related response, growth, and development of termites. Treatment with miR-701 agomir, either through injection or ingestion, resulted in a notably reduced survival rate of termites infected with M. anisopliae compared to the control group infected with M. anisopliae alone. Additionally, termites injected with miR-701 agomir exhibited a significant decrease in the expression of antimicrobial peptide genes termicin and lysozyme, alongside a notable increase in the colony-forming units of M. anisopliae in the infected termites. Subsequent investigations demonstrated that miR-701 suppressed the expression of the target gene Toll4, consequently inhibiting the Toll signaling pathway and diminishing the expression of antimicrobial peptides. These findings suggest that termites can combat M. anisopliae by downregulating miR-701 expression to activate the Toll signaling pathway and enhance antimicrobial peptides synthesis. This discovery improves our comprehension of the role of miRNAs in termites’ immune responses and the mechanism of termites managing miRNAs to boost their pathogen resistance. Additionally, it reveals a new molecular target for termite biological control.

Urbanization significantly impacts genetic connectivity and imposes selective pressures on insect populations, particularly in urban habitats. Although these effects are well documented at the scale of individual cities, the impact of mega-urbanization on species occupying large urban agglomerations remains poorly understood. Here, we investigate the population structure and detect selection signals associated with local adaptation in the white butterfly Pieris latouchei across the Yangtze River Delta Megalopolis (YRDM), a region comprising 27 contiguous cities and adjacent areas. Using genome-wide 2b-RADseq data, we demonstrate that urbanization reduces genetic diversity but not gene flow in the pest populations. Compared to peripheral populations, we observed population homogenization and identified selected genes related to detoxification, immune response, and thermal stress resistance in YRDM populations. Mega-urbanization, driven by habitat modifications and an extensive transport system, facilitated the expansion of a specific population with adaptive genes, leading to the loss of genetic diversity in YRDM. This study provides genomic evidence for low genetic divergence and the genetic basis of adaptation to megalopolitan environments in an urban insect pest.

Desiccant dusts have been shown to be effective materials in the management of bed bugs (Cimex spp.). Past research primarily focused on exploring the direct lethal effect of dusts against bed bugs, whereas information on their sublethal effects on bed bugs are limited. In this study, we evaluated the lethal effect of 3 dust products (Johnson's^® Baby powder, Vazor DE powder^™, and CimeXa™; abbreviated as Baby powder, DE, and CimeXa) against the tropical bed bug, Cimex hemipterus (F.), under laboratory conditions. Results showed that the 3 dust products caused 97%–100% mortality to tropical bed bugs within 14 d, both in no-choice and choice experiments. However, in a brief exposure experiment (crossing a 2.5-cm-wide band), Baby powder exposure caused a significantly lower mortality (23%) than DE (88%) and CimeXa (100%). The more effective dusts (DE and CimeXa) were selected for further evaluation of their sublethal effects on C. hemipterus. DE and CimeXa caused significantly higher mortality (48%–58%) through horizontal transfer than that of control (6%). Brief exposure to DE and CimeXa dusts did not affect tropical bed bug activity and their response to CO₂. However, CimeXa-exposed tropical bed bugs exhibited reduced feeding and lowered survival rate after feeding. Moreover, both DE and CimeXa dust bands showed strong barrier effects on the crawling of tropical bed bugs. Our results indicate that both DE and CimeXa have great potential for preventing C. hemipterus from reaching protected areas and for controlling C. hemipterus.

Wild bees are ecologically vital but increasingly threatened by anthropogenic activities, leading to uncertain survival and health outcomes in urban environments. The gut microbiome contains features indicating host health and reflecting long-term evolutionary adaptation and acute reactions to real-time stressors. Moving beyond bacteria, we propose a comprehensive analysis integrating diet, bacteriome, virome, resistome, and their association to understand the survival status of urban lives better. We conducted a study on mason bees (Osmia excavata) across 10 urban agricultural sites in Suzhou, China, using shotgun gut metagenome sequencing for data derived from total gut DNA. Our findings revealed that most ingested pollen originated from Brassica crops and the unexpected garden tree Plantanus, indicating that floral resources at the 10 sites supported Osmia but with limited plant diversity. Varied city landscapes revealed site-specific flowers that all contributed to Osmia sustenance. The gut bacterial community, dominated by Gammaproteobacteria, showed remarkable structural stability across 8 sites but suggested perturbations at 2 sites. Antibiotic resistance gene profiles highly varied across 10 sites with prevalent unclassified drug classes, highlighting environmental threats to both bees and humans. The virome analysis identified honeybee pathogens, suggesting potential virus spillover. Many unknown bacteriophages were detected, some of which targeted the core gut bacteria, underscoring their role in maintaining gut homeostasis. These multifaceted metagenomic insights hold the potential to predict bee health and identify environmental threats, thereby guiding probiotic development and city management for effective bee conservation.

Our previous study shows that Coptotermes formosanus (Blattodea: Rhinotermitidae) preferred to stay on filter paper treated with ethyl 2,4-dioxovalerate, a metabolite in the soil fungus Trichoderma virens. Here, we hypothesized that adding ethyl 2,4-dioxovalerate in sand could trigger aggregation and tunneling preferences of C. formosanus and improve the effectiveness of liquid termiticide. In aggregation-choice tests, significantly more termites were found on/in sand blocks containing ethyl 2,4-dioxovalerate (250 µg/g) than untreated blocks throughout the 24-h experiments. In the tunneling-choice tests, termites also excavated significantly more tunnels in the sand treated with ethyl 2,4-dioxovalerate (2.5, 25, or 250 µg/g) than untreated sand. However, in no-choice tests, ethyl 2,4-dioxovalerate (2.5, 25, or 250 µg/g) did not significantly affect tunneling activities, termite survival, wood consumption, or activities of detoxification enzymes (peroxidase, superoxide dismutase, and catalase) compared to controls. Interestingly, in aggregation- and tunneling-choice tests, termites preferred to stay and made more tunnels in sand treated with both ethyl 2,4-dioxovalerate (250 µg/g) and fipronil (1 µg/g) than untreated sand. In addition, in choice tests, sand treated with the combination of ethyl 2,4-dioxovalerate (250 µg/g) and fipronil (1 µg/g) caused significantly higher termite mortality than the sand treated with only fipronil (1 µg/g). Our study showed that ethyl 2,4-dioxovalerate may enhance the effectiveness of fipronil (1 µg/g in sand) by triggering aggregation and tunneling preferences of termites, thereby increasing the contact between termites and fipronil.

Carbon dioxide (CO₂) plays a crucial role in the host search/localization process of bed bugs, Cimex lectularius L., and Cimex hemipterus (F.). Current research on the CO₂ perception by bed bugs mainly focuses on their behavioral responses, and the molecular mechanisms are still unclear. In addition, existing research has mainly been conducted on C. lectularius with very little research on C. hemipterus. In this study, we investigated the behavioral response of C. hemipterus to CO₂ and analyzed the role of antennae in the CO₂ sensing process. Then, potential CO₂ receptor genes were screened through antennal RNA sequencing and tissue-specific expression profiling analysis. Finally, the function of CO₂ receptors was further validated using RNA interference. Results indicated that increasing the CO₂ concentration in the air not only stimulated the activity of C. hemipterus, but also presented a directional attraction effect on them. CO₂, with a concentration of 0.06%–1.8%, had a significant attraction effect on C. hemipterus. Removal of the antennae led to the loss of bed bugs’ preference for CO₂. Four candidate CO₂ receptor genes (ChGR1, ChGR2, ChGR3, and ChGR4) were identified through transcriptomic analysis of the bed bug antennae, and 3 of them (ChGR2, ChGR3, and ChGR4) were highly expressed in the antennae. Silencing ChGR2 or ChGR4 individually led to a significant reduction or even loss of CO₂ sensing ability in C. hemipterus. In conclusion, CO₂ can induce the host searching behavior of C. hemipterus; moreover, ChGR2 and ChGR4 are crucial for detecting CO₂ in C. hemipterus.

Pathogens are shared between wild bees and wasps but little is known about how urbanization affects their occurrence. Here, the role of temperature and fragmentation of green areas, both associated with urbanization, in modulating pathogen loads was investigated. Twelve pathogens were investigated in the bees Anthophora plumipes Pallas, 1772, Halictus scabiosae (Rossi, 1790), Osmia cornuta (Latreille, 1805), and the wasp Polistes dominula (Christ, 1791) sampled across an urbanization gradient in a metropolitan area of northern Italy. Overall, the relative presence/abundance of the pathogens were found to be species specific, as were the responses to urbanization. Anthophora plumipes and O. cornuta had a higher occurrence probability of the neogregarine protozoan Apicystis bombi in more fragmented urban areas. In the same bee species, both temperature and the fragmentation of green areas reduced the number of copies of the deformed wing virus (DWV). In H. scabiosae and P. dominula, higher temperature increased respectively the likelihood of occurrence of DWV and chronic bee paralysis virus (CBPV). In addition, the viruses were found to be replicative in all samples tested. The results show a consistent presence of pathogens in the four target species, and that urbanization plays a role in modulating the pathogen load. Although transmission pathways could not be considered here, it may be suggested that appropriate management of urban areas may buffer wild insects from potentially harmful pathogens. Whether the presence of such pathogens also results in symptomatic phenotypes remains to be determined in laboratory experiments.

Urbanization, one of the most significant global environmental issues of our time, causes significant environmental and structural changes in natural or seminatural habitat patches. These urbanization-related changes trigger significant impact on ecological interactions and functioning. Predation is one of the most important ecological interactions, and urbanization-related changes on predation pressure may have substantial ecological consequences. We studied predation pressure over a full season (from April to October) in rural versus urban forests using the sentinel approach in and around a large city (Debrecen) in the eastern part of the Great Hungarian Lowland. Model caterpillars made of nondrying green plasticine were readily attacked by arthropods, birds and mammals. From attack marks left by potential predators, a relatively high predation pressure was documented: up to 36% of the caterpillars exposed for 24 h showed attack marks. Seasonal differences were also obvious, with predation pressure during summer being significantly higher than in spring or autumn. This trend held for overall attack rates, also for attacks by arthropods and mammals but not birds. Surprisingly, attack rates were often higher in urban than rural habitats, contradicting the general hypothesis that predation pressure is lower in urbanized areas. As attack rates depend on both predator abundance and activity, and general data indicate lower predator abundances in urban habitats, this phenomenon may have been caused by hungrier predators in urban forest fragments or by the predator relaxation/safe habitat hypothesis that argues that a reduced need for vigilance allows more time to search for prey.

Urban green spaces (UGS) are vital habitats for maintaining insect diversity within cities. However, the key factors driving insect biodiversity under accelerating urbanization remain poorly understood. This study systematically investigates insect diversity and its environmental drivers across 200 sampling sites distributed along an urban-to-rural gradient in 4 climatically distinct cities of China: Changchun, Beijing, Hangzhou, and Haikou. We identified 102 common insect species across 8 orders, with Diptera dominating overall abundance. Species richness was highest in farmland and well-managed urban parks, while roadside and residential habitats exhibited reduced or variable diversity. Insect abundance and composition showed significant spatial variation, strongly influenced by UGS type and urbanization intensity. Diptera emerged as key indicator taxa, responding sensitively to urbanization gradients. A generalized linear mixed-effects model framework was developed to integrate large-scale urban environmental variables with local habitat characteristics, enabling a comprehensive assessment of how urbanization and climate combinedly influence insect diversity across cities. Principal coordinate analysis and non-metric multidimensional scaling revealed that both land use and climate zone contributed to community differentiation, with the strongest effects observed in transitional habitats. Mantel tests and random forest models demonstrated that the key environmental drivers of diversity were taxon- and city-specific, including geographic location, particulate matter, wind speed, and floral diversity. Maximum ecological niche model analysis revealed divergent trends in future habitat suitability, driven by distinct climatic variables in each city. Overall, our findings underscore the importance of region-specific conservation strategies in sustaining insect diversity in urban ecosystems.

The invasive white-footed ant Technomyrmex difficilis has emerged as a rising pest in several regions, yet its invasion dynamics remain underexplored. This species outcompetes native ants and causes agricultural losses by tending pest insects, including aphids and mealybugs. This study provides the first integrated analysis of the species’ behavioral, chemical, and genetic variation across Texas and Florida populations. Observations suggest that the recently discovered Texas population of white-footed ants originated from Florida. Microsatellite and mitochondrial DNA analyses revealed low genetic diversity in both populations, with a shared haplotype consistent with the Texas population originating from Florida. STRUCTURE analysis further supported genetic clustering between the two regions. Despite similar within-colony coefficients of relatedness for workers, the populations differed in reproductive strategy: Florida colonies showed signs of inbreeding and high inter-colony aggression, whereas Texas colonies exhibited potential localized outbreeding, low aggression, and more uniform cuticular hydrocarbon profiles. Aggression was positively correlated with chemical divergence but not with genetic differentiation. This study establishes a foundational understanding and highlights the importance of integrating multiple types of data to understand the invasion biology of Technomyrmex difficilis.

The western flower thrips (Frankliniella occidentalis) is a significant agricultural pest, causing severe global yield losses due to extensive feeding damage and the transmission of plant pathogenic viruses. Despite recent advancements in RNA interference (RNAi) in thrips species, its application has been mostly limited to the adult stage. Given the crucial role of first instar larval thrips in acquiring and transmitting orthotospoviruses, achieving gene silencing in these larvae is critical for studying virus entry and acquisition. While thoracic and abdominal injections have proven effective in adult thrips, the low post-injection survival rate hinders their use in larval thrips. This study addresses this challenge by presenting a microinjection methodology to deliver dsRNA into the hemolymph of first instar larval thrips through the coxa, the first proximal segment of the foreleg. This method significantly improved larval survival rate by preventing detrimental damage to the internal tissues. Significant knockdown of V-ATPase-B, cytochrome P450 (CYP3653A2), and apolipophorin-II/I (ApoLp-II/I) transcripts was confirmed after 48 and/or 72 h post injection (hpi), corresponding to the first and second instar larval stages, respectively. Silencing CYP3653A2 or ApoLp-II/I significantly increased larval mortality. These findings demonstrate proof-of-principle of gene silencing and associated silencing phenotype (mortality) for first instar larval thrips and highlight the essential role of CYP3653A2 and ApoLp-II/I in larval vitality. Our RNAi-based tool offers an opportunity to investigate the molecular mechanisms of thrips-orthotospovirus interactions, as the virus must be acquired by young larval thrips for successful transmission to plants, thus presenting potential targets for thrips pest management.

The regulation and maintenance of immune homeostasis are essential for animal survival, but the molecular mechanisms are not fully understood. Here, we used the model organism Drosophila melanogaster to uncover a potential mechanism by which the nuclear factor-κB transcription factor Relish and miR-100 cooperatively regulate innate immune homeostasis. We first demonstrated in vitro and in vivo that miR-100 can negatively regulate the immune responses of the Imd pathway by inhibiting the expression of TAK1-associated binding protein 2 (Tab2) gene. Second, we found that Relish, an important transcription factor in the Drosophila Imd pathway, could not only modulate the expressions of antimicrobial peptides (AMPs) to promote immune responses, but also bind to the promoter region of miR-100 and activate its transcription to inhibit immune responses. Third, the dynamic expression of genes profiling indicated that the Relish/miR-100/Tab2 regulatory axis could contribute to innate immune homeostasis in Drosophila. Together, our findings reveal the dual role of Relish in immune regulation, that is, Relish promotes the expression of AMPs to resist pathogen infection in the early immune response, while in the late immune stages, Relish readjusts the expression of miR-100 to negatively control immune responses to avoid excessive immunity thus maintaining immunohomeostasis. Meanwhile, our study provides a new perspective for further understanding the complex regulatory mechanism of immune homeostasis in animals.

Olfactory plays an important role in insect behaviors. Pheromone binding proteins (PBPs) are thought to play a certain role in the transport of pheromone molecules in the olfactory recognition process for courtship and mating. Mythimna separata is one of the most serious cereal pests in Asia. The sexual pheromone components of M. separata were clarified; however, to date, little evidence in vivo or in vitro has disclosed the binding properties of PBPs toward the pheromone components of M. separata. To address this research gap, the functional characterization of PBPs in M. separata, spectroscopic investigations were conducted by using recombinant MsepPBPs. Subsequently, MsepPBP1 and MsepPBP3 were selected for RNA interference to assess changes in behavioral responses of male mutants toward normal females. Fluorescence displacement binding assays, combined with fluorescence quenching assays, revealed that MsepPBP3, among the 3 MsepPBPs, exhibited the strongest affinity for Z11-16:Ald, the primary component of sex pheromone in M. separata. Static quenching was observed only between MsepPBP1 and Z9-16:Ald, as well as between MsepPBP3 and Z11-16:Ald or Z9-16:Ald. Transcript levels of MsepPBP1 or MsepPBP3 of male adults were significantly reduced compared to the control when injected with dsMsepPBPs. Both dsPBP1- and dsPBP3-treated males displayed a notable decrease in successful calling behaviors, with this reduction being more pronounced in dsMsepPBP3 injected groups than in dsMsepPBP1 injected groups. These experiments indicated the specificity of MsepPBP1 and MsepPBP3, with both contributing to the sensitivity of female detection. MsepPBP3 appeared to be a key protein for recognizing the sex pheromones of M. separata.

Fungal pathogens produce secretory ribonuclease (RNase) T2 proteins during infection, which contribute to fungal virulence via their enzyme functions in degradation of host cell RNA. However, the details of those proteins entering the host cells are unclear. Our previous study demonstrated that the two secretory RNase T2 members, BbRNT2 and BbTrv, produced by the insect fungal pathogen Beauveria bassiana, caused cytotoxic damage to insect cells and contributed to fungal virulence. Here, the Spodoptera frugiperda ovarian epithelial cells (sf9 cells) were used as models to investigate the interactions of the two fungus-produced RNase T2 proteins with the insect cells. Two transmembrane proteins, an ABC transporter (SfABCG) and an Innexin 7-like protein (Sfinx), were identified from the sf9 cells as interacting with BbRNT2 and BbTrv, respectively, through protein immunoprecipitation, yeast-two hybrid tests and protein pull-down assays. Although a slight decrease in the sf9 cell viability was examined by transfection of RNA interference of SfABCG or Sfinx, the transfected cells displayed a dramatically decreased sensitivity to BbRNT2 or BbTrv, suggesting the requirement of the two transmembrane proteins for BbRNT2 and BbTrv to enter the insect cells. These results reveal a mechanism of the cytotoxic molecules, T2 RNases, produced by the fungal pathogen, entering the insect cells via interaction with specific insect cell transmembrane proteins and causing cytotoxic damage.

Antimicrobial peptides (AMPs) are critical components of innate immunity in diverse organisms, including plants, vertebrates, and insects. This study identified and characterized a novel Lepidoptera-specific AMP, named lepidoptin, from the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae). Lepidoptin is a 116-amino acid protein containing a signal peptide and a novel β-sandwich domain that is distinct from previously reported AMPs. Temporal and spatial expression analyses revealed a significant upregulation of the lepidoptin gene in vivo and in cultured SF9 cells in response to pathogens. Molecular docking analysis identified a specific binding cavity. Enzyme-linked immunosorbent assay and binding assays confirmed that lepidoptin can bind to pathogen-associated molecular patterns, bacteria, and fungi. Recombinant lepidoptin exhibited potent antibacterial activity by inducing bacterial agglutination, inhibiting bacterial growth, increasing bacterial membrane permeability, and preventing biofilm formation. Lepidoptin also showed antifungal activity against the entomopathogenic fungus Beauveria bassiana by inhibiting spore germination, increasing fungal cell permeability, and increasing reactive oxygen species. Injection of recombinant lepidoptin into S. frugiperda larvae increased survival after B. bassiana infection, whereas knockdown of lepidoptin by RNA interference decreased larval survival. In addition, lepidoptin showed antimicrobial activity against the plant pathogen Fusarium graminearum by inhibiting spore germination and alleviating disease symptoms in wheat seedlings and cherry tomatoes. This study demonstrates the remarkable dual functionality of lepidoptin in enhancing S. frugiperda immunity and controlling plant pathogens, making it a promising candidate for biocontrol strategies in both pest management and plant disease prevention.

As the catalytic subunit of the Elongator complex, Elongator protein 3 (Elp3) plays a crucial role in multiple physiological processes, including growth, development and immune responses. Previous studies on Elp3 have focused on Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens (human) or Mus musculus (mouse), whereas there are few reports on Elp3 in agricultural pests. Here, the role of TcElp3 in reproduction in the red flour beetle, Tribolium castaneum, was investigated, and the underlying mechanisms were explored. The results showed that RNA interference (RNAi)-mediated knockdown of TcElp3 in female pupae led to decreased fecundity in female adults. Consistently, the injection of dsTcElp3 into female pupae decreased the mRNA levels of the vitellogenin (Vg)-encoding genes TcVg1 and TcVg2 in female adults. Notably, knockdown of TcElp3 upregulated the expression of forkhead box protein O (FOXO) at both the mRNA and the protein level in T. castaneum, and promoted the nuclear translocation of TcFOXO. Additionally, TcElp3 directly interacts with TcFOXO and the silencing of TcElp3 significantly decreased the acetylation level of TcFOXO. Overall, our studies reveal that Elp3 regulates beetle reproduction by interacting with FOXO and modulating its acetylation status.

Lipids perform a diverse and unique array of functions in insects. Lipases are key enzymes in lipid metabolism, and their metabolic products are crucial for development and reproduction of insects. Here, a total of 110 lipase genes were identified in the genome of Spodoptera frugiperda. Cluster analysis indicated that neutral lipases constitute the majority of lipases. Tissue expression profile analysis displayed that most lipase genes were highly expressed in the larval gut of S. frugiperda. Some lipases exhibited a diet-specific expression pattern, which implied their roles in host adaptation. Key domain analysis proved that none of the neutral lipases highly expressed in the gut has an integrated lid domain, while most lipases highly expressed in the fat body contained both the integrated lid domain and β9 loop, indicating the activity loss of neutral lipases in guts. The assay of triacylglycerol (TAG) hydrolytic activity confirmed that the gut had the lowest activity when compared to that of fat body and epidermis. Interestingly, the opposite TAG hydrolytic activity trends across mating were observed between adult males and females, implying that lipase played different roles in the reproduction of both sexes. In conclusion, neutral lipases lost TAG hydrolytic activity in S. frugiperda guts, but retained the activity in fat body. Neutral lipases would play vital roles in many physiological processes in insects, especially in insect reproduction, which provides palpable targets for novel insecticide development to control insect population growth.

Juvenile hormone (JH) plays a pivotal role in regulating post-emergence development and metabolism in previtellogenic female Aedes aegypti mosquitoes. In contrast, yolk protein precursor production and egg maturation after a blood meal are regulated by the steroid hormone 20-hydroxyecdysone, the insulin-like growth factor (IGF)/insulin signaling (IIS) pathway, and the mammalian target of rapamycin (mTOR) pathway. The role of IIS/mTOR signaling in female adults prior to blood feeding has not been thoroughly investigated. In this study, we identified a significant increase in the phosphorylation of key effector proteins in the IIS/mTOR signaling pathway, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K) and forkhead box protein O1 (FoxO1), in previtellogenic females. In vitro fat body culture experiments suggest that JH induces these phosphorylations through rapid nongenomic signaling mediated by the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR network. RNA interference experiments demonstrated that activation of IIS/mTOR signaling in previtellogenic females modulate metabolic gene expression, promoting the accumulation of energy reserves (glycogen and triglycerides), which influence mosquito fecundity. Additionally, depletion of either the insulin receptor (InR) or the JH receptor Methoprene-tolerant (Met) in adult mosquitoes abolished the phosphorylation of these proteins, indicating that both receptors are involved in JH-induced membrane-initiated signal transduction. Although the precise mechanisms remain unclear, this study uncovers a novel function of the IIS/mTOR pathway in adult mosquitoes before blood feeding, as well as a new mode of JH action through its crosstalk with the IIS pathway.

Insecticide contamination and climate change are key factors driving the global decline in insect populations. However, how these factors interact to impact insect survival remains uncertain. In this study, we examined the effects of sex and genotype on the response to long-term low insecticide exposure at two temperatures, 18 °C and 28 °C, using the Drosophila melanogaster model. We focused on a polymorphic gene, Cyp6g1, known for conferring broad insecticide resistance. We found that while temperature and insecticide have a synergistic effect on mortality of susceptible flies (Cyp6g1—M allele), they act additively on resistant flies (Cyp6g1—BA allele). And whereas the mortality of BA flies exposed to insecticides is strongly dependent on sex at 18 °C, no sex bias is found at 28 °C. Under no insecticide exposure, BA females showed shorter median lifespan than males regardless of temperature, possibly reflecting a cost associated with the resistant allele. Surprisingly, across all genotypes, females showed lower Cyp6g1 gene expression levels than males, which contrasts with their higher insecticide tolerance. Temperature and insecticide exposure had small effects on Cyp6g1 expression levels, suggesting the presence of additional mechanisms of resistance. Our results indicate that the effect of high insecticide doses on insect mortality cannot be used to predict how insects will respond to low contaminating doses, especially when considering the strong interactions between sex, temperature, and genotype. The combined effects of temperature and long-term low insecticide exposure are complex and can have major impacts on insect population dynamics and survival.

The use of synthetic pesticides carries a significant risk of pests developing resistance, leading to decreased pesticide effectiveness. ATP-binding cassette (ABC) transporters, especially the ABCC subfamily members, have been suggested to act as efflux pumps for various pesticides, thereby contributing to pesticide resistance. So far, the identification of potential pesticide substrates of insect ABC transporters is most often based on the quantification of transcript in arthropods. Here, we screened and identified the potential pesticide substrates of ABCC-9C from Tribolium castaneum based on an in vitro ATPase activity assay. Together with affinity evaluation-, cytotoxicity analysis-, and RNA interference-based bioactivity tests, we revealed that the insecticides, carbofuran, and buprofezin, are potential substrates of TcABCC-9C. Additionally, we identified an amphipathic translocation channel in the transmembrane domain of TcABCC-9C formed by 8 transmembrane helices. Molecular docking suggested that both carbofuran and buprofezin bind at the same site within the translocation channel via hydrophobic interactions. These findings indicate that TcABCC-9C might play a critical role in multi-pesticide resistance, providing a potential target for managing pesticide resistance and laying the groundwork for future pest control strategies. Given the conservations among ABCC subfamily members, the experimental model we developed in this study can be also applied to identify the potential substrates of other ABCC transporters, as well as to predict insecticide resistance mediated by ABCC transporters.

Distinct lineages of the grasshopper Chorthippus parallelus (Orthoptera: Acrididae) form well-known hybrid zones (HZs) both in the Pyrenees and the Alps mountain ranges in South Europe. These HZs represent unique experimental systems to identify “key genes” that maintain genetic boundaries between emerging species. The Iberian endemism C. p. erythropus (Cpe) and the subspecies C. p. parallelus (Cpp), widely distributed throughout the rest of Europe, overlap and form the Pyrenean HZ. Both subspecies differ morphologically, as well as in behavioral, mitochondrial, nuclear, and chromosomal traits, and in the strains of the maternally transmitted bacterial endosymbiont Wolbachia infecting them. This results in either unidirectional and bidirectional cytoplasmic incompatibility between both grasshopper subspecies, pointing out that Wolbachia clearly affects gene expression in the infected individuals. Here we explore how Wolbachia may modify the expression of some major genes involved in relevant pathways in Cpp in the Pyrenean HZ. We have analyzed, through molecular biomarkers, the physiological responses in C. parallelus individuals infected by Wolbachia, with particular attention to the energy metabolism, the immune system response, and the reproduction. qPCR was used to evaluate the expression of selected genes in the gonads of infected and uninfected adults of both sexes, since this tissue constitutes the main target of Wolbachia infection. Transcriptional analyses also showed differential sex-dependent responses in most of the analyzed biomarkers in infected and noninfected individuals. We identified for the first time new sensitive biomarkers that might be involved in the reproductive barrier induced by Wolbachia in the hybrid zone.

Resource partitioning among sympatric species is crucial for assembling ecological communities, such as caterpillar–ant assemblages in tropical forests. Myrmecophilous caterpillars use behavioral and chemical strategies to coexist with ants, avoiding attacks. While these strategies are well-understood in single pair of interacting species, such as those involving myrmecophiles and ants, their role in complex multitrophic interactions that include several species of plants, herbivores and ants remains unclear. We aimed to identify the role of cuticular hydrocarbons and specialized morphological structures that caterpillars use to interact with ants (called ant organs) in the recognition process between two riodinid caterpillar species and their respective ant–plant systems. We hypothesized that caterpillars’ cuticular profiles would be conspicuous, possessing cues of rewards to ants, allowing specific ants to recognize and not attack them on plants. We performed experiments exposing caterpillars to ants to assess the role of larval ant organs and the specificity of caterpillar–ant interactions on plants. We analyzed cuticular hydrocarbons of caterpillars, ant workers and plants using gas chromatography/mass spectrometry. Our experiments showed that larval ant organs were activated according to each treatment and caterpillars were consistently accepted by their associated ants when transferred to host plants occupied by the same ant species. However, caterpillars transferred to plants with a non-associated ant species that do not tend them were often killed. This highlights the specificity of these interactions. Caterpillar cuticular hydrocarbon profiles, while present in far lower amounts than those of ant workers and plants, were distinctive, suggesting a strategy of chemical conspicuousness that helps caterpillars to be recognized by ants and prevents attacks in specific ant–plant systems. Our results indicate that ants recognize conspicuous cuticular hydrocarbons, while caterpillars convey multimodal signals from ant organs during interactions, which are essential for caterpillar survival in these specific interactions.

Due to the rise in global temperatures with climate change, insects, as ectotherms, critically depend on their heat tolerance for survival and reproduction. Heat shock proteins (HSPs) are essential for heat tolerance by averting protein denaturation; however, whether HSPs contribute to reproduction-related heat tolerance remains largely unexplored. The study investigated the reproductive heat tolerance and recovery of Monochamus alternatus, a major forestry pest, in response to heat stress. Alongside impairing the development and viability of reproductive organs and sperm, heat stress was also found to reduce fecundity, fertility, mating, and oviposition behaviors. Remarkably, all reproductive parameters of M. alternatus recovered within 4 weeks postexposure. To investigate the recovery mechanisms, we identified 10 reproduction-related proteins as candidate substrate proteins of an HSP protein in M. alternatus using immunoprecipitation coupled with mass spectrometry analysis. Heat stress inhibited the transcription of these reproduction-related genes, thereby adversely affecting reproductive parameters. However, the induction of HSP20s transcription in response to heat stress appeared to facilitate the refolding of these critical reproduction-related proteins during the recovery phase, thus preventing lasting reproductive damage. Overall, this study suggests that while M. alternatus populations might be vulnerable to climate-induced temperature increases, their fertility can recover, mediated by the interaction of HSPs with reproduction-related genes. These findings offer profound insights into insect heat tolerance and recovery, expanding our understanding of HSP20 proteins’ biological functions.

Ultraviolet (UV) radiation, an environmental stressor, is crucial for the survival and adaptation of organisms. Myzus persicae, a global pest, is exposed to sunlight year-round, making it unable to avoid UV rays in its environment. MicroRNAs (miRNAs) are important posttranscriptional regulators of gene expression and mediate various biological processes. However, the role of miRNA in aphids in response to UV-B stress is unclear. In this study, Mpp53 expression level significantly increased with an increase in the duration of UV-B radiation, peaking at 2 h; knockdown of Mpp53 decreased the survival rate of aphids under UV-B stress, suggesting that Mpp53 is involved in aphid responses to UV-B. Here, we first predicted 8 miRNAs targeting Mpp53, and then screened for miRNAs related to UV-B resistance in aphids; of these, 5 miRNAs (miR-305-5p, novel_50, novel_80, novel_166, and novel_61) were found to target Mpp53. Luciferase reporter assays demonstrated that novel_61 binds to the noncoding region of Mpp53 and downregulates its expression. Overexpression of novel_61 in aphids decreased Mpp53 expression and caused significant mortality under UV-B irradiation. Furthermore, the aphids exhibited lower reproductive capacity, lower body weight, and shorter body length and width. This is the first study to systematically screen and identify miRNA related to aphid responses to UV-B stress and deepens our understanding of the molecular mechanism of insect responses to environmental stress, which may eventually aid in developing better control strategies.

Haug, T.J., Linhart, S., Baranov, V., and Haug, C. (2024) Eocene and modern entomofauna differ—a Cretaceous-like larva in Rovno amber. Insect Science, 32, 712–718. https://doi.org/10.1111/1744-7917.13410.

The article type, Letter to Editor, was incorrect. It should have been Short Communication.

The online version of this article was corrected.

We apologize for the error.