Abstract: Insects touch their proximal environment with their tarsi. The immediate contact with xenobiotics occurs with the tarsal cuticle surface that is covered with cuticular hydrocarbons (CHCs). In this work, we tested the hypothesis that xenobiotics entry through the tarsi depended on CHC amounts and composition. Applying RNA interference, we suppressed the expression of genes coding for the key enzymes of CHC production Cyp4G1 (total CHC), desat1 (unsaturated CHCs) and FASN2 (branched CHCs) in lipid producing oenocytes and analyzed the penetration efficiency of the insecticides DDT and chlorantraniliprole and of the inert dye Eosin Y in the respective flies. As expected, in walking experiments, reduction of CHC amounts (cyp4G1RNAi) enhanced insecticide and dye penetration into the tarsi. In the same assay, we identified unsaturated CHCs as the main CHC component attenuating DDT and chlorantraniliprole at low concentrations. Likewise, tarsal adhesion and uptake of Eosin Y depended rather on unsaturated than on branched CHCs. Extrapolating from our data, we propose a two-step model of xenobiotics penetration through the tarsal cuticle: first, modulated by unsaturated CHCs, the molecule is repelled or adheres to the cuticle surface; upon adhesion, the molecule penetrates the cuticle and accumulates in the tarsal lumen in a second step. Whether these mechanisms apply to molecules other than Eosin Y remains to be investigated. Taken together, the tarsal cuticle constitutes a selective bipartite barrier against uncontrolled uptake of contact xenobiotics.

Abstract: Monochamus alternatus Hope, commonly known as the pine sawyer beetle, is a devastating pest in coniferous forest ecosystems. While insect olfactory-related genes have been implicated in pathogen defense, the potential involvement of chemosensory protein (CSP) genes in M. alternatus immune responses to the entomopathogenic fungus, Beauveria bassiana remains largely unknown. In this study, we identified 15 CSP genes in M. alternatus based on transcriptome, seven of which exhibited altered expression following B. bassiana infection. Quantitative real-time PCR (RT-qPCR) analysis showed significant upregulation of MaCSP8 and MaCSP15 at 3 d post-infection. Tissue-specific profiling revealed predominant expression of MaCSP8 in larval heads and epidermis, whereas MaCSP15 expression was highest in the fat body. Crucially, RNA interference (RNAi)-mediated silencing of MaCSP8 and MaCSP15 significantly increased larval susceptibility to B. bassiana infection. Furthermore, knockdown of the two MaCSPs resulted in significant downregulation of key immune effectors such as Dorsal, Defense, Attacin, and Coleoptericin, suggesting modulation of the Toll pathway. Taken together, MaCSP8 and MaCSP15 were defined as critical components of the antifungal immune response in M. alternatus larvae, likely functioning through the regulation of antimicrobial peptide (AMP) production. This study provides novel insights into CSP-mediated innate immunity, establishing it as a promising target for developing next-generation bio-insecticides and enhancing fungal biocontrol strategies against forestry pests.

Abstract: We find a specialized Pachyteles-like larva from mid-Cretaceous Kachin amber, which represents a rare insight into the behavioral and morphological adaptations of early beetles. In our finding, its terminal disk morphology suggests a highly specialized ambush predation strategy, integrating both prey capture and phragmotic defense-traits that were already well developed over 99 million years ago. This fossil not only reveals the antiquity of complex larval predation and defense behaviors in Carabidae but also pushes back the evolutionary origin of phragmosis among insects. It also allows for a functional morphological interpretation of ecological interactions in Cretaceous terrestrial ecosystems. This functional approaches offer a valuable lens for decoding the ecological complexity of Mesozoic food webs, revealing that intricate predator-prey relationships were already well established in the mid-Cretaceous.

Abstract: Dominance contests have important implications for the allocation of limited resources among insect individuals. Prior experiences, particularly winning and losing have been well-documented to influence the performance and outcomes of future contests across various species. This experience effect is believed to arise either from alterations in an individual's self-assessment of its fighting ability based on past experiences or from social cues generated by previous experiences that are subsequently recognized by opponents. However, the influence of previous fighting experiences on dominance contests and the underlying cognitive mechanisms in insects remains underexplored. This study aims to investigate how previous fighting experiences influence the performance and outcomes of dominance contests among bumblebee workers (Bombus terrestris) and the underlying cognitive process. Our research findings indicate that prior winning experiences significantly influence individuals' behavioral decision-making during the pre-escalation phase of contests. Specifically, individuals with previous victories demonstrate a pronounced tendency to initiate and escalate contests, providing compelling evidence that such experiences enhance their self-perceived fighting ability. Furthermore, despite conflict resolution being largely contingent upon escalated contests, prior winners consistently exhibit a markedly higher probability of securing victories. This finding suggests that previous winning experiences improve individuals' actual fighting ability. This enhanced fighting ability enables smaller individuals to overcome larger opponents. Notably, the winner effect persists for a minimum duration of 3 d. In contrast, losers did not show significant changes in performance, suggesting the absence of a pronounced loser effect. These findings provide new insights into the mechanisms underlying the winner effect in insects.

Abstract: The venom gland is a remarkable organ of the parasitoid wasps, which produces venom that controls the host and is important for the survival and reproduction of the parasitoid. But little is known about how venom gland cells precisely regulate venom synthesis. Here, we used single-nucleus RNA sequencing to construct the cell atlases of the venom gland and ovary of the Pteromalus puparum, a natural enemy of vegetable pests. The cells in the venom gland were clustered into six subsets, and the main cell types in the ovary were follicle cells, germline cells and nurse cells. The cellular function of venom gland subsets named VgC1 to VgC6 was enriched in the production and secretion of venom. In addition, the expression pattern of identified venom genes that 77 genes expressed across all six VgCs and 46 genes in five VgCs, demonstrated that most venom genes expressed widely in VgCs. Finally, hdWGCNA analysis of venom gland cells identified a set of co-expressed venom and non-venom genes that include putative regulators of venom production in P. puparum. Our analysis of transcription factor (TF) binding sites within the venom modules revealed that TFs such as AP-1 and EHF are potential regulators of venom genes. This work reveals the cellular heterogeneity, venom gene expression patterns, and transcriptional regulatory networks in the venom gland of P. puparum at single-cell resolution. These findings provide fundamental insights into the biology of venom-producing organs and establish a molecular foundation for developing novel biopesticides based on venom proteins.

Abstract: Pheromone-binding proteins (PBPs) play pivotal roles in the moth olfactory system by assisting the neuronal response to pheromone components. Thalassodes immissaria is a major notorious defoliator of lychee orchards in China. In this study, we demonstrated that (3Z,6Z,9Z,12Z)-icosa-3,6,9,12-tetraene (Z3,Z6,Z9,Z12-20:H)-a principal sex pheromone component previously identified in the Japanese population of T. immissaria-elicits significant electroantennogram (EAG) responses and robust behavioral attraction to male moths of the Chinese population. Subsequently, we identified three PBP-encoding genes, among which TimmPBP1 and TimmPBP2 were highly expressed in the antennae of adult male T. immissaria. Further fluorescence competitive binding assays revealed that recombinant TimmPBP1 and TimmPBP2 exhibited strong binding affinities to Z3,Z6,Z9,Z12-20:H. Additionally, the antennae of male moths treated with dsTimmPBP1 or dsTimmPBP2 exhibited significantly reduced EAG responses to Z3,Z6,Z9,Z12-20:H. Finally, the expression profiles of TimmPBP1 and TimmPBP2 exhibited circadian oscillations, which aligned with the diurnal rhythms of T. immissaria mating behavior and the males' EAG responsiveness to Z3,Z6,Z9,Z12-20:H. Our study elucidates the physiological roles of PBPs in the recognition of Type II sex pheromone and offers novel molecular targets for monitoring and sustainable control of T. immissaria in orchard ecosystems.

Abstract: Group-living insects like the pea aphid, Acythosiphon pisum, use population density as a key signal to regulate behavior, but the underlying mechanisms of individual responses remain unclear. This study employed a novel dual-color system to precisely track individual aphid behavior across a density gradient. We found that aphid behavioral responses are not linear but are triggered at critical density thresholds, which differ depending on the aphid's initial state (settled or active). As density increased, aphids exhibited significantly greater movement and reduced reproductive output, with a key behavioral shift toward dispersal occurring above 1.25 individuals/cm² (1: 11 group). To investigate the molecular basis for this shift, we analyzed the expression of genes related to three key monoamine neurotransmitters. The results showed a strong positive correlation between population density, locomotor activity, and the expression of the tyramine β-hydroxylase (TBH) gene, which is critical for octopamine synthesis. Conversely, the expression of the serotonin-related gene tryptophan hydroxylase (TPH) decreased, while the dopamine-related gene Tyrosine hydroxylase (TH) showed no clear trend. These findings suggest that aphids utilize a "threshold-triggered" model to rapidly shift from a settled to a dispersal state, a process primarily mediated by the monoamine neurotransmitters system. This provides a new perspective on the self-regulation mechanisms of insect populations.

Abstract: Bursicon is indispensable for wing expansion in Bactrocera dorsalis, acting through PKA-mediated regulation of the wing-development gene Bdhh. Although cuticular proteins are recognized as critical regulators of insect wing development, it remains unclear whether they directly respond to the bursicon signaling pathway. Here, we conducted a functional dissection of cuticular protein genes during wing expansion. Transmission-electron-microscopy (TEM) analysis revealed that bursicon controls endocuticle development in the wing cuticle. Genome-wide mining uncovered sixteen CPR-type endocuticle structural glycoprotein genes, of which three cuticular protein genes (BdABD-4a, BdABD-4b, and BdABD-4c) were selected for RNAi on the basis of their expression profiles. Silencing of each gene via dsRNA injection at the 5-d-old pupal stage produced wing malformations in 37.5%, 32.5%, and 40% of adults, respectively, and reduced cuticle thickness by 40.4%, 42.4%, and 44.2% relative to controls. TEM confirmed the presence of thinner endocuticle in malformed wings. Furthermore, expression levels of all three endocuticle structural glycoprotein genes were markedly altered following knock-down of Bursicon genes, Bdhh and after PKA inhibition, indicating that these proteins operate downstream of the bursicon signaling cascade. The results in this study demonstrated that the functional role of BdABD-4a, BdABD-4b, and BdABD-4c in wing expansion, and provide new insights into the molecular mechanism underlying bursicon regulated wing expansion in B. dorsalis.

Abstract: Coastal dunes are highly vulnerable ecosystems that require adequate bioindicators for effective biodiversity monitoring. Although moths are recognized as good bioindicators in many contexts, their diversity patterns in these habitats remain understudied. This research examines the taxonomic and functional diversity of noctuoid moths in a well-preserved, protected dune system in Central Italy (Adriatic coast). Specifically, the study aimed at investigating how vegetation composition, and hence dune zonation, influences noctuoid diversity and traits. Moth sampling was carried out on an annual basis using UV LED traps in both shifting and fixed dune zones. Vegetation surveys were conducted in spring using 4 m × 4 m plots. We compared noctuoid species richness, species abundance distribution patterns, and trait attributes between the two dune zones in relation to vegetation characteristics. We also evaluated the congruence in species composition and abundance between dune zones for both moths and plants. Sampled noctuoids included 98 species (78 Noctuidae, 18 Erebidae, 1 Nolidae, and 1 Notodontidae). The presence of habitat-exclusive species underscores the influence of both abiotic and biotic filtering processes in shaping noctuoid assemblages across the seashore-inland zonation. Trait-based analyses and diversity patterns showed clear ecological links between noctuoid assemblages and dune zones. This congruence supports the use of noctuoid moths as effective bioindicators and stresses the need to conserve the entire dune system as an integrated ecological unit.

Abstract: Thrips are economically important pests in the global agriculture. Thrips cause direct damage to crops through feeding and also act as vectors for numerous plant viruses, particularly tomato spotted wilt virus (TSWV). TSWV, a member of the genus Orthotospovirus in the family Tospoviridae (order Elliovirales, class Bunyaviricetes), poses a threat to various crop plants in the world. This review summarizes the transmission mechanisms of TSWV by western flower thrip (WFT) and the effects of TSWV on WFT behavior and fitness. By synthesizing current literature, this review aims to offer novel insights into the complex relationships between thrips, viruses, and plants, and to establish a theoretical foundation for developing molecular-based strategies for virus prevention and control.

Abstract: During insect oogenesis, follicular cells (FCs) typically undergo the endocycle to become polyploid, thereby supporting oocyte development. The brown planthopper (BPH, Nilaparvata lugens), one of the most destructive rice pests, exhibits remarkable fecundity. However, the polyploidization of FCs and its regulatory mechanisms remain poorly understood. Here, we demonstrate that 92.3% of FCs become binucleate via endomitosis at stage 4 of oogenesis, followed by a significant increase in DNA content through endoreplication at stage 5. Knockdown of fizzy-related protein (Fzr), a key regulator of the mitosis-to-endocycle transition, disrupted the expression of cell cycle-related genes and caused a marked reduction in both binucleate FC numbers and DNA content, resulting in ovarian malformation and impaired egg development. Gene expression analyses revealed that Fzr knockdown caused aberrant expression and alternative splicing of genes related to spliceosome function and energy metabolism. Furthermore, these alterations appear to be at least partially independent of the polyploidization process. Our findings reveal a two-step polyploidization mechanism in BPH reproduction, offering insights into the evolutionary adaptation of reproductive strategies in insect pests. Additionally, this work advances our understanding of the molecular mechanisms underlying cell cycle transitions and establishes a foundation for future studies on insect reproduction and pest management strategies.

Abstract: Heavy metals are widespread environmental pollutants. This study investigated the growth toxicity of cadmium (Cd) stress on Chouioia cunea offspring during the parasitism period within a Cd-contaminated food chain of artificial diets-Hyphantria cunea pupae-C. cunea. After parasitizing Cd-accumulated H. cunea pupae, the offspring exhibited a significant reduction in both body length and the expression levels of most growth regulatory genes. Transcriptome analysis revealed that, during the early parasitism stage, genes responsive to Cd stress predominantly involved the endoplasmic reticulum (ER) stress, mitochondrial dysfunction, apoptosis, and calcium homeostasis. In the subsequent stages of parasitism, Cd exposure induced substantial damage to the ER and mitochondrial ultrastructure, disrupted the antioxidant defense system, activated the mitochondrial apoptotic and ER stress pathways, and decreased the expression of SLC24A3 and SLC24A4. Overexpression of SLC24A3 and SLC24A4 in Sf9 cells mitigated Cd toxicity by alleviating the Ca2+-mitochondrial permeability transition pore (MPTP)-mitochondrial membrane potential (MMP) collapse/apoptosis and oxidative stress cascade reaction. Overall, Cd exposure impaired the growth of C. cunea parasitic offspring through the SLC24A3/SLC24A4-mediated apoptosis and oxidative damage signaling pathways.

Abstract: This study aimed to explore the reason for Haemaphysalis longicornis restricting Borrelia burgdorferi colonization and transmission from the perspective of gut microbiota, and to investigate the impact of different infection statuses on the Haemaphysalis longicornis microbiota and its potential role in pathogen transmission. Pathogen-free Haemaphysalis longicornis ticks and IFNAR1^-/- mice were used to establish infection models. Ticks fed on pathogen-infected mice, and their midguts were analyzed at day 4 and 10 post-feeding. DNA was extracted from the midguts, and the V3-V4 region of bacterial 16S rRNA gene was amplified and sequenced. Analysis of microbiome data were analyzed using QIIME2 and R. After digesting the bloodmeals, the gut microbiota of ticks that ingested bloodmeal with Borrelia burgdorferi exhibited minimal structural changes, while the ticks with uninfected bloodmeal or bloodmeal with Langat virus showed the dysbiosis. The Borrelia group showed minimal temporal shifts in β-diversity, with stable co-occurrence networks and increased core microbial interactions. Neutral model analysis revealed a hybrid niche in the Borrelia group. Potential biomarkers were identified that may suppress Borrelia burgdorferi transmission. Our findings reveal that Borrelia burgdorferi infection is associated with a stabilized and functionally distinct gut microbiota in Haemaphysalis longicornis. The gut microbiota of Haemaphysalis longicornis functions as a barrier against Borrelia burgdorferi colonization and transmission through microbial regulation and resource limitation, thus providing a potential mechanistic explanation for the observed vector incompetence. These findings highlight the potential of microbiome-targeted strategies to block pathogen transmission and offer new insights into vector-borne disease.

Abstract: The widespread use and improper disposal of polypropylene (PP) facemasks have resulted in persistent environmental pollution, posing urgent challenges for waste management. This study added bran-PP mixture, which increased the survival rate, consumption rate and removal rate of Tenebrio molitor feeding solely on PP. The average consumption of the larvae fed with the edible bran-PP mixture was 61.54% higher than those fed only PP. The removal rates were 39.26% and 36.14%, respectively. Fourier-transform infrared spectroscopy, thermal gravimetric analysis, gel permeation chromatography, and nuclear magnetic resonance confirmed the production of oxygenated compounds in the larval gut, indicating partial oxidation and degradation of PP masks within the intestinal tract. HT-GPC analysis revealed significant reductions in molecular weight parameters, with the number-average (Mn), weight-average (Mw), and Z-average (Mz) molecular weights decreasing by 51.07%, 33.60%, and 32.99%, respectively. High-throughput 16S rRNA sequencing revealed that feeding on PP enhanced gut microbiota richness and diversity. The bran-PP mixture group exhibited significantly higher relative abundances of Enterobacter and Spiroplasma, whereas the bran group was dominated by Lactobacillus. PICRUSt functional predictions indicated upregulation of plastic degradation-associated oxidases (alkane 1-monooxygenase, cyclohexanone monooxygenase) and hydrolases (chitinase, carboxylesterase) in larvae fed PP or bran-PP diets. Metabolomic profiling revealed significant enrichment in pathways related to histidine, glycerophospholipid, choline and steroid hormone metabolism in PP-fed larvae. These findings demonstrate that PP can be biodegraded in T. molitor larvae through gut microbe-mediated depolymerization involving a diverse microbial community.

Abstract: The cotton aphid (Aphis gossypii) is a major pest of greenhouse peppers in southern Spain. Biological control using the parasitoid wasp Aphidius colemani is limited because the ant Tapinoma ibericum, engaged in a mutualistic relationship with aphids, protects them from parasitoid attack and thereby reduces parasitism success. To assess the impact of ants on pest biological control, an ant-exclusion experiment was conducted over two consecutive years in four experimental greenhouses. Pepper plants were infested with aphids and then treated with Aphidius colemani. The presence of natural enemies and fruit production were also evaluated. Excluding ants significantly increased the abundance of mummies by 22.2% and reduced the number of aphid colonies, while total aphid abundance showed variable, year-dependent effects and was not affected by ants. Ant presence negatively affected some natural enemies such as Chrysoperla carnea s.l., the mirid Nesidiocoris tenuis, and the ladybird Scymnus sp., had no effect on hoverflies and spiders, and increased the abundance of Aphidoletes aphidimyza, indicating contrasting responses among natural enemies. Fruit weight was not affected by ant presence. Overall, these findings confirm that T. ibericum reduces parasitism by A. colemani, and demonstrate that it modifies aphid spatial distribution, and reshapes the natural enemy community, but does not necessarily diminish crop production. These results suggest that combining A. aphidimyza with A. colemani could improve control of A. gossypii in ant-infested crops.

Abstract: Chemosensory proteins (CSPs) are small soluble proteins that play various roles in insects. The non-sexual parthenogenetic females (virginoparae) of Rhopalosiphum padi can transition to sexual reproduction and produce sexual females (oviparae) when exposed to short photoperiods and low temperatures. To date, research on the distinct roles of chemosensory proteins in sexual versus parthenogenetic female aphids remains scarce. In this study, we investigated the roles of RpCSP8 in R. padi. Among the eight RpCSPs, RpCSP8 exhibited a 3.81 fold higher expression in the oviparae compared to virginoparae. Across developmental stages and tissues, RpCSP8 showed the highest expression in the fourth-instar nymphs and adults, as well as in the salivary glands of oviparae. In contrast, it exhibited the highest expression in adults and the antennae of virginoparae. In oviparae, knockdown of RpCSP8 significantly impaired reproductive performance: fecundity decreased by over 80%, and lifespan was reduced by more than 50%. RpVg (vitellogenin) expression declined by 37.1%, although no significant differences in egg morphology were detected. Electrical penetration graph (EPG) recordings revealed that RpCSP8 knockdown in oviparae significantly reduced phloem ingestion duration, indicating impaired feeding behavior. In virginoparae, knockdown of RpCSP8 showed no significant changes in fecundity, lifespan, or feeding behavior. These results demonstrate different roles for RpCSP8 in regulating both reproductive output and host plant utilization in the oviparae and virginoparae of R. padi. This study reveals the non-chemosensory functions of insect CSP and provides new insights into the adaptive mechanisms underlying reproductive mode transitions and host switching in R. padi.

Abstract: V-ATPases are crucial for animal development and survival, but their functions in fertility are largely unknown. Here, we found that knockdown of VhaM9.7-d in germ cells induced complete sterility in male Drosophila melanogaster, but had no effects on female fertility. Depletion of VhaM9.7-d did not severely impair spermatogenesis, as the mature sperm appeared in the seminal vesicles (SVs) of the testes. However, the sperm released from the SVs of the VhaM9.7-d-knockdown males rapidly lost their motility and were unable to move over long distances. These sperm could be transferred to the female's uterus during copulation, but failed to be stored in the seminal receptacle (SR) and fertilize the egg. Tandem mass tag (TMT) proteomic analyses of SVs, including their contents, identified 434 differentially expressed proteins (DEPs) when comparing the control group to the VhaM9.7-d-knockdown group. Many downregulated proteins were enriched in phagosome and oxidative phosphorylation (OxPHOS) pathways. Subsequent experiments, encompassing the LysoSensor Probe assay, CMXRos staining, and ATP measurement, confirmed that the knockdown of VhaM9.7-d significantly disrupted phagolysosomal and mitochondrial functions, leading to diminished acidity, heightened levels of reactive oxygen species (ROS), and a decrease in both mitochondrial membrane potential and ATP contents in the SVs. These results suggest that VhaM9.7-d plays an essential role in maintaining the homeostasis of sperm energy metabolism by regulating phagolysosomal activity and mitochondrial OxPHOS. Our data provide valuable insights for the further study of the mechanisms of related diseases such as human asthenospermia.

Abstract: Micromelalopha sieversi (Staudinger) (Lepidoptera: Notodontidae) is a destructive defoliator of poplar (Populus sp.) trees in China. In prior study, (13Z,15E)-octadeca-13,15-dienal (Z13,E15-18:Ald) (I) and (13Z,15Z)-octadeca-13,15-dienal (Z13,Z15-18:Ald) (II) were identified from the sex pheromone glands of M. sieversi females. Although traps baited with Z13,E15-18:Ald (I) captured M. sieversi males, the attractiveness was inferior to that of unmated females. Moreover, male moths exhibited distinct mate-selection and mating behaviors, suggesting the production of unidentified bioactive components by the sex pheromone glands of females. Gas chromatography-electroantennographic detection and gas chromatography-mass spectrometry were used to identify two additional minor components, (13E,15Z)-octadeca-13,15-dienal (E13,Z15-18:Ald) (III) and (13E,15E)-octadeca-13,15-dienal (E13,E15-18:Ald) (IV), in the sex pheromone gland extracts from M. sieversi females. The four geometric isomers of 13,15-octadecadienal were present in a relative ratio of 100 : 13.43 : 5.27: 20.04. A wind tunnel assay and field tests demonstrated that E13,Z15-18:Ald (III) played a pivotal role in the "short-range" localization of females by male moths, and exhibited a synergistic effect with Z13,E15-18:Ald (I). In contrast, Z13,Z15-18:Ald (II) and E13,E15-18:Ald (IV) had antagonistic effects. Gas chromatographic analysis revealed that the proportion of Z13,Z15-18:Ald (II) in the sex pheromone gland extracts increased markedly after mating. Furthermore, application of exogenous Z13,Z15-18:Ald (II) to the terminal segment of unmated female moths reduced mating rates, suggesting its potential role in the mating decision-making processes of M. sieversi. By clarifying the specific functional roles of minor pheromone components, this study provides a practical approach for development of precise pheromone-based control strategies against M. sieversi.

Abstract: The industrial scaling of Hermetia illucens production for waste bioconversion and alternative protein provision is severely constrained by logistical challenges in breeding stock preservation and long-distance transport. To address this, we developed and optimized stage-specific cold storage protocols to significantly prolong the shelf-life of H. illucens. Through a multi-stage experimental approach, we determined that 1-d-old eggs can be successfully stored at 17 °C for 10 d with 50.0% hatchability, larvae retained a 72.8% survival rate after 30 d at 15 °C, and prepupae maintained a 75.3% eclosion rate following 60 d at 14 °C. In addition, a key innovation of this study was the strategic use of dietary cryoprotectants to markedly enhance cold tolerance. Under a severe discriminating temperature (-5 °C), supplementation with 4% proline and 0.5% trehalose elevated larval survival to over 80%. Furthermore, at the chronic stress of the developmental threshold (12 °C), 3% glycerol, nearly doubled larval survival rates compared to the control. The application of these optimized cryoprotectants with stage-specific storage temperatures effectively mitigated sublethal fitness costs, ensuring high post-storage performance in survival, pupation, eclosion, and reproductive output. Our findings provide a robust, comprehensive framework for synchronizing H. illucens supply chains, enabling viable long-distance transport, and facilitating the reliable industrial scaling of H. illucens production for the circular bioeconomy.

Abstract: Insects, the most diverse group of animals, inhabit almost all environments on Earth. They are susceptible to a wide range of parasites, including entomopathogenic protozoans, nematodes, and ectoparasitic mites. These parasites manipulate host physiology via immunomodulation, endocrine disruption, and metabolic reprogramming. The long-term coexistence of insects and parasites has driven the evolution of intricate survival strategies. Insects deploy morphological, physiological, and behavioral adaptations to mitigate infection risks, whereas parasites counter with sophisticated mechanisms enhancing transmission and reproductive success. Emerging evidence indicates symbiotic microbiota as critical mediators in this evolutionary arms race, modulating infection outcomes through microbial-host-parasite crosstalk. Here, we review recent research progress on the effects of parasites on the development, reproduction, immunity, and behavior of insect hosts; the evolutionary dynamics between insects and parasites; and the interactions of host-parasite-microbiota in insects. Compared to mammals, insects provide a simple model system for elucidating conserved molecular mechanisms underlying host-parasite-gut microbiota interactions. This paradigm not only advances fundamental understanding of evolutionary parasitology but also pioneers microbial-based biocontrol approaches, offering sustainable alternatives for agricultural pest management and economic insect conservation.

Abstract: The insect olfactory system is essential for survival, enabling the detection of chemical cues critical for feeding, reproduction, and avoiding threats. Semiochemicals, including pheromones and allelochemicals, are processed through specialized organs, primarily the antennae and maxillary palps, which contain sensilla housing olfactory receptor neurons (ORNs). Odorant-binding proteins (OBPs) transport volatile compounds to odorant receptors (ORs) on sensory neurons, initiating precise signal transduction. Rapid signal termination, vital for sensitivity, is achieved by odorant-degrading enzymes (ODEs) that prevent receptor saturation. Evolutionary adaptations optimize OBPs and ORs for species needs, such as the detection of foreign odors. Environmental factors, including temperature, nutritional state, and circadian rhythms, further modulate olfactory sensitivity. In this review article, we underline the interaction between olfactory proteins and insect immunity. Reports coming from different laboratories, point to the role of olfactory proteins in defense response, including its cellular, humoral, and behavioral aspects. Beyond chemosensitization, the olfactory system contributes to insect immunity by regulating pathogen recognition and immune signaling. OBPs interact with Toll-like receptors, regulating antimicrobial responses and gut microbiota stability. Symbiotic bacteria influence OBP expression, linking olfaction to systemic immunity. Finally, some odorant-binding proteins and chemosensory proteins possess direct antimicrobial activity. In conclusion, the insect olfactory system integrates sensory and immune functions through molecular and neuronal components, reflecting its evolutionary versatility. Blood-feeding insects, for example, Aedes aegypti or Rhodnius prolixus, exhibit heightened detection of host odors during reproductive cycles, while starved insects prioritize food-related cue.

Abstract: The tortoise scale insect (Toumeyella parvicornis) is rapidly spreading in stone pine (Pinus pinea) forests and urban parks of Mediterranean Europe. Its current distribution and potential spread is concerning as, so far, it already includes three different European countries. Pest management strategies based on endotherapic treatments have a limited time coverage and are unfeasible on large scales. Biological control can be a valuable alternative to contain the spread of T. parvicornis, as highlighted by some recent studies conducted with predators under controlled conditions. Although promising outcomes, open-field efficacy is still poorly explored. This study aimed to fill this gap in knowledge through an open-field assessment of the predation impact of Exochomus quadripustulatus and its capability of reducing T. parvicornis infestation level. Adult ladybug females were released on stone pine groups divided as follows: (i) plants treated only with ladybugs, (ii) plants pre-treated with bio-insecticide prior the release of the ladybugs, and (iii) an untreated control. The stone pine groups that received the ladybugs application, showed a lower infestation level (in terms of T. parvicornis adult females) than the untreated control, for most of the evaluation period. Results showed, for the first time, evidence of E. quadripustulatus efficacy in open-field applications, confirming the previously positive outcomes observed under laboratory and semi-field conditions. The outcomes of this study, accordingly, open the door to future biological control programs.

Abstract: Farnesoic acid O-methyltransferase (FAMeT) and juvenile hormone acid O-methyltransferase (JHAMT) are key enzymes in the isoprene branch pathway, a specialized downstream biosynthetic pathway for juvenile hormone (JH). Both enzymes play crucial roles in insect egg production. While their mechanisms have been well characterized in model insects, this study aims to elucidate their specific functions in the reproductive process of the small brown planthopper (SBPH), Laodelphax striatellus. Here, we cloned the full-length cDNA encoding a putative FAMeT (LsFAMeT) from SBPH. The deduced protein sequence has a conserved Methyltransf_farnesoic acid domain and shares high identity with other insect FAMeTs. To gain further insight, we silenced LsFAMeT and LsJHAMT in SBPH and found that LsJHAMT expression increased when LsFAMeT was knocked down, and vice versa, suggesting a potential coordinated relationship between LsFAMeT and LsJHAMT in JH biosynthesis. In addition, RNAi-mediated silencing of LsFAMeT, LsJHAMT, or both genes significantly reduced female fecundity while simultaneously decreasing JH III titers. This was associated with reduced levels of total protein, cholesterol, triacylglyceride, and four carbohydrates (glucose, fructose, sucrose, and trehalose), as well as decreased ovarian cell mitosis, increased ovarian cell apoptosis, and downregulation of the JH receptor Met and its early-responsive gene Kr-h1. Together, these data suggest that LsFAMeT- and LsJHAMT-mediated JH biosynthesis regulates the reproductive capabilities of SBPH through energy mobilization, and ovarian cell activity. Our findings help elucidate the role of JH in insect reproduction and provide insights into key enzymes that may serve as potential targets for the development of selective insect growth regulators.

Abstract: Disruption of reproduction is an important pest control tactics for management of high-fecundity pests like Helicoverpa armigera (Hübner). In this study, we investigated the role of the testis-biased miR-252a-5p in regulating gonad development and reproduction. Through a combination of bioinformatics prediction, dual luciferase reporter assays, functional injections (agomir, antagomir, and siRNA), and phenotypic evaluations, we identified OVOL as a key target gene of miR-252a-5p. We found that miR-252a-5p negatively regulates OVOL expression. Injection of miR-252a-5p agomir or OVOL siRNA into newly emerged females delayed egg maturation and reduced hatching rates. In contrast, inhibiting miR-252a-5p or knocking down OVOL in larval stages did not affect testis development or fertility. These results demonstrate that miR-252a-5p specifically targets OVOL to modulate ovarian development and reproductive success in H. armigera. Both miR-252a-5p and OVOL represent promising targets for reproduction-based control of this and other lepidopteran pests.