Insect Science

Salivary secretions of herbivorous insects contain proteins that modulate plant defence and facilitate herbivory. Despite the broad host range of Spodoptera frugiperda, a major agricultural pest, its salivary molecular arsenal involved in interactions with different host plants remains poorly understood. In particular, the salivary proteins induced during feeding on different host plants and their potential interactions with plant immune components remain largely unexplored. To address this gap, we generated salivary gland transcriptomes from larvae reared on an artificial diet as well as larvae fed on tomato or chickpea plants. Comparative transcriptomic analysis identified salivary genes that were differentially expressed in response to plant feeding compared with the artificial diet. The encoded proteins were subsequently analysed using a secretory protein prediction pipeline to identify host-induced putatively secreted proteins with potential effector functions. Candidate salivary effectors were further investigated using large-scale in silico protein-protein interaction analyses with AlphaPulldown to predict their interactions with immune-pathway associated proteins of tomato and chickpea. This analysis identified multiple putative salivary effector candidates whose expression was induced by plants feeding, with distinct candidate repertoires associated with tomato and chickpea. Predicted interactions between these candidates and plant immune-associated proteins further identified potential host targets. Together, these findings suggest that S. frugiperda may adjust its salivary molecular repertoire in response to the host plant and provide a transcriptome-wide framework for validating effector-target interactions and elucidating the molecular mechanisms underlying S. frugiperda-plant interactions.

In Germany, insecticide use is recommended during mosquito-borne disease outbreaks, yet nationwide data on insecticide resistance in key vector species are scarce. This is particularly relevant for Culex pipiens s.l., a vector of West Nile virus. Here, we provide the first broad-scale assessment of molecular insecticide resistance (kdr and ace-1 mutations) in Cx. pipiens f. pipiens across Germany. We associated insecticide resistances with environmental conditions, potential links with mitochondrial variation of the cytochrome c oxidase I (COI) gene as well as phenotypic resistance (CDC bottle assay with permethrin) of the Frankfurt population with elevated kdr frequencies. Kdr resistance alleles were detected in German Cx. pipiens f. pipiens reaching frequencies of up to 50% in the metropolitan areas of Berlin and Frankfurt am Main. Annual precipitation was negatively associated with kdr allele frequency. Interestingly, ace-1 resistance mutations were only present in the metropolitan area of Frankfurt am Main and in Muggensturm (Rhine valley, southwestern Germany). COI haplotype analyses revealed low genetic variability within German Cx. pipiens f. pipiens populations with no association to the insecticide resistance genotype. Phenotypic assays of the Frankfurt population showed early signs of permethrin resistance, but unmatched genotypic and phenotypic resistance state. This study provides the first broad-scale assessment of molecular insecticide resistance in Cx. pipiens f. pipiens across Germany, establishing a baseline for resistance surveillance and vector control. Further research is needed to identify factors shaping phenotypic resistance, as molecular markers alone may not reliably predict resistance. Since sampling was restricted to urban green spaces, broader sampling across environmental gradients is needed to identify additional environmental effectors.

Over the course of evolution, many species have adapted to the environmental conditions found at higher latitudes. This is likely also true of the circadian system, which regulates daily behavior and helps measure day length to facilitate adaptation to the seasons, though how the circadian clock adapts to latitude remains largely unknown. Here, we investigated daily activity patterns under long photoperiods and in constant darkness, as well as the underlying circadian clock network, in 52 Drosophila species from around the world. We found a strong, significant positive correlation between the clock's plasticity, measured as the phase difference between morning and evening activity, and the average geographical latitude from which the species originate. The mechanisms underpinning this increase in plasticity differed between species: in the subgenus Siphlodora, the clock weakened with increasing latitude, accompanied by a reduction of the neuropeptide Pigment-Dispersing Factor in the small ventrolateral clock neurons (s-LNv), whereas in the subgenus Drosophila the opposite was true. The subgenus Sophophora, by contrast, showed a negative correlation between circadian plasticity and PDF levels in the s-LNv axons. These results suggest that Drosophila species have evolved diverse, independent strategies to adapt their circadian clock to higher latitudes.

Aim To investigate how repeated historical connections between continental islands and the mainland have shaped the phylogeographic structure and genetic diversity of populations, using the widespread water strider Aquarius paludum as a model. Location Japan Taxon Water strider Aquarius paludum (Insecta: Hemiptera: Gerridae) Methods We analysed mitochondrial (COI and COII) and nuclear (ITS1) DNA sequences from populations across the Japanese archipelago and the Eurasian continent. Phylogenetic and population genetic analyses were used to reconstruct phylogeographic structure and infer demographic history. Results Western Japanese populations exhibited nucleotide diversity comparable to that of continental populations despite their insular distribution. Bayesian phylogenetic analyses identified two major mitochondrial haplogroups (A and B) that frequently co-occurred across the Japanese archipelago. Haplogroup A, which is rare on the Eurasian continent, occurred at high frequency in Japan and showed pronounced isolation by distance, whereas haplogroup B showed only weak but significant isolation by distance and was significantly more frequent in populations closer to the coast. These contrasting spatial patterns within species indicate distinct demographic histories for the two mitochondrial lineages. Main conclusion Repeated colonization from the Eurasian continent and the long-term coexistence of mitochondrial lineages with different demographic histories have shaped the pattern of genetic diversity of A. paludum in the Japanese archipelago. Our findings exemplify how continental islands can accumulate genetic diversity through lineage accumulation and secondary contact, which produces an intraspecific phylogeographic pattern comparable to those observed during an early phase of the taxon cycle between species.

Fluctuating asymmetry (FA) is widely used as a proxy for developmental instability, although reduced rather than increased FA, has occasionally been reported under particularly challenging conditions, possibly because environmental filtering removes less developmentally stable individuals. Here, we investigated temporal variation in wing shape asymmetry in males of Drosophila sturtevanti collected in 2022, 2024, and 2026 at the same sampling site, an urban Atlantic Forest fragment. Sampling in 2022 and 2026 were completed during single field expeditions in early April, whereas in 2024 repeated collections were required until mid-May because of markedly low capture success. We therefore examined whether this difference was associated with unusual environmental conditions and whether FA was higher in 2024, as expected under greater developmental perturbation. Daily temperature and relative humidity were examined from early March onward, encompassing approximately one month before adult emergence. The 2024 sampling period was warmer and drier than the corresponding periods in 2022 and 2026 and coincided with the final phase of the strong 2023-2024 El Ni&ntildeo event. Contrary to our prediction, FA was significantly lower in 2024. The magnitude of directional asymmetry remained similar among years, although the spatial pattern of the DA vector differed between 2024 and 2026. The coincidence of reduced capture success and reduced FA is consistent with the hypothesis that adverse conditions may impose demographic filtering, such that individuals expressing greater developmental instability are less likely to contribute to the sampled adult population.

A complete reference genome for species with chromosomally-determined separate sexes should contain scaffolds for all sex chromosome homologs. However, sex chromosomes present distinct computational challenges compared to autosomes. Here we present a k-mer based analysis that utilizes whole-genome sequencing of a few sex-identified isolates: Cytogenetics-By-Sequencing (CBS) tools. Unlike other approaches that typically address one aspect of the sex chromosomes, CBS-tools strives to guide users from the discovery of the heterogametic sex through identifying the sex-determination region (SDR). The core of CBS-tools is automated quantification of sex-specific k-mers in order to predict the heterogametic sex. Using publicly-available datasets, CBS-tools correctly identified the known sex-system of the 31 species tested. Additionally, we used these k-mers to verify and correct phasing of sex chromosomes between haplotypes in species representing different sex-systems. Finally, we used these k-mers to delimit the SDR boundary using an interactive web platform. CBS-tools was developed with previously unexplored sex chromosome systems in mind, but is also suitable for well-examined sex chromosome pairs.

Whole genome analysis of spittlebug populations, an Aphrophora species (Hemiptera: Aphrophoridae) known to spread Pierce's disease, from various vineyards in Napa and Sonoma Counties demonstrates that these insects are conspecific with Aphrophora permutata found in quasi-natural populations. The genomes analyzed fall in three autosomal clusters with two sympatric populations (based on admixture) that diverged around 40,000-90,000 years before present according to demographic models. We also recover three related but distinct mitochondrial clusters. There is no genetic differentiation between Napa and Sonoma County vineyard populations and very modest differentiation among vineyards and quasi-natural Berkeley/Oakland Hills population. These vineyard and quasi-natural populations have distinct life histories, including divergent oviposition hosts (understory herbs versus pines) and different patterns of adult seasonal migration (out-migration and return versus simple movement from the understory to the pine canopy). Analysis of the mitochondrial gene COI indicates that A. permutata, Aphrophora fulva and Aphrophora maculosa are intermingled in the same mitochondrial clade, along with the more distantly related species Aphrophora gelida. Further, all three autosomal genetic clusters show evidence of selection for insecticide resistance. We also find that endosymbionts Wolbachia and Rickettsia were both present in a substantial fraction of specimens analyzed, but neither appears to influence population structure. Overall, our findings elucidate an intriguing degree of life history divergence between geographically proximate, conspecific populations linked by gene flow and may have implications for the continued management of vineyard populations.

Stink bugs (Hemiptera Pentatomidae) are major agricultural pests worldwide, including both native and invasive species. In France, for instance, Halyomorpha halys has rapidly expanded its range, affecting key crops such as hazelnuts, apples, and vegetables. Meanwhile, several native, polyphagous species - Nezara viridula, Palomena prasina and, to a lesser extent, Graphosoma italicum - are re-emerging and impacting diverse crops because of changes in agricultural practices and climatic conditions. Egg parasitoids represent promising biological control agents for these pests, be it through conservation, augmentation or introduction biological control. However, their actual potential is not precisely documented due to a lack of ambitious surveys and possible taxonomic issues. This study aims to address this gap by compiling results obtained from various of egg parasitism surveys of Pentatomidae in France and implementing a large-scale DNA Barcoding approach (2000 individuals) for the emerged parasitoids. Our results indicate a high diversity of egg parasitoids, and detected some taxonomic issues and molecular inconsistencies that warrant further study. Among the three families of egg parasitoids recovered (Encyrtidae, Eupelmide, and Scelionidae), Scelionidae was the most abundant and diverse with nine species of Trissolcus (including the two adventive species, Tr. japonicus and Tr. mitsukurii) and four Telenomus species (including two unidentified to species). Scelionidae dominated the parasitoid assemblages associated with most of the Pentatomidae host species, except for H. halys for which the eupelmid Anastatus bifasciatus was the most common species.

Abstract Background Many ecologically important small invertebrates are preserved in ethanol, which compromises DNA integrity and severely limits their potential to be utilized in further genomic research. The lack of soil arthropod representation ultimately results in limited availability of reference genomes and population genomic inference from pooled sequencing (Pool-seq). Results We introduce PoLoCo (Pooled Low-Coverage), a reproducible workflow integrating pooled specimen draft genome assembly from ethanol-preserved specimens with population-level allele-frequency analysis. We assembled a 411 Mb draft genome of Entomobrya nivalis from pooled short reads, achieving 97.7% BUSCO completeness despite notable fragmentation. Comparison with the published reference genome showed partial genomic correspondence while also revealing reference-dependent differences in the retained SNP datasets. The application of PoLoCo to 82 retained pooled populations produced a final distance-thinned allele-frequency dataset suitable for population-scale genomic inference. Case-study execution documented the computational requirements of the major workflow components on standard HPC infrastructure, with the workflow, documentation, and software environments openly available in Zenodo. Conclusions PoLoCo demonstrates that draft genome assemblies and reproducible Pool-seq allele-frequency analyses can be performed with long-preserved sample material from the field. PoLoCo integrates reference construction, sync-based allele-frequency analysis, and benchmarking into a free and open workflow. It establishes a reproducibility benchmark for biodiversity genomics and yields a transferable workflow for ecological and evolutionary initiatives where high-quality DNA or long-read sequencing is not feasible.

Ethanol occurs naturally in sugar-rich foods used by insects, but its effects in bees have been studied primarily in honeybees. We tested whether dietary ethanol affects survival and locomotor performance in workers of the bumblebee Bombus terrestris (L.). In the survival experiment, workers were fed ethanol (0, 1, or 10%) for 21 days; in the locomotor experiment, they were fed the same doses for one or seven days, after which we measured spontaneous movement using an open-field assay. We measured the proportion of time in movement, speed of movement, path meander (curviness), and arena exploration. The 10% ethanol concentration greatly increased the risk of mortality, whereas the estimated effect of 1% ethanol was smaller and statistically inconclusive. In terms of locomotion, the largest differences occurred after seven days of exposure to 10% ethanol. Workers in this treatment showed greater path meander and lower exploration than control workers. Speed was also lowest in this group. Evidence that exposure for seven days was worse than for one day at the same concentration was detected only for exploration. No clear locomotor effect was detected at 1% ethanol exposure. These findings provide comparative data on sustained ethanol exposure in bumblebees and show a strong concentration-dependent contrast, with substantial effects under high-concentration challenge but no clear effects on the measured endpoints at 1%.

Morphological variation in insects can indicate responses to habitat change and land use intensification, with potential consequences for trophic interactions. In insect host-parasitoid systems, host body is often a key determinant of offspring development and emergence success, as well as parasitoid host-selection behavior. Yet, most studies in agricultural landscapes have focused on size variation in crop-associated insects (i.e.: pests, pollinators and enemies), leaving those relying on non-crop resources largely unexplored. Here, we investigated how agroenvironmental contexts and trophic resource availability influence puparial size in Protocalliphora sialia (Diptera: Calliphoridae), an obligate hematophagous ectoparasite of Tree Swallow (Tachycineta bicolor) nestlings. We further tested whether puparial size affects emergence probability and risk of parasitoidism by Nasonia spp. (Hymenoptera: Pteromalidae) jewel wasps. Using 1,513 P. sialia puparia from 88 nests within a nest-box monitoring system spanning a 10,200-km^2 east-west intensification gradient in southern Quebec, Canada, we evaluated how agrointensive contexts, avian host availability, and ectoparasite densities influence puparial size, parasitoidism risk, and emergence success. The agricultural context appears to shape puparial size through survival biases and/or altered growth rates. In agroextensive contexts (i.e.: pastures, forages with high tree cover), where Protocalliphora are more numerous, P. sialia puparia were smaller and exhibited greater size variation. Conversely, in agrointensive environments (maize and soybeans monocultures with higher doses of pesticides), where Protocalliphora are fewer, small individuals were underrepresented, suggesting a selective mortality in P. sialia population. However, puparial size explained less than 10% of the variation in emergence or parasitism, likely reflecting consistently high parasitic pressure from Nasonia wasps, which infested 50-90% of puparia, depending on site and year. Our findings highlight the importance of selective mortality in structuring trait distributions and modulating multi-trophic interactions in human-modified landscapes.

The intracellular bacterium Wolbachia pipientis alters the biology of its arthropod hosts in many ways, and can increase resistance to RNA viruses in both Drosophila and mosquitoes. Wolbachia-induced pathogen blocking has generated much interest because of its potential to restrict insect vector transmission of human diseases caused by RNA viruses. However, the molecular mechanisms by which Wolbachia affects host viral resistance are still mostly elusive. We used dilp2-3,5 mutant Drosophila, which are long-lived, but only in the presence of Wolbachia, to show that the presence of Wolbachia also increased the resistance of the mutant flies to infection with Drosophila C virus (DCV), relative both to mutants lacking Wolbachia and to wild type flies with and without Wolbachia. The insulin mutant flies had higher Wolbachia titres than wild type flies. By RNA-seq analysis of the Wolbachia transcriptome, we identified Wolbachia genes that were more strongly expressed in dilp2-3,5 mutant flies. Ankyrin-domain-containing proteins were among the most strongly up-regulated and they are predicted to be secreted effector proteins. To address their effect on host physiology, we generated 4 transgenic fly lines each with inducible expression of a different genes encoding ankyrin-domain-containing proteins. Expression of 3 of these did not cause obvious effects, but expression of WD0754 at high levels severely shortened fly survival. Interestingly, however, chronic low-level induction of WD0754 increased the resistance of the flies to DCV infection. Proteomics analysis showed a robust, tissue-specific, anti-viral response upon WD0754 induction, and identified the NFkB-like IMD pathway as a potential mediator of the antiviral activity of WD0754. Consistently, WD0754 expression activated Relish, a key downstream transcriptional mediator of IMD signalling, while loss-of Relish blocked the antiviral effect of WD0754. In summary, we identified a Wolbachia-derived ankyrin domain containing protein that modulates host immunity through the IMD pathway.

Many animals can sense the Earth's magnetic field and use it for orientation. However, the mechanism underlying magnetoreception has not been identified, partially due to the lack of reproducible and genetically tractable model systems. Here, we establish a directional assay for planaria to test their responses to different magnetic fields. Girardia dorotocephala showed a preferred magnetic directionality in the horizontal plane in an Earth-strength magnetic field in the absence of visible light, which shifted if the azimuth was rotated and was absent when the magnetic field was cancelled. This directional preference differed between labs. Furthermore, the directional response was affected by a short but strong magnetic pulse, resulting in a random distribution. The effect of the pulse was temporary, with directional preferences recovering over several days. Taken together, these data are consistent with a magnetoreception mechanism in planarians that functions independently of light and relies on permanently magnetised material, such as single-domain magnetite. As planarians are genetically tractable and well-established, they are a promising invertebrate model organism that might offer insights into the mechanisms underlying magnetoreception in animals.

The honey bee ectoparasitic mite Tropilaelaps mercedesae is an emerging threat to Western beekeeping, as it is expanding its range from native Asia. In this study, the first record of the presence of Tropilaelaps mercedesae in Turkiye, in the Artvin province, in the north-east of the country is documented. Following reports of weakening colonies, eight apiaries were inspected using the rapid brood decapping method. All apiaries were found positive, and infestation was detected in 22 of 24 examined colonies. Mite samples were analysed with morphometry and barcoding and were confirmed as T. mercedesae. In the paper, we discuss the impact of imports and migration practices on the mites occurrence in Turkiye. In addition, we forecast the potential corridors and means of further invasion and address the implications for European and global apiculture.

Background: The yak (Bos grunniens) is an essential livestock species in high-altitude alpine ecosystems, serving key roles in meat, milk, and wool production as well as ecological maintenance. However, traditional yak breeding systems face several limitations, including fragmented herd data management, inefficient phenotypic collection, missing pedigree records, and high technical barriers to applying genomic technologies. These factors constrain the widespread application of modern genetic evaluation methods in yak breeding programs. Description: We developed the Yak Genomic Evaluation Platform (YGEP, https://yakbreeding.swun.edu.cn/en/), an integrated digital system for yak genetic selection and herd management. YGEP integrates three core analytical modules: pedigree reconstruction, genotype imputation and genomic breeding value estimation. Benchmarking on 706 yaks showed that GBLUP achieved a prediction accuracy of r = 0.96 for cannon bone circumference (CCB), and KBeagle reached 84.0% imputation accuracy with the combined reference panel. Conclusion: YGEP bridges the gap between advanced genomic analytics and practical yak breeding. By integrating pedigree reconstruction, genotype imputation, and GEBV estimation into a user-friendly cloud platform, YGEP effectively eliminates technical and data bottlenecks. Overall, it provides an accessible, scalable, and standardized solution to advance digital herd management and precision genetic improvement in alpine livestock.

Microbiome has co-evolved with insects over millions of years, establishing symbiotic relationships that influence their fitness and shape evolutionary trajectories. These long-term associations are shaped by the hosts ecology, diet, and physiology which determines microbial diversity. In turn, external stresses such as pathogenic infections can adversely affect the host microbiome, further compounding the effects of pathogenic infections on the host. Using a computational pipeline and publicly available transcriptomics datasets, we analyzed the effect of viral infections on transcript derived microbiome diversity and composition in various insects including Drosophila melanogaster, Acyrthosiphon pisum, Antheraea pernyi, and Culex pipiens. The meta-transcriptomic reanalysis provides an indirect but comprehensive comparison of infection-associated transcriptional microbiome changes across hosts with distinct life histories. Our study reveals that viral infections influence the host-associated microbial transcripts, leading to shifts in transcript-derived microbial profiles and offering insights into host-microbe dynamics. In Cx. pipiens, microbial transcript profiles remained relatively stable following RVFV exposure and were dominated by the bacterial endosymbiont Wolbachia. Our study highlights viral infections as key drivers of infection-associated shifts in microbial profiles and suggests a potential association between Wolbachia dominance and transcriptional microbiome stability in insects, with age as a contributing factor in shaping microbial transcript profiles in Culex. This cross-species framework uncovers conserved and host-specific patterns of virus-microbiome interactions, expanding our understanding of microbial resilience mechanisms in insects.

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

Chemotaxis assays are widely used to quantify sensory-driven behavioral responses in Caenorhabditis elegans. Sodium azide is commonly used to immobilize animals at assay endpoints, although concerns have been raised that diffusion beyond the endpoint region could affect behavior before a choice is completed. We compared sodium azide-mediated immobilization with post-assay cooling using the repellent 1-octanol and attractant diacetyl. Wild-type animals showed robust avoidance of 30% 1-octanol and attraction to 0.5% diacetyl with either method, across multiple assay durations, and with or without worm food Escherichia coli OP50. These results support post-assay cooling as an alternative endpoint-scoring method.

Crickets are gaining attention as sustainable protein sources because of their high nutritional value and low environmental impact. Growth models are essential tools for optimising production by predicting growth characteristics. However, previous studies have not compared multiple non-linear growth models for crickets or explicitly considered environmental factors. We analysed the growth patterns of the two-spotted cricket (Gryllus bimaculatus) and the Asian cricket (Teleogryllus occipitalis) at multiple temperatures (25.0, 27.5, 30.0, 32.5 and 35.0 {degrees}C) using three non-linear models (Gompertz model, logistic model, and von Bertalanffy model) and combined the predicted body mass with observed survival rates to predict the optimal harvest time and temperature. All three models fitted the data well (R2 [≥] 0.97 in both species). The asymptotic body mass was highest at 30.0 {degrees}C, and the growth rate constant increased with temperature, reaching its maximum at 35.0 {degrees}C except for the logistic model in G. bimaculatus (32.5 {degrees}C). In addition, the predicted value of harvest, calculated by combining predicted body mass and observed survival probability, was highest at 30.0 {degrees}C in both species, reaching 13.82 kg/m3 at 38 days in G. bimaculatus and 7.12 kg/m3 at 62 days in T. occipitalis. These results suggest that 30.0 {degrees}C is the most suitable constant-temperature condition for maximising predicted productivity, although the optimal harvest timing differs between species.

1. Organisms rely on environmental cues, such as moonlight illumination and weather, to modulate nocturnal behaviors that reduce predation risk and conserve energy during demanding periods. Urbanization can alter the reliability of cues, potentially impairing organisms' ability to respond appropriately with consequences in increasingly urbanized landscapes. 2. We leveraged the Motus Wildlife Tracking System to identify roost initiation, roost departure and nocturnal restlessness of dark-eyed juncos (Junco hyemalis) overwintering along an urban to rural gradient to determine if activity patterns are affected by weather, moonlight illumination, and urbanization. 3. Nocturnal behavior of overwintering juncos varied with environmental factors, including moonlight illumination and temperature. However, the strength of these responses differed among sites, suggesting that natural cues of moonlight illumination were masked in urban areas, where individuals were more responsive to temperature. 4. Our results suggest that urbanization weakens birds' responsiveness to natural light cues, likely because artificial lighting reduces the visibility of moonlight; representing a key mechanism by which human-altered landscapes can disrupt nocturnal ecology in birds.