Insect Science

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

BACKGROUNDThe two-spotted spider mite, Tetranychus urticae Koch, is a major agricultural pest with a rapid propensity for developing acaricide resistance. Bifenazate targets mitochondrial cytochrome b (CYTB). While the G126S mutation is frequently associated with resistance, its independent role remains unclear as it often occurs with other substitutions. This study explores the molecular basis of bifenazate resistance in a Russian laboratory strain derived from a St. Petersburg greenhouse population. RESULTSDisruptive selection with increasing bifenazate concentrations generated resistant and susceptible isofemale lines. AlphaFold2 structural modeling of CYTB indicated that G126S causes a steric clash, leading to conformational destabilization, whereas other reported mutations primarily affect the ligand-binding pocket. Oxford Nanopore sequencing revealed a very low initial frequency of the G126S allele (<1%; 226/35,895 reads) in the unselected population. After one year of stepwise selection (0.00005-0.031% a.i.), the mutant allele frequency surged to 90% (7,272/8,056 reads). No other known resistance-associated mutations were found in the analyzed cytb fragment. CONCLUSIONWe report the first identification of the G126S mutation in a Russian T. urticae population and demonstrate its rapid fixation under bifenazate selection. Within this genetic background, G126S alone appears sufficient to confer high-level resistance, emphasizing the population-specific nature of resistance evolution and the critical need for local monitoring.

Kissing bugs are the primary vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Kissing bugs are exposed to thermal variability, including short periods of heat stress, which can induce mortality or exert sublethal effects. This study investigated Rhodnius prolixus following brief periods of high thermal stress with respect to survival, blood feeding, developmental processes, and T. cruzi infection, with a focus on sublethal effects. Our results demonstrated a significant decrease in survival for R. prolixus at 42 {degrees}C for 8 hours. When exposed to sub-lethal thermal stress (40{degrees}C for 8 hours), blood ingestion (amount and proportion) was reduced after 24 hours of recovery from thermal stress. Among the bugs that fed after 24 hours, molting was not impacted by temperature exposure. The infection rate decreased after heat exposure, likely due to reduced blood volume ingested when feeding 24 hours after heat stress. A week of recovery after exposure to higher temperatures improved feeding and increased infection rates to levels comparable to those of kissing bugs not exposed to thermal stress. Our findings offer insights into how extreme temperature events may influence Chagas disease. Specifically, these studies highlight the need to clarify how temperature, particularly at sublethal levels, interacts with vector biology to alter parasite transmission.

Life-history traits play an important role in insect population dynamics and ecological processes. The azuki bean beetle Callosobruchus chinensis is a common pest of stored legumes and is also widely used as a model species in ecological and evolutionary research. In this study, we tested whether machine learning models could be used to estimate several traits of C. chinensis, including elytral length, development time and adult lifespan. Experimental data were obtained from laboratory populations. The dataset included biological and environmental variables such as strain, treatment condition, developmental day, sex, temperature, and CO2. Six different machine learning models were tested, including linear regression, random forest, support vector machine (SVM), neural network, gradient boosting and AdaBoost. Model performance was evaluated using cross-validation. The coefficient of determination (R2) and root mean square error (RMSE) were used to measure prediction accuracy. Prediction accuracy differed among traits. Elytral length showed relatively higher predictability than the other traits, while development time was difficult to estimate in most models. Lifespan was easier to predict than the other traits, and the neural network produced one of the highest prediction accuracies among the tested models. Feature importance analysis also showed that factors such as sex and treatment condition contributed to variation in several traits. Machine learning models therefore helped reveal relationships among biological variables and life-history traits in C. chinensis. Combining ecological experiments with machine learning analysis may help improve our understanding of insect traits and may support future studies in insect ecology and pest management.

Nocturnal migration is a remarkable phenomenon observed in many insect species, including moths. Migratory moths are capable of maintaining precise directional orientation during migration, as demonstrated in both laboratory and field studies, suggesting that they use multiple environmental cues for orientation and navigation. Recent studies on Australian Bogong moths revealed that these animals can use stellar cues and likely the geomagnetic field (in conjunction with local visual cues) to select and maintain population-specific migratory direction. However, the underlying orientation mechanisms used by most other migratory moths are still largely unresolved. Further, it remains unclear whether migratory moths can adjust their orientation using Earths magnetic field parameters for determining their position relative to the goal (i.e. location or map information) - an ability clearly shown in some migratory birds which respond to virtual magnetic displacements by correcting their orientation (experiments when animals are exposed to magnetic cues corresponding to other geographic locations). Here, we present results from virtual magnetic displacement experiments conducted on red underwings (Catocala nupta). In addition, we tested their orientation under simulated overcast conditions and in a vertical magnetic field to get indications whether this species relies on geomagnetic or celestial cues to maintain its population-specific migratory direction. Our results show that (1) red underwings did not compensate for virtual magnetic displacement, indicating the absence of a magnetic map; (2) they remained significantly oriented in the absence of geomagnetic information, suggesting the use of a stellar compass; and (3) there was no evidence of magnetic compass orientation in absence of any visual cues.

Local temperatures can shape the ability of introduced species to flourish and disrupt novel environments. The emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an invasive beetle that threatens ash trees in North America and Europe. To assess the role of temperature on EAB reproduction, we reared groups of adult beetles at one of four temperatures (12, 15, 18, and 21 {degrees}C) and measured reproductive success (laying fertilized eggs and egg hatching). There was no effect of rearing temperature on EAB female lifespans but no eggs laid at 15 or 18 {degrees}C hatched, suggesting these temperatures disrupt the reproductive process of EAB. Females reared at 21 {degrees}C, however, consistently laid eggs that hatched. We then used these results to assess the likelihood of reproductive success over the previous ten years in eight cities in Canada that host EAB. All locations experienced temperatures of [&ge;] 21 {degrees}C, but the number of hours and the number of days above this critical temperature were highly variable. There were ample opportunities in all locations for EAB to reproduce, but EAB in cooler cities would experience thermal limitations thus slowing the spread of EAB populations.

It is commonplace in East Africa for 100% of cassava fields to be infected with Cassava mosaic disease (CMD) and/or Cassava brown streak disease (CBSD), resulting in annual losses of more than US$1.25 billion and reduced food and economic security for farming households. The vector of both diseases is the African cassava species of the whitefly Bemisia tabaci. Since the late 1990s, there has been an unprecedented increase in whitefly populations, to the extent that they are referred to as "super-abundant". Research efforts since the late 1990s has focused mainly on developing plant resistance to the viral pathogens and paid scant attention to understanding the root causes of disease epidemics or the control of whitefly infestation. Here, we aimed at developing long-term whitefly-control solutions using an in-planta RNA interference (RNAi) approach. First, transcriptome analysis identified candidate genes that play key roles in whitefly biology: osmoregulation, sugar metabolism and transport, symbiosis with endosymbiotic bacteria and detoxification of phytotoxins. Then, fifteen RNAi inverted repeat constructs were produced, designed to target the candidate genes and 140 independent transgenic lines were generated in cassava variety NASE 13. Whole plant bioassays showed insecticidal activity of transgenic plants, reaching 58% lethality for adults within 7 days and 75-90% lethality of nymphs after 25 days, compared to control plants. Target genes were confirmed to be downregulated by up to 2.5-fold in adult whiteflies and nymphs. We used population dynamics modelling to predict the potential of the RNAi technology to control whiteflies under field conditions in East Africa.

Amphibians are threatened worldwide by various environmental and anthropogenic factors, making non-invasive conservation studies particularly valuable. Newts are one example of a thus challenged amphibian group. In Austria, local population declines of newts have been observed, with the smooth newt (Lissotriton vulgaris) being strongly affected. In this study, skin swabs were used as a non-invasive method to gather DNA, combined with established microsatellite markers. We sampled 139 L. vulgaris individuals at ten sites in North Tyrol, Austria, and, for comparison, 22 L. vulgaris meridionalis individuals in Brixen, Italy. We genotyped all individuals and analysed their population structure. We demonstrate the presence of three distinct L. vulgaris population clusters and find differences in population structure between supposedly introduced allochthonous L. vulgaris individuals and autochthonous populations, as evidenced by differences in Bayesian clustering and elevated values of the fixation index FST. A captive population in a zoological garden, with origins in the Kramsacher Loar in the Tyrolean Unterland (eastern part of Tyrol), performed poorly in terms of conservation genetics, with low genetic diversity (number of alleles) and clear genetic differentiation from populations in the wild (high pairwise FST values with wild individuals, clear separation in cluster analysis). Habitat restoration programs are a crucial aspect of amphibian conservation, as they restore ecosystems that are critical to the animals survival. While breeding programs can play an additional role in the future, they must carefully consider genetic diversity to ensure resilient and viable populations, especially in the face of climate change and chytrid fungus infection. This study emphasizes the significance of considering the geographic origin and genetic diversity of newts in conservation efforts. It also serves as a foundation for future population genetic studies of newts in Austria.

Melanophryniscus moreirae is a diurnal species endemic to the high-altitude grasslands of the Serra da Mantiqueira mountain range, currently classified as "Near Threatened" (NT) by the IUCN. Knowledge about its distribution and natural history is fundamental for conservation plans, especially in the face of the threats of climate change. This study presents a new record of the species in the state of Sao Paulo, at an altitude of 1,904 m, expanding the known distribution in the southwestern portion of the Serra da Mantiqueira. In addition to fieldwork, a spatial and temporal review of data available on the GBIF (Global Biodiversity Information Facility) platform was carried out. The temporal analysis confirmed observation peaks in November, coinciding with the reproductive period, and an absence of records in the colder months, consistent with the species dormancy behavior. The study demonstrates that the integration of citizen science data, when properly validated, is an effective tool to fill knowledge gaps about biodiversity and assist in the monitoring of threatened species.

O_LIEctotherms in thermally variable environments mediate energy expenditure through both physiological and behavioural responses. However, many studies focus on constant temperature acclimation, and few consider behaviour and physiology in unison. It is unclear how acclimation to thermal variability affects locomotory choices, activity timing, and performance across daily thermal cycles. C_LIO_LIWe investigated the effects of thermal variability in the temperate dung beetle Onthophagus taurus. Following acclimation to a low amplitude (22{degrees}C {+/-} 2{degrees}C) or a high amplitude (22{degrees}C {+/-} 10{degrees}C) temperature regime, we measured behaviour and metabolic rate across temperatures. We hypothesised that O. taurus adjusts its locomotive strategy and search window when kept in high amplitude fluctuating temperatures to reduce energy loss associated with high temperature exposure. C_LIO_LIWe found that differences in energy expenditure were determined by propensity for flight which differed between acclimation treatments, particularly at intermediate temperatures. We also found that, following acclimation to a high amplitude of thermal variability, O. taurus exhibited a greater intensity of activity over a narrower window of time, and O. taurus acclimated to a low amplitude of thermal variability showed nocturnal activity. C_LIO_LIWe then used the data to model activity through the growing season over five years. Biophysical models were built using NicheMapR Microclimate and Ectotherm functions to test the length of potential searching time across seasons, the temperatures individuals are exposed, and locomotive strategy. Model outputs showed that acclimation to higher amplitudes of thermal variability increased accumulated degree-hours of activity relative to the low variability acclimation group. Individuals acclimated to higher amplitudes of thermal variability showed greater accumulated degree-hours in spring and fall, but exhibited shorter periods of activity during summer, with the model predicting increased opportunities for flight. Comparatively, O. taurus from the low variability acclimation treatment showed increased night activity in summer but did not fly. C_LI

Dung beetles (Coleoptera: Scarabaeinae) are ecologically important insects, yet genomic resources for this diverse lineage remain limited. Here, we present a high-quality genome assembly for Onthophagus orpheus, an understudied species that is abundant in urban forests in the eastern United States. The assembled genome is highly contiguous and exhibits strong completeness, as assessed by Benchmarking Universal Single-Copy Ortholog (BUSCO) analyses, indicating robust representation of conserved protein-coding genes. Structural and functional annotation recovered a comprehensive gene set consistent with expectations for coleopteran genomes. This genome assembly provides an important resource for future work on the behavioral ecology and population genetics of Onthophagus orpheus, specifically, and Scarabaeinae more broadly.

Istocheta aldrichi (Diptera: Tachinidae) is a specialist parasitoid of the invasive Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae). Research and releases for biological control depend on field collecting parasitized hosts and rearing the parasitoid through diapause to obtain I. aldrichi adults. This study investigated how rearing practices before, during and after the seasonal overwintering period affected the proportion of I. aldrichi pupae that emerged as adults, the timing of parasitoid emergence, and their longevity. Increasing cold exposure duration during overwintering increased adult I. aldrichi emergence from puparia and reduced development time after transfer to warm conditions. Adult I. aldrichi emergence from overwintered puparia depended on interactions between overwintering environment (indoors vs. outdoors), spring thermal regime, and the timing of host collection in the previous season. Burying puparia in the soil in late summer/early fall resulted in higher subsequent adult I. aldrichi emergence. Manipulating spring temperatures in controlled environments allowed parasitoid emergence to be staggered over several weeks without reducing emergence success. Emergence under outdoor spring conditions was unreliable. Adult longevity was affected by temperature and diet: cooler conditions extended lifespan, honey-water increased longevity relative to pollen alone or honey-water and pollen together. These results provide a foundation to further improve I. aldrichi rearing techniques for use in experimental research and applied biological control of P. japonica.

Seasonal rainfall shapes biological responses in tropical ecosystems, yet how tropical organisms integrate behavioral and physiological responses to cope with seasonality remains poorly understood. We assessed how four poison frog species with contrasting reproductive strategies respond to dry and wet season environmental conditions. We quantified spatial behavior, microhabitat use, hormone concentrations, and chemical defenses in two seasonal breeders (Allobates femoralis and Ameerega trivittata) and two year-round breeders (Ameerega macero and Ameerega shihuemoy). Seasonal breeders exhibited pronounced sex-specific shifts in space use, where males expanded their space use during the wet season, likely to track reproductive opportunities, while A. femoralis females increased their spatial use during the dry season, likely responding to foraging demands when prey resources are sparse. Year-round breeders maintained similar space use across seasons, likely reflecting their ability to access key resources within the same space to reproduce year-round. Microhabitat use was flexible, as seasonal breeders shifted toward humid refugia during the dry season and reproduction-associated microhabitats during the wet season, whereas year-round breeders selected microhabitats that facilitate continuous reproduction across seasons. Despite these behavioral responses, corticosterone, testosterone, and chemical defenses showed no consistent seasonal variation, suggesting that behavioral flexibility is decoupled from seasonal variation in these measured physiological responses. Our study suggests that poison frogs are able to buffer environmental fluctuations through behavioral flexibility. However, given the increasing unpredictability in rainfall timing and intensity as a result of climate change, how these coping strategies will function in the long term is uncertain.

The connection between female host plant preference and offspring performance is important for understanding how relationships between plants and phytophagous insects have evolved. According to the preference-performance hypothesis, female insects should evolve to oviposit on host plants on which offspring performance is the highest. Here, we examined the preference-performance hypothesis in the northern brown argus (Aricia artaxerxes) butterfly in the province of Uppland, Sweden, by comparing female host plant preference and larval growth between the host plant species wood cranesbill (Geranium sylvaticum) and bloody cranesbill (G. sanguineum). We also investigated if host plant preference in A. artaxerxes was related to the geographic distribution of A. artaxerxes and its host plants in the province Uppland. We found that the A. artaxerxes females, contrary to the preference-performance hypothesis, preferred ovipositing on G. sylvaticum, even though larvae feeding on G. sylvaticum were slightly smaller than those feeding on G. sanguineum. Since G. sylvaticum is more abundant and probably more utilized than G. sanguineum in Uppland, an explanation for this negative preference-performance connection may be that there are advantages associated with utilizing a more common host plant species, even though larvae feeding on this plant show reduced growth rates. Overall, the results show that factors other than offspring performance, such as geographic distribution, may influence female host plant preference in A. artaxerxes.

Eusociality in bees represents a major evolutionary transition and understanding its molecular basis is fundamental for sociogenomic studies. Comparative genomics has revealed correlations between transcription factor binding site (TFBS) abundance and social complexity; however, when and where these TFBSs function in a eusocial context remains largely unclear. In this study, we performed cap analysis of gene expression (CAGE) during worker metamorphosis in the honeybee Apis mellifera to identify TFBSs within active enhancers and decipher the regulatory relationships between these enhancers and their target genes. We identified 17,349 transcription start sites (TSSs) and 842 candidate enhancers. Using CAGE, we identified five clusters based on expression patterns. Notably, genes associated with the canonical metamorphic regulators, Broad complex (Br-c) and E93, were found within specific clusters. By integrating the correlations between enhancer and TSS activities with motif enrichment analysis, we identified 15 transcription factor-enhancer-TSS regulatory relationships. Among these, tramtrack (ttk)-binding sites were identified in five enhancers associated with four target genes, including Br-c. The number of target genes regulated by ttk was the highest in our dataset. To examine whether this regulatory relationship is conserved across bee species with varying levels of sociality, we analyzed the sequence conservation of ttk-binding sites in Br-c enhancers and found that perfect sequence conservation of ttk-binding site was restricted to the Apis genus. The ttk-binding sites of other target genes exhibited the same Apis-specific conservation pattern. Our findings suggest that gene regulatory relationships during worker metamorphosis occur in a lineage-specific manner in the Apis genus. SignificanceHoneybees produce distinct castes--queens and workers--from genetically identical larvae via differences in gene regulation. Although enhancers have been computationally predicted, their actual activity during bee development has rarely been measured directly, and the CAGE technology has never been applied for this purpose. We identified active enhancers during worker metamorphosis and discovered that the transcription factor ttk may regulate Br-c, a key developmental gene. This study provides the first direct evidence of active enhancers and their regulatory roles in honeybee worker metamorphosis.

Honeybee breeding plans are relatively recent in most countries. In France, diverse small-scale breeding groups are emerging. Beekeepers are highly diverse in their motivations, farm productions and services, practices and management techniques. Yet, little is known about what beekeepers would consider as relevant breeding goals in the design of breeding plans. We therefore conducted an online survey answered by about 250 French beekeepers, mostly professionals, to assess their perceived importance of including 20 pre-defined traits in breeding goals and to identify how beekeeping profiles might influence these priorities. Respondents rated each trait as essential, useful, or useless, and indicated if they wished useful or essential traits to be genetically improved or merely maintained at their current level. Results indicated a strong preference for multi-trait selection, with a median of 13 traits considered useful or essential. Honey yield, disease resistance, swarming tendency, gentleness, and summer feed autonomy, emerged as the main traits of interest with about 90% of beekeepers finding them at least useful. About 40% or more only wished to maintain these traits at their current level rather than to directionally improve them. A major exception to this was disease resistance, that 75% wanted to improve. Bees genetic background influenced the most the importance attributed to breeding goal traits, while other beekeeping profile characteristics only had a marginal effect on breeding goal trait priorities. Some poorly studied traits, such as summer and winter feed autonomy, winter diapause, and longevity, were considered at least useful in a breeding goal by over 70% of beekeepers. Future research is needed to explore possible selection criteria for these traits and estimate the potential for their genetic improvement. ImplicationsOur survey shows that French beekeepers wish to improve or maintain through selective breeding usual colony production and behavioral traits, but also colony resilience, especially disease resistance and feed autonomy. However, trait priorities differ depending on the genetic background of the bees used. This knowledge is essential for designing breeding programs that truly match beekeeper needs and for identifying which traits deserve research attention. In France, beekeepers are increasingly starting breeding efforts to adapt their bees to current conditions, facing growing pressures from climate change, diseases, invasive species, and pesticides. Well-designed breeding programs can support sustainable beekeeping and essential pollination services.

Istocheta aldrichi (Diptera: Tachinidae), a specialist parasitoid of the invasive Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae), was released to eastern North America in the 1920s as part of a classical biological control program. Further releases are being considered in different regions of North America and Europe where P. japonica is establishing. Successful releases of the biocontrol agent depend on identifying efficient techniques for collecting parasitized hosts from the field and rearing the parasitoid through diapause to obtain I. aldrichi adults. In this study, we evaluated how the collection date, the collection method (hand-picking vs. regular traps vs. modified traps) and rearing conditions (food provision and substrate type) of parasitized hosts influence I. aldrichi pupariation and emergence. The proportion of parasitized beetles yielding I. aldrichi puparia decreased considerably as the season progressed. Rearing conditions immediately after collection influenced both puparium yield and quality: withholding food from parasitized P. japonica slightly increased puparium yield but reduced puparium weight, while the effect of food provision on subsequent overwintering survival depended on rearing substrate. Finally, simple modifications to commercial traps (larger, ventilated, containers with added food source and substrate) collected more beetles than regular traps and promoted successful development of the parasitoid to the puparium stage. Our results are used to suggest basic guidelines for collecting and rearing I. aldrichi in experimental research and applied biological control of P. japonica.

Recent advances in sequencing technology have made the sequencing of non-model organisms significantly more streamlined and feasible. Using these technologies, we begin to address the lack of data on non-model organisms, by sequencing the genome of one such species, Phymata mystica (Evans 1931), an ambush bug (Hemiptera: Heteroptera: Reduviidae: Phymatinae) specialized for floral sit-and-wait style predation. Our genome assembly is 710 Mb, in which 99.7% of this sequence is assembled into 14 chromosomal scaffolds. We found that repetitive elements accounted for 58.85% of the sequence. We report 26,760 protein-coding genes in a preliminary annotation of the genome. Using these new resources, we explored both macrosynteny and gene conservation. Starting with chromosome structure, we found that P. mystica has a single X chromosome, unlike other well-assembled Reduviids in which the X apparently split into two linkage groups. Exploring this new annotation, we found a number of venom proteins conserved between P. mystica and the other venomous Heteroptera with reference genomes, primarily serine proteases, metallopeptidase and heteropteran venom family proteins. These results provide a new framework for the evolution of venom in this group of insects and further demonstrate the ease with which non-model species can be studied using modern genomic methods.

O_LIPhenological shifts are a major ecological consequence of climate change, yet studies often focus on single life stages meaning that the potential for carryover effects between life stages remains poorly understood. Failing to account for these effects may lead to inaccurate estimates of phenological shifts, with consequences for predicted synchrony among interacting species. This is especially relevant for temperate systems where climate warming is occurring unevenly across the year. C_LIO_LIHere, we investigated how temperature experienced the previous autumn and winter (during the pupal and egg stage) influences spring phenology in the winter moth (Operophtera brumata), a herbivorous insect with distinct life stages. Using 50 years of local climate data to create five experimental temperature regimes, we first quantified phenotypic plasticity in the duration and temporal variability of pupal and egg development. We then examined how timing of adult moth emergence affects timing of offspring hatching. C_LIO_LIWe found divergent effects of temperature on different life stages; pupal development time was shortest at intermediate temperatures while egg development time decreased linearly with increasing temperature. Furthermore, phenological shifts due to the conditions experienced by the mother were carried over to influence the phenology of her offspring. While this carryover effect was partially compensated during subsequent stages, compensation decreased under warming conditions. C_LIO_LIThese results refine our understanding of the sensitivity of the annual cycle of winter moth phenology to variation in temperature with potential implications for population dynamics and interspecific interactions. Overall, our findings highlight the need to consider the impacts of warming across multiple life stages so that carryover effects can be properly accounted for. Doing so will improve predictions of phenological shifts under future climates. C_LI