Insects

The mass-rearing of black soldier fly (Hermetia illucens) larvae (BSFL) is a promising solution for converting organic waste into high-quality insect protein, but preventing larval escape from open-top rearing containers remains a major management challenge. Conventional escape-control methods are often unreliable or impractical. To address this, we developed and evaluated a novel physical barrier, the anti-climbing tape, featuring regularly arranged macroscale protrusions designed to disrupt larval locomotion on vertical surfaces. We conducted a series of experiments to examine the design parameters of the anti-climbing tapes, including the gap size between protrusions and the number of protrusion rows. Our results demonstrate that the anti-climbing tape prevents escape via a dual mechanism: (1) physical obstruction, in which gaps narrower than the larval body width block larvae from passing through, and (2) adhesion reduction, in which the elevated protrusion array decreases the effective contact area for wet adhesion while increasing gravitational torque acting on the larval body. The effectiveness of these mechanisms was dependent on larval size. A design featuring 0.5-mm gaps and a 15-row protrusion array completely prevented the escape of later-instar larvae (>10 mm) in a 20-day large-scale trial, whereas the method was less effective for smaller larvae. In conclusion, the anti-climbing tape provides a robust and chemical-free approach to BSFL escape in mass rearing. To ensure reliable performance, its design parameters, both gap size and array width must be optimised to suppress the mechanical and adhesive components of larval climbing according to the target larval size. Conflict of interestS. Jang and M. Shimoda are inventors on a Japanese patent application (No. 2022-172252, filed November 27, 2022) related to the method described in this manuscript. FundingThis study was supported by Korea-Japan Joint Government Scholarship Program for the Students in Science and Engineering Departments, the Korean Scholarship Foundation, and the University of Tokyo Foundations Support Fund for International Students.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

The global spread of invasive insects poses serious ecological and economic threats to forest ecosystems. Euwallacea fornicatus and E. perbrevis are cryptic ambrosia beetles native to Southeast Asia that have invaded multiple regions worldwide, damaging diverse woody hosts through gallery formation and fungal symbiont inoculation. We compiled confirmed and novel occurrence records to describe their global distributions, reconstruct invasion histories and likely origins using mitochondrial COI phylogenies, and compare their potential distributions through models based on bioclimatic variables. Euwallacea fornicatus has expanded rapidly over the past decades, establishing in North America (2003), Israel (2009), South Africa (2016), South America (2020), Australia (2021), Europe (2022) and Turkey (2024). In contrast, E. perbrevis has an earlier but slower invasion history, with establishments in Hawaii (1918), Central America (1979), Oceania (1982), and North America (2004). Phylogenetic analyses revealed at least six independent introductions for each species. Euwallacea fornicatus primarily originated from native populations in China, Taiwan and Vietnam, whereas E. perbrevis from Indonesia and Thailand, with additional introductions from unknown sources. Secondary spread from invaded regions is also likely. Distribution models indicated distinct climatic niches. Euwallacea fornicatus tolerates broader thermal ranges and drier conditions, enabling establishment from subtropical to temperate regions, whereas E. perbrevis appears restricted to tropical climates. Only 32% of predicted suitable habitat overlapped, indicating low coexistence potential. The broader climatic tolerance and faster recent spread of E. fornicatus highlights a higher invasion risk and greater management challenges. These findings provide key insights to strengthen biosecurity strategies aimed at preventing further spread. Key messageO_LIWe updated the global distribution and likely invasion routes of two cryptic ambrosia beetles. C_LIO_LIWe identified multiple independent and secondary introductions worldwide. C_LIO_LIWe revealed distinct climatic niches and limited habitat overlap. C_LIO_LIWe showed higher invasion risk for E. fornicatus due to broader climatic tolerance. C_LIO_LIWe identified high-risk regions for surveillance based on climate suitability. C_LI

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

The tropical house cricket, Gryllodes sigillatus, is a mass-produced insect that is used as a protein source for pets and livestock. However, intensive mass-rearing conditions, coupled with high genetic relatedness, create an ideal environment for the spread of pathogenic microbes that severely impact production. Cricket iridovirus (CrIV) is a pathogen that impedes cricket growth and causes significant losses for cricket farmers. Interestingly, recent studies have shown that CrIV is often present asymptomatically, yet the molecular basis of the emergence of disease symptoms remains unknown. To address this, we sampled healthy and diseased crickets and examined differences in cricket and CrIV gene expression via RNAseq. Using differential gene expression analysis and functional enrichment analysis, we found significant differences in host and viral gene expression between healthy and diseased crickets, including genes involved in immunity. Interestingly, while we observed high CrIV gene expression across the entire CrIV genome in sick populations, healthy asymptomatic populations showed elevated expression at a single viral locus. Our results shed light not only on the cricket immune response to CrIV infection but also identify a viral gene that is highly expressed during covert infections, suggesting its potential role in suppressing the hosts immune response. These findings enhance our understanding of how CrIV interacts with our cricket host, providing essential insights for developing targeted strategies to manage CrIV outbreaks in cricket mass-rearing facilities.

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

Identifying the sensory cues that enable insects to find host plants, and understanding the neurobiology underlying their selection, provide solid foundations for developing state-of-the-art pest management strategies. Our work was aimed at identifying the main sensory cues attracting the leaf-cutting ant Acromyrmex ambiguus to alternative host plants in commercial willow plantations in the Lower Delta of the Parana River (Argentina), with a focus on native plant species. Eight plant species were selected and evaluated as potential hosts under field and laboratory conditions, allowing the establishment of a robust hierarchy of preference based on individual and collective behaviour. As a result, Senna corymbosa emerged as the most preferred species, whereas Blepharocalyx salicifolius was the least preferred. Video analyses of ant foraging in controlled indoor nests revealed a sequential decision-making process underlying plant preference and consumption. This included an initial approach driven by olfactory cues, followed by a second step involving contact-dependent cues that elicited leaf-cutting and carrying the leaf fragments to the nest. Volatile compounds and leaf cuticular components potentially involved in plant preference were identified. In addition, physicochemical analysis of both plant species - including total sugars, organic matter, polyphenols, leaf hardness, total proteins and lignin-revealed differences, particularly in polyphenol content, which may contribute to preference patterns. These findings provide insights into the sensory ecology of host preference and inform management strategies based on the reintroduction of native plants as alternative resources in willow plantations, potentially reducing pesticide use and promoting environmental sustainability. Summary statementThis work shows how leaf-cutting-ants rely on olfactory and contact cues, sequentially, for foraging decision-making of plant species. Thus, revealing how sensory cues shape their foraging decisions.

Evidence-based decision making on malaria vector control strategies increasingly rely on triangulation of data which requires informatics systems that can integrate data from complex, multi-stage studies involving mosquitoes. This manuscript describes a performance evaluation of an extended version of the generic schema underpinning the VBDs360 platform, specifically improved to accommodate multiple distinct entomological assays spanning the field, insectary and laboratory. The utility of this extension, with respect to high-fidelity data linkage and robust sample traceability across complex entomological workflows, was evaluated through a case study conducted in southern Tanzania. Wild female mosquitoes were collected from 40 locations across a >4,000 km{superscript 2} area and then reared through multiple generations in an insectary before derived iso-female lineages were tested for phenotypic susceptibility to a pyrethroid insecticide. Such multi-generational lineages (F to F where n [≥] 2) were propagated to prevent non-heritable maternal effects on phenotype and produce enough progeny for standard WHO susceptibility assays. All samples were subsequently archived in a molecular laboratory, where all F specimens were tested for sibling species identity. A paper-based implementation of the extended schema enabled successful integration of 77,017 lines of data distributed across 6 different tables that spanned 3 distinct field, insectary, and laboratory workflows, implemented by three different teams working in different locations. At each step, fully independent and redundant primary and secondary keys enabled high fidelity error correction and sample tracing. Consistently perfect linkage between assay design and sample sorting data was achieved for F0 wild-caught adults, with 100% of 66,108 record successfully linked between field capture and morphological categorization. This complete traceability extended to the propagation of derived Fn lineages, with all 100 and 243 records from 9 adult-derived and 13 larval-derived lineages, respectively, correctly linked. Insecticide susceptibility phenotype further confirmed 100% linkage for 5,654 records between exposure history and recorded mortality outcome data in the insectary. Although such cross-cleaned linkages to sample analysis and storage data recorded by the laboratory team were not entirely perfect and could be improved, they were nevertheless of very high fidelity (97.3% (1967/2,022) for F0 samples and 99.3% (437/440) for Fn samples). Overall, this pilot application of the extended generic schema ensured robust data provenance and minimized transcription errors in this complex study distributed across multiple teams and locations. These findings demonstrate how this generic informatics framework may be scaled and adapted to support data integrity across diverse, large-scale, multi-team entomological research workflows.

Xenoglossa cressoniana, also known as Tetraloniella cressoniana or Xenoglossodes cressoniana, is a eucerine bee known mainly from the US Great Plains. The species was described from a female collected somewhere in Texas in the early 1900s. Here, we report rediscovery of this species in Texas after over a century with no intervening observations. While surveying north Texas ranches, we collected six specimens, including both males and females, at four sites northwest of the Dallas-Fort Worth Metroplex. Xenoglossa cressonianas range, the Great Plains and parts of the deep South, covers a large proportion of the United States. The southern and northern Great Plains, and deep South, have been historically overlooked by most bee researchers. Our results show the urgent need to increase data from under-sampled regions, even within a heavily sampled country such as the US.

Introduced to Hawaii in 2016, Lema equestris has become a garden pest commonly reported on Solanum americanum, which is grown as a native and cultural plant in Hawaii and supports native vertebrates elsewhere across Oceania. Originally identified as L. solani, the species was later found to have been misidentified. Here, molecular and morphological evidence is used to discriminate Hawaiian specimens from L. solani and support the updated identification of L. equestris. As a new invasive species, it is important to confirm host associations and determine whether it will prey on important species, such as endemic or endangered plants, in its new range or any potential range to which it could spread. To this end, feeding assays were performed with adults, first-instar larvae, and newly hatched naive larvae on 11 potential hosts, comprising mostly Solanum species: Solanum americanum, potato, tomato, tomatillo, poha (gooseberry), chili pepper, eggplant, tobacco, tree tobacco, cabbage, and Brazilian nightshade. While feeding was attempted on cabbage, poha berry, and Brazilian nightshade, no host besides S. americanum supported survival. Rearing was used to further characterize the biology and life history of L. equestris, including instar length and distinctive morphological traits for identifying each life stage. While many basic biological traits are confirmed here, much remains to be studied to better understand this species and why it has begun to spread.

Over the past decade, the global rise in invasive species has accelerated at an unprecedented rate, intensifying threats to ecosystems, human health, and economies worldwide. Newly invasive taxa, such as Tropilaelaps mites, are of particular concern for apiculture and agroecosystems. Despite growing concern about the spread of Tropilaelaps mites and other arthropods, limited resources are available to assess their invasive potential. We characterized 118 invasive arthropod species using available literature to identify key biological and ecological traits associated with invasive potential. We developed predictive generalized linear mixed models (GLMMs) to determine the traits most important for predicting invasive potential (number of invaded regions), and the top-performing models were subsequently applied to Tropilaelaps mercedesae. Several traits were identified as significant predictors of invasiveness, including the degree of human association, resilience at small population sizes, diet breadth, maximum annual number of generations, altitude range, and the interaction between human association and temperature range. Notably, T. mercedesae was predicted to be capable of invading 160 regions, ranking it within the top 10% most invasive species among those evaluated (12th out of 119), ranked just below the cosmopolitan Varroa destructor mite. These findings position T. mercedesae as a high-risk, yet under-recognized, invasive threat. Collectively, this demonstrates the power of predictive trait-based modeling to inform invasion risk prior to widespread establishment and underscores the urgency of reallocating resources toward surveillance, research, and proactive management strategies rather than relying on costly, often ineffective post-establishment eradication.

Biting behavior is an important natural defense mechanism in honeybees (Apis mellifera) against Varroa destructor. Significant variation in this behavior exists across genetic lines of honeybees, with certain colonies exhibiting higher mite-biting activity than others. Selective breeding for enhanced biting behavior provides a promising strategy for sustainable mite control and colony resilience. However, successful implementation of such breeding programs requires a comprehensive understanding of the genomic mechanism underlying this trait. In this study, RNA-seq analysis of mandible transcriptomes of 1-day and 8-day old worker honeybees from high mite biting (HB) and low mite biting (LB) colonies were performed. A total of 9,345 genes (97.30%) showed a significant differential expression between LB and HB honeybees across different ages (one-way ANOVA, FDR < 0.05). Comparison of LB vs. HB workers collected on day 1 detected 166 down-regulated and 403 up-regulated genes, whereas workers collected on day 8 identified 82 down-regulated and 46 up-regulated genes. Furthermore, the Weighted Gene Coexpression Network Analysis (WGCNA) exhibited the brown and yellow modules with significantly higher expression in HB compared with LB on both day 1 (FC = 1.52, FDR = 0.0019 and FC = 1.47, FDR = 2.7 x 10-4, respectively) and day 8 (FC = 1.27, FDR = 0.056 and FC = 1.16, FDR = 0.073, respectively). Gene Ontology (GO) enrichment analysis identified over-representation of biological processes involved in muscle contraction, chitin binding, neural signaling, oxidative phosphorylation, neuron development, electron transport chain, mitochondrial ATP synthesis, stress response, sensory perception and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis identified significant enrichment of various pathways including oxidative phosphorylation, cytoskeleton-related pathways, carbon metabolism, motor proteins, ribosome-associated pathways, and citrate cycle (TCA cycle). The present findings demonstrate mite-biting behavior is associated with coordinated activation of neural, energetic and muscular system rather than a single molecular mechanism. These findings provide a basis to improve honeybee health, enhance resistance to Varroa and other ectoparasite, and eventually support sustainable beekeeping and agricultural pollination systems.

The multivoltine non-native box tree moth, Cydalima perspectalis (Lepidoptera: Crambidae), was accidentally introduced in several continents and exhibited a very rapid expansion in its invaded ranges, colonising around 40 countries in less than twenty years. Initially observed in urban areas, it quickly spread to forest stands, causing significant damage to boxwood trees. If the role of the ornamental plant trade in its fast invasion has been investigated, the insects flight capabilities has thus far been overlooked, limiting estimations of its natural dispersal. In this context, we assessed the flight performance of the adults and investigated potential effects of sex, age and generation using computer-monitored flight mills. Under these controled conditions, we estimated the distance flown per night and over the adults lifespan in both sexes for each generation. The adults were able to fly on average 18 km within their lifespan, even though distances flown were highly variable, including several females capable of performing long-distance flights (up to 150 km). The distances covered were partly correlated with the age and body mass of the adults. Mated females flew longer distances than virgin ones, but long dispersal seemed to limit their fecundity. Finally, the overwintering generation presented the highest flight capabilities with individuals able to cover 22 km on average while the late summer generation covered only 10 km. The box tree moth showed good dispersal abilities that likely played a significant role in its rapid local expansion, while human-mediated dispersal favoured its long-distance dispersal.

Nociception, the capacity to detect tissue-damaging stimuli such as noxious chemicals or high heat, is increasingly studied across insect orders and life stages, informing our understanding of its adaptive value and molecular mechanisms. The black soldier fly (Hermetia illucens; Diptera: Stratiomyidae) is widely recognized as the star of the growing insects as food and feed industry. Black soldier fly larvae (BSFL) are reared at high densities that, combined with their extraordinary metabolism, can generate lethal overheating on farms. Data on the thermal nocifensive capabilities of BSFL could inform our understanding of larval behaviors during overheating events (and potential welfare impacts of thermal slaughter), and provide a comparative datapoint to the well-studied vinegar fly (Drosophila melanogaster; Diptera: Drosophilidae). Accordingly, we adapted global and local thermal nociception methods from larval vinegar flies for use with BSFL. We find that global assays (akin to boiling) adapt easily to both first and sixth instar BSFL, that BSFL exhibit slightly different nocifensive behaviors than last instar vinegar fly larvae, and that BSFL have higher thresholds (thrashing begins at 39.70 {degrees}C in last instar BSFL versus 26.6 {degrees}C in D. melanogaster). In contrast, the classical local nociception assay did not adapt easily to BSFL; a modified version generated a unique, gradual pattern of increasing responsiveness to the probe in sixth instar BSFL (>95% responsiveness above 66 {degrees}C) rather than the sharp cutoff in responsiveness at 52 {degrees}C demonstrated in D. melanogaster. Altogether, these protocols open the door for standardized research on BSFL thermal nociception for fundamental and applied purposes.

Conophthorus coniperda (Coleoptera: Curculionidae; Schwarz), known as the white pine cone beetle, is a pest in white pine seed orchards. The insect was primarily studied in the United States of America during the late 1960s to 1992. No contemporary studies have been performed since, despite the devastating damage the beetle causes to white pine seed orchards, and the seeds they produce for reforestation purposes. To help future research on potential biological control, this work revisits the ontogeny of the white pine cone beetle. The biology of the insect was studied over two years in 2022 and 2023 in a seed orchard (Quebec, Canada). Observations were complemented with data collected from the same orchard and other sites in 2009 and in 2012. Except in 2022, the emergence of the insect occurred around 53.6 {+/-} 1.98 {degrees}C.d above a threshold of 6.5 {degrees}C. A shorter developmental cycle was observed compared with the ones described in 1965 and 1976. The relationship between cone size and the number of insects per cone was statistically significant but explained only a small proportion of the variance and showed high variability. These results will help improve survey timing and sampling strategies for this species.

Honeybees face increasing threats from biotic stress of viral pathogens that can severely impact colony health and contribute to global colony decline. However, comprehensive studies of biotic stress and composition of bee viruses across different environmental contexts and seasons remain scarce. This study aims to characterize and compare the diversity, abundance and composition of the Apis mellifera L. virome across three different landscapes (conventional, organic, and roadside) and seasonal gradients (early vs. late season) to better understand how environmental and temporal factors affect viral communities in honeybees. A. mellifera were collected from three different habitats (conventional farm, organic farm, and roadside habitat) during the spring and summer of 2024. Total RNA was extracted individually from whole honeybees and mRNA libraries were prepared, which were subsequently used for sequencing on an Illumina NovaSeq X Plus platform using paired-end 150 bp reads. Several bacteriophages, putative novel viruses, plant-, insect- and bee-associated viruses were detected in the honeybee viromes including Sacbrood virus, Black queen cell virus, Deformed wing virus-B (previously known as Varroa destructor virus-1) and Deformed wing virus. Furthermore, both habitat types and seasons influence viral abundance as majority of detected viruses showed higher abundance in the conventional farm and late season samples. The present findings provide novel insights into the ecological and seasonal dynamics of honeybee-virus interactions and contribute to strategies for improving honeybee health and resilience.

Xylosandrus crassiusculus (Motschulsky), the granulate ambrosia beetle, was one of the first highly-destructive ambrosia beetles introduced into the southern U.S in the 1970s where it was found in South Carolina (Kovach 1986). The Redbay ambrosia beetle, Xyleborus glabratus Eichhoff, was first detected in the U.S. in South Georgia in 2002. This beetle and its associated fungi, the laurel wilt fungus Raffaelea laurelensis and others have caused substantial destruction to native redbay (Persea borbonia) in GA, SC, FL and elsewhere. This beetle-pathogen complex also poses a threat to commercial avocado production in the U.S., Central and South America as well as to valuable other Persea spp. and related plants (Laureacea) that are known hosts. As an addition here, 10 years of the spring appearances (Fig.1) of X. crassiusculus in North Florida is offered for future comparisons. A second unusual appearance is the finding and working with UV mulch and ethanol, as a surprising attraction of X. crassiusculus and other ambrosia beetles including X. glabratus. It was also found that the ambrosia beetles do not respond to yellow and green as expected by most. Also, adding burlap was found to be attractive (increases dead and dying appearing trees) as is silver metallic like UV mulch, while camouflage (camo) was found to work like yellow and green. These occurrences led to the invention and development of UV mulch with new traps to better monitor ambrosia beetles. New traps led to new uses for yellow, green and camo to monitor and decrease damage and losses from ambrosia beetles. The data are presented as evaluated and appear in the figures, discussion and a supplemental section. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733798v1_fig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1006101org.highwire.dtl.DTLVardef@1e0a3d6org.highwire.dtl.DTLVardef@1244d1borg.highwire.dtl.DTLVardef@423cb7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Relative timing of annual emergence of Xylosandrus crassiusculus in north Florida. Collected over 10 years using 5 Baker traps with a 10% ethanol/water solution. Data are from years as marked. Note: data from year 2003 was not collected. C_FIG

Seasonal variation in day length provides a reliable cue that allows insects to anticipate upcoming environmental challenges. Here, we demonstrate that photoperiod induces pronounced, sex-specific immune priming in the linden bug Pyrrhocoris apterus. Females exposed to short-day, diapause-inducing conditions exhibited broadly enhanced immune activity compared with long-day females, whereas immune parameters in males were largely unaffected by photoperiod. Short-day females showed increased immune cell abundance, elevated expression of immune-related genes, enhanced humoral immune activity, and increased resistance to bacterial infection. Importantly, photoperiod-induced immune priming depended on a functional m-cryptochrome gene, linking seasonal immune regulation to the photoperiodic timer. Consistent with laboratory results, females collected under natural short-day conditions also displayed enhanced immune parameters despite increased environmental variability. Together, our findings identify photoperiod as a key regulator of immune preparedness in female insects and reveal a sex-specific anticipatory immune strategy associated with seasonal timing.

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to pesticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including a splice-junction target site. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes, likely a consequence of reduced cleavage efficiency due to inefficient gRNA production. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings highlight that the approach used for multi-gRNA expression, target site choice and sex-specific germline environments can impact gene drive efficiency, and emphasize the need to optimize construct design within the biological context of the target species.

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the genes encoding TuPDS and TuLCPS markedly reduced orange pigmentation as well as {beta}-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene restored reproduction even under diapause-inducing conditions and selectively reduced TuPDS and TuLCPS protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.