Insect Biochemistry and Molecular Biology

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

The black soldier fly (Hermetia illucens, Diptera) undergoes an atypical metamorphic program in which a distinct non-feeding prepupal instar precedes pupation, but its developmental and molecular basis remains poorly understood. Here, we investigated this unusual metamorphic program through integrated developmental and RNAi-based analyses. Postembryonic staging confirmed that the 6th instar feeding larva molts into a non-feeding 7th instar prepupa, whose hardened cuticle subsequently serves as the puparium during intra-puparial development. Expression profiles of four key metamorphic genes revealed a stage-specific Chinmo/Kr-h1-Br-c-E93 regulatory shift corresponding to larval, prepupal, and pupal/adult development, with sustained Br-c expression defining the 7th instar prepupal stage. RNAi-mediated knockdown of Kr-h1 and/or Chinmo induced precocious larval-prepupal metamorphosis, supporting their roles in larval stage maintenance, whereas depletion of Br-c or E93 disrupted prepupal-pupal transition and adult differentiation, respectively, consistent with their functions as pupal and adult specifiers. These results support generally conserved functions of the metamorphic gene network while indicating the absence of a repressive effect of Br-c on E93 in H. illucens prepupae. Together, these findings establish the 7th instar prepupa as an independently regulated transitional stage, providing insight into how metamorphic programs are reorganized to diversify life-history strategies in holometabolous insects.

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

Background and AimsPlants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. MethodsLarvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC- qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key ResultsLarvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. ConclusionsInsect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Issue SectionOriginal article

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Rhodnius prolixus is a primary insect vector of Trypanosoma cruzi, the causative agent of Chagas disease, a neglected parasitosis endemic in Latin American countries. It has been estimated that Chagas disease affects 7-8 million people worldwide and is responsible for approximately 1000 deaths per year. Genetic and molecular studies in this species remain challenging due to its life cycle and feeding habits, thus hindering the development of new strategies to control their populations and reduce the diffusion of Chagas disease. Recently, two stable cell lines - RPE/LULS53 and RPE/LULS57 - were derived from Rhodnius embryos, which represent promising new tools to investigate the genetics of this insect vector. Here, we describe their gene expression landscapes through transcriptomic approaches. We show that 8,968 expressed genes are shared between the two cell lines, whereas 391 and 1,088 genes are uniquely expressed in RPE/LULS53 and RPE/LULS57, respectively. Although key components of primary developmental, immune and redox signaling pathways are expressed in both cell lines, some genes such as Frizzled-10-a-like and catalase show marked differences in expression. Our results strongly suggest that RPE/LULS53 and RPE/LULS57 likely represent two different cell phenotypes. Consistent with this, gene ontology analysis reveals that RPE/LULS53 is enriched for animal organ morphogenesis and stress response, while RPE/LULS57 for DNA-directed RNA polymerase activity, among others. Despite these differences, both cell lines express comparable levels of transcripts from resident transposable elements, including the highly abundant Mariner and LINE/I elements, as well as horizontally transferred transposons. Our findings shed light on the nature of the RPE/LULS53 and RPE/LULS57 embryo-derived cell lines and provide valuable transcriptomic resources for future genetic and functional studies in Rhodnius and other triatomine insect vectors. Author summaryRhodnius prolixus is a blood-feeding insect and a major vector of Chagas disease, a parasitosis endemic in Latin America and affecting millions of people worldwide. In the absence of effective drugs and vaccines, the control of the insect population represents a promising strategy to reduce the diffusion of the disease. Yet, genetic and functional studies in Rhodnius are extremely challenging due to its feeding habit and life cycle. To overcome these limitations, researchers have previously developed two stable cell lines derived from Rhodnius embryos. In this study, we provide the first characterization of the genes expressed in these cell lines. We found that, while the two cell lines share many expressed genes, each of them also has distinct gene expression patterns pointing to two different cell types with specialized functions. These differences likely affect the way they respond to stress and regulate biological processes. Our findings provide an important resource for researchers studying Rhodnius prolixus and other insect vectors, helping advance our understanding of the genetic and molecular mechanisms that control the insect development and mediate the interactions between insect vectors and the parasites they transmit

Adult mosquitoes rely on plant-derived sugars for survival, reproduction, and flight, yet the plant taxa that mosquitoes encounter in nature and the odors that make those plants attractive remain poorly understood. Most studies of mosquito attraction to plant odors have focused on candidate plants selected a priori, rather than plants linked to field-collected mosquitoes. Here, we combined plant DNA barcoding, semi-field behavioral assays, and volatile profiling to identify field-associated plant resources relevant to Aedes aegypti. Plant DNA recovered from mosquitoes collected across three Florida counties revealed broad plant associations, including 90 genera spanning 37 families, with several taxa recurring across counties or appearing prominently within particular localities. Behavioral experiments in semi-field sticky-trap assays found that five field-associated plant taxa were significantly attractive relative to blank controls, indicating that taxa associated with mosquitoes in nature can also function as attractive cues under semi-field conditions. GC-MS analyses of headspace collections from 42 plant taxa detected 211 volatile compounds and revealed substantial variation in both total emission rate and odor composition among taxa. Although several compounds, including -pinene, limonene, 4-ethylacetophenone, 2-ethyl-1-hexanol, 4-ethylbenzaldehyde, and caryophyllene, were broadly distributed across plant groups, volatile profiles differed significantly among taxa and shared compounds often occurred at markedly different proportional abundances. The five behaviorally tested taxa likewise showed both overlap and divergence, sharing 17 compounds across all five taxa while differing in dominant constituents and total emissions. Together, these results show that Ae. aegypti interacts with a diverse set of plants in the field, and suggests nectar-seeking is shaped not simply by plant identity or total odor abundance, but by the composition and proportional structure of plant odors.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

Sleep is a ubiquitous, yet highly variable, behavior across species. The duration and timing of sleep are influenced by ecological demands and dietary context. In the mosquito Aedes aegypti, a blood-feeding insect with specialized nutritional requirements, the relationship between feeding and sleep remains poorly understood. Here, we investigated how blood-derived dietary protein influences sleep regulation. Using postural analysis, videography, and arousal-threshold assays, we established that immobility bouts of greater than 10 minutes reliably define sleep in Ae. aegypti. Mosquitoes lacking the circadian clock gene cycle still maintained daily sleep rhythms but exhibited reduced sleep duration and heightened overall activity. Infrared activity monitoring revealed that blood-fed females showed a marked increase in sleep beginning immediately after feeding and persisting for several days, accompanied by reduced locomotor activity. Notably, this sleep elevation lasted well beyond the cessation of previously reported host-seeking phases, raising the possibility of distinct phases of opportunistic versus targeted host pursuit. To determine the dietary basis of this effect, we tested mosquitoes fed a bovine serum albumin (BSA)-based diet. BSA feeding alone was sufficient to mimic the sleep-promoting and activity-reducing effects of blood, suggesting dietary protein is a major nutritional regulator. Moreover, RNAi-mediated knockdown of the leucokinin receptor (Lkr), which has previously been associated with fluid homeostasis and feeding behavior, resulted in enhanced sleep and reduced activity, implicating mosquito LK signaling in the modulation of postprandial sleep. Together, these findings demonstrate that blood-derived proteins drive sustained increases in sleep and reductions in locomotor activity in Ae. aegypti. This work positions Ae. aegypti as a model for dissecting nutrient-specific regulation of sleep and highlights potential adaptive functions of protein-induced quiescence, such as energy conservation and predator avoidance. More broadly, it provides insight into how specialized diets shape the neural and behavioral architecture of sleep.

Drosophila melanogaster serves as a valuable model system for advancing our understanding of adipose tissue given its analogous organ systems, conserved metabolic, endocrine, and nutrient-sensing functions, and well-established genetic tools. Among the most widely used genetic tools is the Gal4/UAS system. Several Gal4 driver lines are reported to control expression in the D. melanogaster fat body, but secondary expression sites and responses to physiological changes have not been fully characterized. In this report, we describe fluorescent reporter expression for 31 fat body Gal4 transgenic lines in larvae and adults, males and females, and in multiple tissues, brain, indirect flight muscle, gut, ovary, testis, and fat body. This screen highlights expression differences with respect to level, sex, and stage. Moreover, we find that several lines drive expression in tissues that had not been previously described. Together, we present a comprehensive expression atlas of fat body driver lines, serving as a resource to advance adipose tissue research.

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.

The yellow fever mosquito, Aedes aegypti, is a vector of Zika, Dengue, Chikungunya and yellow fever, disease-causing viruses impacting millions of people annually across the globe. Female mosquitoes transmit pathogens through serial blood-feeding, while both male and female mosquitoes feed on plant resources. Contact chemosensation (taste) guides these two feeding modes in both positive ways, feeding readily on sugar in nectar or ATP in blood, and negative ways, demonstrating aversion to chemically diverse bitter compounds in blood or sugar meals. Here, we identify a receptor, AaegGr14, that is expressed in neurons in the labellum and cibarium of the mosquito. Activation of Gr14-expressing neurons causes reduced feeding, while Gr14 and Gr14-expressing GRNs are required for aversion to bitter compounds during nectar- but not blood-feeding. This work provides a molecular and cellular on-ramp to understand bitter taste in mosquitoes and establishes that there are feeding context-dependent differences in how taste mechanisms influence blood- and nectar-feeding. Understanding the molecular and cellular basis of bitter taste in mosquitoes during nectar- and blood-feeding will help inform vector control strategies and elucidate shared principles and unique aspects of insect taste systems.

BackgroundAttractive Targeted Sugar Baits (ATSBs) are a promising, environmentally friendly approach for mosquito control, but the direct field application, scalability and long-term effectiveness of ATSBs across diverse mosquito species remain significant challenges. Methodology/Principal FindingsWe assessed the efficacy of a genetically engineered RNA interference (RNAi) yeast strain (Sh.463_56.10R) formulated in three sugar baits, soda (Coca-ColaTM), 10% sucrose, and a commercial mosquito bait (BaitStabTM), on Aedes, Anopheles and Culex mosquitoes. All RNAi yeast bait formulations induced significantly higher mean mortality (87-100%) compared to the control groups (0-9%; P<0.0001), but mosquitoes exhibited a higher feeding preference for RNAi yeast-soda baits, which induced mortality rates of 94-100% (P < 0.0001) recorded across all mosquito species. Additionally, to assess the competitiveness of the RNAi yeast-soda bait to other tropical sugar sources, semi-field choice assays were conducted in Trinidad, West Indies using competing flowering plants and fruits typically found in residential environments. The RNAi yeast-soda ATSB continued to perform well in the presence of competing floral and fruit sugar sources during both Aedes albopictus and Culex quiquefasciatus trials, though the presence of several fruits and flowers did reduce A. aegypti mortality, suggesting that further field testing will be necessary. The residual activity of the Sh.463_56.10R + soda formulation was retained for at least 14 months, with sustained 100% mortality in C. quinquefasciatus and 93-100% mortality in Aedes aegypti, Anopheles gambiae and Anopheles stephensi. The RNAi yeast-soda ATSB also performed well in semi-field studies performed with a prototype soda bottle feeder. Conclusions/SignificanceThis study demonstrates the potential of soda-baited RNAi yeast as a potent, long-lasting, and scalable platform for ATSB-based mosquito control as a component of integrated vector management programs. Author SummaryMosquito-borne diseases continue to affect millions of people worldwide, and current mosquito control methods face challenges such as low public uptake, insecticide resistance and environmental concerns. Here we evaluated a new and environmentally friendly approach to mosquito control using ATSBs. We tested genetically engineered species-specific yeast producing RNAi molecules capable of killing mosquitoes that feed on it. We mixed the yeast with three different sugar baits, including soda (Coca-ColaTM), 10% sucrose, and the commercial mosquito bait BaitStabTM formulation, and evaluated how well they worked against different mosquitoes. The results showed that the RNAi yeast mixed with soda was the most effective, killing up to 100% of mosquitoes in laboratory and outdoor tests. The bait remained effective in the presence of many competing natural tropical fruit and floral sugar sources. Remarkably, the bait, which can be delivered in a soda bottle feeder, stayed active for at least 14 months under simulated field conditions. These findings suggest that soda-based RNAi yeast baits could provide a practical, long-lasting and scalable tool for mosquito control and may help strengthen future strategies to reduce mosquito-borne diseases.

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.

The electrophysiological process of auditory transduction in insects remains largely conjecture due to the unknown role of ion channels localised to the cilia, but experimental evidence supports either NompC or Nan-Iav as the auditory mechanotransduction ion channel. Here, we knocked down two key genes that code for the two candidate sound-activated ion channels using dsRNA-mediated RNA interference. We measured sound-evoked activity of the auditory nerve and intracellular electrical currents from the ciliated ending of individual auditory receptors. We found that the sound-evoked nerve activity was reduced in nompC, nan and ift88 knockdown. Using whole-cell patch clamp recordings we found that nompC and nan knockdown resulted in reduced sound-evoked transduction current. Stochastic depolarisations hypothesised to be mediated from one of the candidate mechanotransduction ion channels, either NompC or Nan-Iav, where not affected by knockdown of either channel. The discrete depolarisations are therefore mediated through another unidentified ion channel. We test the hypothesis that discrete depolarisations are graded action potentials that travel toward the soma through noise analysis of the transduction current and analysis of discrete depolarisations to voltage-steps. As a positive control we also knocked down ift88, a protein essential for transporting proteins, including ion channels, along the cilium and found both the transduction current and the discrete depolarisations decreased. Key pointsO_LIInjection of dsRNA decreased RNA of nompC and nan C_LIO_LISound-evoked nerve activity is reduced for RNAi-mediated knockdown of nompC and nan C_LIO_LINompC and Nan both contribute to the transduction current C_LIO_LIThe stochastic discrete depolarisations are not due to NompC or Nan-Iav ion channel but to a third unidentified ion channel. C_LIO_LINoise analysis of the transduction current and the discrete depolarisations suggests they are graded action potentials that travel in the direction of the soma. C_LIO_LIKnockdown of ift88 reduced both the transduction current and discrete depolarisations. C_LI Significance StatementInsects are important to understand, economically, agriculturally and medically. However, we still do not understand fundamental aspects of how insects detect their own body movements, vibrations and sound. These senses are detected by insect chordotonal organs, specialised miniaturised mechanoreceptors that convert movements into electrical signals through specialised ion channels. Previous experimental work has advocated either NompC or Nan-Iav as the mechanosensitive ion channel. Here, for the first time, we reduced the expression of both nompC and nan and measured the sound-evoked transduction current directly from neurons in a specialised auditory chordotonal organ. In contradiction to previous studies, we show that both ion channels contribute to the transduction current and find that electrical signals termed "discrete depolarisations" travel toward the soma.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with poorly understood sublethal effects on insects. Perfluorooctanoic acid (PFOA), one of the most widely distributed legacy PFAS is increasingly recognized for altering organismal physiology beyond traditional toxicity endpoints. Here, we use the fruit fly Drosophila melanogaster as a model to examine how PFOA exposure during larval (juvenile) development reshapes insect life-history progression and metabolic homeostasis. Our studies reveal that at environmentally relevant concentrations (nM to low {micro}M), PFOA induces precocious expression of developmentally-regulated genes and leads to metabolic changes that persist into adulthood. At higher concentrations used to probe mechanism, PFOA accelerates larval development, disrupts mitochondrial membrane potential, and increases whole-organism metabolic heat production - results that suggest altered mitochondrial energetic efficiency. Consistent with this tradeoff, PFOA-exposed larvae that develop faster under permissive conditions exhibit heightened sensitivity to environmental stressors, including elevated temperature and reduced food hydration. Together, these findings demonstrate that PFOA disrupts metabolic and developmental processes in a dose- and context-dependent manner, highlighting sublethal effects that may influence insect resilience under environmental stress. SYNOPSIS STATEMENTHere we describe how PFOA alters the growth, development, and metabolism of the fruit fly Drosophila melanogaster. Specifically, we find that PFOA accelerates Drosophila juvenile growth while also rendering exposed larvae sensitive to environmental stress. These observations suggest that widespread PFOA contamination may impair the developmental fitness of insect populations.

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.

BackgroundSynonymous mutations, once considered neutral, can affect translation efficiency through mRNA folding and splicing, generating codon usage bias. This bias is often linked to genomic GC content, which also influences gene regulation. In the parasitoid wasp Lysiphlebus fabarum, GC content was previously shown to shift between developmental stages, with larvae showing higher GC than adults. Whether this phenomenon is widespread among insects remains unknown. ResultsTranscriptomic data from six insect species spanning Diptera, Hymenoptera, and Lepidoptera was used to compare GC content between expressed genes in larvae and adults. In five species, larval transcripts exhibited higher GC content than adult transcripts. Differential expression analysis revealed that stage-biased genes displayed consistent GC shifts, and orthologous gene families with representatives across species showed particularly GC-rich larval-biased genes in Hymenoptera and Diptera. At the genome scale, modeling in 317 insect species demonstrated an association between parasitic lifestyle and reduced mean GC content in Hymenoptera and Diptera, providing a possible ecological explanation for AT-rich genomes. ConclusionsOur results show that GC content is dynamic across developmental stages, independent of overall genome composition. Stage-specific GC enrichment may reflect adaptive codon usage optimizing translation during energetically demanding life-history stages such as larval development. Furthermore, the association between parasitism and reduced genomic GC highlights how ecological lifestyle might with genome content and evolution. Lastly, this work identifies candidate genes underlying stage-specific GC bias and provides new insights into the interplay between molecular evolution, development, and parasitic adaptation in insects.