Insect Science

The global population, which is expected to reach 10.4 billion by 2086, will significantly increase the demand for sustainable food sources. Edible insects such as Tenebrio molitor are promising alternatives because of their nutritional value, low environmental footprint, and suitability for mass rearing. However, the efficiency of industrial production depends on the optimization of reproductive processes. Moreover, T. molitor is also a pest species that contributes to grain loss, highlighting the dual need for strategies that increase reproduction under farming conditions and suppress fertility in pest populations. Neuropeptides, including tachykinin-related peptides (TRPs), are known regulators of metabolism and immunity, but their role in reproduction remain unclear. Here, we investigated whether TRPs are involved in female T. molitor reproduction. Expression analyses revealed strong correlations between TRP, TRPR and vitellogenin (Vg) gene expression, suggesting TRP-mediated stimulation of yolk precursor synthesis. The application of Tenmo-TRP-7 affects basic reproductive parameters, including egg production, follicular epithelium permeability, and terminal oocyte volume. These effects are confirmed by the use of dsRNA directed against the gene encoding TRP precursor. These findings show that TRPs regulate reproduction at multiple levels, positioning them as molecular targets for both enhancing insect farming and developing environmentally safe pest control strategies.

After mating, insects always perform mating refusal behavior, termed post-mating refractoriness, due to physiological restrictions. Male crickets, Gryllus bimaculatus, characteristically exhibit 1-hour post-mating refractory stage, controlled by terminal abdominal ganglion. The molecular mechanisms underlying the male-specific precisely timed refractory stage remain elucidated. Here we show that among 28 neuropeptide precursors expressed in the terminal abdominal ganglion, DH31, myosuppressin, allatotropin, and sNPF exhibited male-specific expression based on RT-qPCR and in situ hybridization. However, RNA interference experiments showed that only knockdown of allatotropin and sNPF changed the duration of the refractoriness. Furthermore, allatotropin and sNPF knockdown influenced functions of male reproductive system by inhibiting seminal fluid secretion from male accessory gland and decreasing sperm storage in seminal vesicles, respectively. Knockdown of their receptors caused similar phenotypes. In conclusion, this study demonstrated the regulation of post-mating refusal behavior and reproductive system by Allatotropin and sNPF signalings in male crickets.

Foraging behavior, as an energy-consuming behavior, is very important for collective survival in termites. How energy metabolism related to glucose decomposition and ATP production influences foraging behavior in termites is still unclear. Here, we analyzed the change in energy metabolism in the whole organism and brain after silencing the key metabolic gene isocitrate dehydrogenase (IDH) and then investigated its impact on foraging behavior in the subterranean termite Odontotermes formosanus in different social contexts. The IDH gene exhibited higher expression in the abdomen and head of O. formosanus. The knockdown of IDH resulted in metabolic disorders in the whole organism, including the impairment of the NAD+-IDH reaction and decreased ATP levels and glucose accumulation. The dsIDH-injected workers showed significantly reduced walking activity but increased foraging success. Interestingly, IDH downregulation altered brain energy metabolism, resulting in a decline in ATP levels and an increase in IDH activity. Additionally, the social context obviously affected brain energy metabolism and, thus, altered foraging behavior in O. formosanus. We found that the presence of predator ants increased the negative influence on the foraging behavior of dsIDH-injected workers, including a decrease in foraging success. However, an increase in the number of nestmate soldiers could provide social buffering to relieve the adverse effect of predator ants on worker foraging behavior. Our orthogonal experiments further verified that the role of the IDH gene as an inherent factor was dominant in manipulating termite foraging behavior compared with external social contexts, suggesting that energy metabolism, especially brain energy metabolism, plays a crucial role in regulating termite foraging behavior. Author summaryForaging behavior plays a key role in collective survival in social insects, as found in termites. Worker termites are responsible for foraging duty and exhibit large foraging areas and long foraging distances, so they need to consume much energy during foraging. It is well established that energy can influence insect behaviors. However, how energy metabolism affects foraging behavior in termites remains unknown. Here, we found that the downregulation of the conserved metabolic gene IDH impaired whole-organism and the brain energy metabolism and further altered foraging behavior, resulting in decreased walking activity but increased foraging success in the termite O. formosanus, which is an important insect pest damaging embankments and trees in China. Additionally, the social context affected brain energy metabolism and obviously changed foraging behavior in O. formosanus, causing a decline in foraging success in the absence of nestmate soldiers and the presence of predator ants. However, the increasing number of nestmate soldiers strengthened social buffering to relieve the negative effect of predator ants on worker foraging behavior. Our findings provide new insights into the underlying molecular mechanism involved in modulating the sophisticated foraging strategy of termites in different social contexts from the perspective of energy metabolism.

Gypsy moth Lymantria dispar (GM) is a polyphagous insect and one of the most significant pests in the forests of Eurasia and North America. Accurate information on GM cold hardiness is needed to improve methods for the prediction of population outbreaks, as well as for forecasting possible GM range displacements due to climate change. As a result of laboratory and field studies, we found that the lower lethal temperature (at which all L. dispar asiatica eggs die) range from -29.0 {degrees}C to -29.9 {degrees}C for three studied populations, and no egg survived cooling to -29.9 {degrees}C. These limits agree to within one degree with the previously established cold hardiness limits of the European subspecies L. dispar dispar, which is also found in North America. This coincidence indicates that the lower lethal temperature of L. dispar is conservative. Thus, we found that the Siberian populations of GM inhabit an area where winter temperatures go beyond the limits of egg physiological tolerance, because temperature often fall below -30 {degrees}C. Apparently, it is due to the flexibility of ovipositional behavior that L. dispar asiatica survives in Siberia: the lack of physiological tolerance of eggs is compensated by choosing warm biotopes for oviposition. One of the most important factors contributing to the survival of GM eggs in Siberia is the stability of snow cover. SummaryWithin the geographical range of Siberian gypsy moth populations, extreme temperatures go beyond the limits of the physiological tolerance of wintering eggs (-29.9 {degrees}C), and their survival depends on the choice of warm biotopes for oviposition.

While the evolution of insecticide resistance is often assumed to come with a fitness cost, there are instances where insect populations that acquired resistance fail to show an evolutionary cost to maintaining the trait in the absence of insecticide. In comparing two populations of tea tortrix moth, Adoxophyes honmai, we found the absence of a cost of resistance but also noted differences in digestive enzyme gene expression. This raised the possibility that insecticide resistance coevolved with enhanced digestive capabilities, potentially offsetting putative costs of resistance. This study explored gene transcript patterns that may influence how traits manifest spatially and temporally, evaluating potential connections between digestion and the costs of resistance. We found that our resistant larvae had constitutively greater transcript levels of multiple putative digestive genes as well as a marker of resistance, CYP9A170. The putative digestive genes were expressed mostly in the digestive tract, whereas the tissue-specific pattern of CYP9A170 expression was strain-dependent. For most genes, the difference in expression between susceptible and resistant larvae remained consistent throughout development. Interestingly, the expression of an ABC transporter was upregulated in response to tebufenozide exposure, but only in the resistant larvae. A comparison of A. honmai transcriptomes suggests that the majority of differentially expressed genes between populations may not be directly contributing to resistance, but rather microevolutionary variations specific to individual populations. Future studies on fitness costs of resistance should consider other physiological systems and their interactions with direct mechanisms of resistance.

Genetic correlations among behavioural traits are often controlled by pleiotropic genes. Many studies suggest the existence of genetic correlations among behavioural traits based on artificial selection experiments in the laboratory. However, few studies have examined whether behavioural correlations in the laboratory are maintained in the field, where natural selection works. Artificial selection experiments showed a behavioural correlation among death feigning, walking movement, and locomotor activity in the red flour beetle (Tribolium castaneum). This study investigated whether this behavioural correlation is observed in wild T. castaneum populations. We also collected beetles from various regions in Japan and investigated the geographic variation in these traits. There was geographic variation in the three behavioural traits. However, these behavioural traits were not correlated. The results suggest that the genetic correlations among behavioural traits are not maintained in the field. Therefore, the results derived from laboratory experiments may be overestimated. The same correlation between traits was not believed to arise in the field, as the indoor results may have been caused by unrealistic selection pressures. Further laboratory and field investigations are both needed.

Intraspecific brood parasitism (IBP), where a parasitic female lays eggs in the nest of another female of the same species, occurs in insects and birds. Also, quasi-parasitism (QP), where a parasitic female copulates with a host male at his nest and lays eggs that are fertilized by the male, has been documented in a few monogamous birds, but QP has not been observed in any insects. Burying beetles, genus Nicrophorus, use small vertebrate carcasses for reproducing and providing biparental care for their offspring. IBP has been observed in one burying beetle by laboratory experiments, but has not been well reported under natural conditions. IBP and QP may occur under natural conditions in burying beetles. Here we focused on a burying beetle, Nicrophorus quadripunctatus. Ten broods, consisting of larvae and their parental female and male, were collected from a deciduous forest. To investigate the kin relationship between parents and larvae, eight microsatellite DNA loci were used. We detected three types of parasitic larvae: 1) larva not related to either its parental female or male, 2) larva not related to its parental female, but unknown regarding its parental male, and 3) larva not related to its parental female, but related to its parental male. These results suggested that IBP and QP can occur with certain frequencies in the reproduction of N. quadripunctatus under natural conditions. QP is thought to have a benefit for a parental male to enhance his paternity within one brood in this species.

Strains of Euschistus heros (Hemiptera: Pentatomidae) with resistance to thiamethoxam (NEO) and lambda-cyhalothrin (PYR), generated by selection with these insecticides in the laboratory, have been recently reported in Brazil. However, the mechanisms conferring resistance to these insecticides in E. heros remain unresolved. We utilized comparative transcriptome profiling and single nucleotide polymorphism (SNP) variant calling of susceptible and laboratory-selected resistant strains of E. heros to investigate the molecular mechanism(s) underlying resistance. The E. heros transcriptome was assembled using approximately 190.1 million paired-end reads, which generated 91,673 transcripts with a mean length of 720 bp and N50 of 1795 bp. Approximately, 54.8% of the assembled transcripts ware functionally annotated against the NCBI nr database, with most sequences (43%) being similar to the pentatomids Halyomorpha halys (43%) and Nezara viridula (29%). Comparative gene expression analysis between the susceptible (SUS) and NEO strains identified 215 significantly differentially expressed (DE) transcripts. DE transcripts associated with the metabolism of xenobiotics were all up-regulated in the NEO strain. The comparative analysis of the SUS and PYR strains identified 204 DE transcripts, including an esterase (esterase FE4), a glutathione-S-transferase, an ABC transporter (ABCC1), and aquaporins that were up-regulated in the PYR strain. We identified 9,588 and 15,043 non-synonymous SNPs in the PYR and NEO strains respectively in comparisons with the SUS strain. One of the variants (D70N) detected in the NEO strain occurs in a subunit (5) of the nicotinic acetylcholine receptor, the target-site of neonicotinoid insecticides. Nevertheless, the position of this residue was found very variable among 5 from insect species. In conclusion, neonicotinoid and pyrethroid resistance in laboratory-selected strains of E. heros is associated with a potential metabolic resistance mechanism mediated by the overexpression of several proteins commonly involved in the three phases of xenobiotic metabolism. Together these findings provide insight into the potential basis of resistance in E. heros and will inform the development and implementation of resistance management strategies against this important pest. HighlightsO_LI419 DE genes were observed in E. heros insecticide-resistant strains C_LIO_LI24,631 SNPs were identified in E. heros insecticide-resistant strains C_LIO_LIE. heros insecticide-resistant strains overexpress metabolic resistance genes C_LIO_LILambda-cyhalothrin-resistant E. heros overexpresses cuticular proteins C_LIO_LIThiamethoxam-resistant E. heros carries the target-site mutation D70N in nAChRalpha5 C_LI

In the era of global warming, much attention has been paid to the possibility that high temperature may influence diverse insects not only directly but also indirectly via effects on their symbiotic microorganisms. The stinkbug Plautia stali develops a midgut symbiotic organ that harbors a specific bacterial symbiont indispensable for its growth and survival. Being maintainable in laboratory and tractable experimentally, P. stali is recently highlighted as a model system to investigate the mechanisms underpinning insect-microbe symbiotic interactions. In this study, we reared newly-emerged adult insects of P. stali under different temperature conditions for 8 days and monitored how their symbiotic organs and symbiotic bacteria are affected. While all insects survived at temperatures from 25{degrees}C to 37{degrees}C, some insects died at 38{degrees}C, 39{degrees}C and 40{degrees}C, wherein mortality rates increased as temperature elevated. While the symbiotic organs of the normal insects exhibited vivid yellow color, the symbiotic organs of the insects reared at 35{degrees}C or higher frequently exhibited abnormal colors such as pale yellow, yellowish white, or white, the extent of which became more severe as temperature elevated. Symbiont quantification revealed that, while the symbiont titers were almost constant for 8 days at 25{degrees}C and 30{degrees}C, the symbiont titers on the 8th day drastically declined to 1/100 at 35 and 1/10000 at 37{degrees}C and 39{degrees}C. Based on these results, we propose that rearing at 37{degrees}C for a week is a recommended treatment regime by which the symbiont is effectively suppressed with minimal damage to the host insect.

Insects have various strategies like mimicry or camouflage to avoid predation. Swallowtail butterfly larvae switch from a black and white pattern mimicking bird droppings to a green camouflage pattern in the fifth (final) instar. This larval pattern switch is regulated during the juvenile hormone (JH)-sensitive period, when JH titer declines rapidly, and clawless (cll), abdominal-A (abd-A), and Abdominal-B (Abd-B) function during this period. However, the molecular mechanism behind the background green color, a crucial aspect of the camouflage pattern, remains poorly understood. Here, we used Papilio memnon, which switches to the camouflage pattern in the fifth instar but is greenish from the third instar, to investigate the mechanism of camouflage pattern formation, particularly the larval green coloration. Through RNA sequencing, we found that BBPs forming a gene cluster are upregulated in the green regions of P. memnon larvae during the fourth instar, whereas P. xuthus larvae, which have not yet turned green, showed minimal BBPs expression. When BBP1 and BBP2, which were particularly highly expressed, were knocked down by RNAi, there was a phenotypic change in green to yellow in both fourth and fifth instar larvae. Expression analysis and knockdown experiments were conducted also for JHBP, which had been previously reported, and confirmed that it is involved in the synthesis of yellow pigment. Furthermore, knockdown of Ubx resulted in no phenotypic change in fourth instar larvae, but in fifth instar larvae, the eyespots pattern characteristic of the camouflage pattern almost entirely disappeared, suggesting that Ubx is also functional only during JH-sensitive period. Our results indicate that the switch from mimetic to camouflage patterns resulted from the function of cll, abd-A, Abd-B, and Ubx prepatterning genes during the JH-sensitive period. And the increased expression of BBPs and JHBPs, independent of the JH-sensitive period, contributed to the development of green coloration.

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera ligustica larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. m. ligustica. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (P < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (P < 0.001), while that in the inhibi- tor-miR-34 group was significantly downregulated (P < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibi- tor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5- and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5- and 6-day-old larval guts were significantly downregu- lated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. m. ligustica larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

Several studies have demonstrated that diapausing insects upregulate glycerol production in response to cold temperatures. However, relatively few studies have investigated whether diapause can independently trigger increased glycerol production. In other words, little research has been dedicated to analyzing whether diapause and glycerol upregulation are linked. If a linkage between these two elements in insects exists, then photoinduced diapausing insects should upregulate glycerol production, even without exposure to cold temperatures. The current study examines whether this linkage exists in the tobacco hornworm pupae (Manduca sexta). Tobacco hornworms will enter a pupal diapause if presented with consistently short photoperiods, most notably 12 hours of daylight followed 12 hours of darkness. Regarding temperature, photoinduction of diapause in the tobacco hornworm is optimal at 26 {degrees}C. Using a spectrophotometric assay, glycerol levels in hemolymph were assessed in non-diapausing pupae shortly after pupation and in diapausing pupae after 16 days of pupation. Interestingly, non-diapausing pupae contained significantly higher glycerol levels in hemolymph compared to diapausing pupae. Furthermore, non-diapausing pupae produced approximately .099 M glycerol and diapausing pupae produced .085 M glycerol. This study provides evidence that glycerol upregulation and diapause are not linked in Manduca sexta, but are rather two separate events with two distinct causes. Nevertheless, the results are valuable since non-diapause specimens produced more glycerol than diapausing specimens, and moderately high concentrations of glycerol were found in both diapausing pupae and non-diapausing pupae.

Trichogramma, a genus of egg parasitoid wasps, are widely used as biological control agents and serve as model organisms in parasitoid research. Despite their significance, the understanding of RNA interference (RNAi) in Trichogramma remains very limited. In this study, we investigated RNAi-associated genes by bioinformatic approaches and experimentally assessed the feasibility of RNAi and the susceptibility of environmental RNAi in Trichogramma. We found that Trichogramma genomes contain a complete set of genes in the RNAi pathway and exhibit extensive gene expansion of dsRNase, which may influence RNAi efficiency by degrading dsRNA. We demonstrated successful RNAi through pupal microinjection in T. dendrolimi Matsumura, providing a technical approach for future gene functional studies. In addition, we observed no evidence of susceptibility to environmental RNAi in either T. dendrolimi adults or larvae, which might be attributed to the extensive expansion of dsRNase. This low environmental RNAi sensitivity in Trichogramma could suggest a reduced risk of RNAi-based pest management strategies affecting nontarget Trichogramma populations. Overall, this study presents a technical approach for conducting gene functional studies in Trichogramma and provides a foundation for evaluating the nontarget effects of RNAi-based pest control strategies on Trichogramma.

The sex determination mechanism for hemipteran species remains poorly understood. During the sex determination of the brown planthopper (BPH), Nilaparvata lugens, one species of Hemiptera, the functions of doublesex (Nldsx) and NlTra-2 (NlTra-2) genes were identified in our previous studies. Here, we identify an upstream gene for Nldsx in the sex determination cascade, NlFmd, which acts as female determinant gene for N. lugens. The sex-specific transcript of NlFmd (NlFmd-F) encodes an arginine/serine-, and proline-rich protein that is essential for female development. The knockdown of NlFmd resulted in the development of pseudomales, with sex-specific alternative Nldsx processing, and maternal RNA interference (RNAi) against NlFmd generates male-only progeny. Moreover, homologous genes for NlFmd have also been identified in two rice planthopper species, the white-backed planthopper (WBPH, Sogotalla furcifera) and the small brown planthopper (SBPH, Laodelphax striatellus), and these genes appear to be involved in the sex determination cascades for these species. Our data suggest that the sex determination cascade in Delphacidae is conserved.

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

Insects are the most abundant described living creatures in the world, and they play important roles in our global ecosystem. Climate change affects global biodiversity, and researchers in many fields are striving to better understand the impact of the climate crisis. One such endeavour is the study of temperature-dependent effects on insects. At present, we know little of how climate affects gene expression in insects of different sexes. Here, we took four species of fruit flies of the genus Drosophila (D. melanogaster, D. virilis, D. pseudoobscura, and D. erecta), and subjected the male and female flies of each species to three different temperatures to test their sex-specific gene expression responses. A total of 144 transcriptomic profiles of protein-coding genes and microRNAs were generated. We found that, at the same temperature, there were more male-biased than female-biased protein-coding genes and microRNAs in all four investigated drosophilid species. Interestingly, upon temperature changes, there were more differentially expressed protein-coding genes in females than in males in all four investigated species, while the microRNAs were highly species- and sex-specific. This study provides the first evidence that sex-biased protein-coding gene and microRNA expression responses to temperature change differ between insect species within the same genus, and demonstrates the complexity of sex-specific responses of insects to climate change. HighlightsO_LIAt the same temperature, protein coding gene and microRNA expression showed a greater bias towards males than towards females in all four tested insect species. C_LIO_LIIn response to increasing temperature, females of all 4 tested species exhibited more differentially expressed genes than did males, and enrichment analyses showed that they are species-specific. C_LIO_LIDifferentially expressed microRNAs did not show a conserved trend between insects upon temperature changes. C_LIO_LISex-specific gene and microRNA expression of insects in response to climate change evidently involves a complex adaptation mechanism. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/591511v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1037b18org.highwire.dtl.DTLVardef@18683f3org.highwire.dtl.DTLVardef@40dee5org.highwire.dtl.DTLVardef@e9a108_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Appropriate hatching time is important for successful brood parasitism in some bird and insect groups. Parental burying beetles typically rear their larvae on carcasses buried in nests; however, they occasionally raise brood-parasitic larvae from eggs dumped by other females. Parasitic larvae must hatch synchronously with host larvae because the host parents indiscriminately accept any larvae during that period. However, parasites in burying beetles are assumed to have more severe restrictions on synchronous hatching than those in birds because of less predictability of reproductive sites, the necessity for ovarian development at the site, and difficulty in adjusting by oviposition. Is the hatching of parasitic larvae synchronized with that of host larvae, even if the reproductive behavior of the parasites lags behind that of the host owing to restrictions? In the burying beetle Nicrophorus quadripunctatus Kraatz, we observed the hatching of host and parasite offspring under experimental conditions in which female parasites (intruders) encountered carcasses that had been secured by female hosts (residents) two days earlier. In 14 of the 40 replicates, defeated intruders dumped eggs, residents laid 36 eggs, and intruders laid six eggs on average. The hatching of intruder offspring was synchronized with that of the resident offspring despite the two-day lag. Egg dumping is regarded as brood parasitism. Notably, the period from contact with carcasses to hatching was significantly shorter for intruders than for residents. Advances in hatching time may contribute to the success of brood parasitism.

The largest insect order, Coleoptera, includes several economically important beetles that also serve as major model species for biological research. Perhaps foremost among these is the red flour beetle, a global pest of stored grains and flour whose genome was sequenced in 2008. However, the currently available reference genome (Tcas5.2) is incomplete, fragmented and contains many gaps, and the Y chromosome is not assembled. Here we present inTcas1, an updated genome assembly and annotation of T. castaneum collected from India, assembled using both short and long read sequencing, and annotated using two transcriptome datasets. We report that inTcas1 has fewer gaps, less fragmentation, and many new genes and new isoforms of previously annotated genes. This new resource provides a useful update, comparison, and reference for new beetle genome assemblies. The first Y chromosome assembly for this species also provides critical data to study the evolution of insect sex chromosomes and sex determination systems. SIGNIFICANCEWe present here an improved T. castaneum genome assembly (inTcas1) for beetles sampled from India, including the first Y chromosome of this important pest and laboratory model species. The new genome should facilitate more comprehensive analysis of Coleoptera genome and transcriptome datasets, especially beetle populations from Asia - the previously available genome, Tcas5.2, was assembled using the GA2 strain from USA. The updated genome should also facilitate analyses of genome evolution, including sex determination and sex chromosome dynamics; and the new gene annotations can expand the genetic toolkit for this beetle.

Mosquitoes within the Culex pipiens complex play a crucial role in human disease transmission. Insecticides, especially pyrethroids, are used to control these vectors. Mosquito legs are the main entry point and barrier for insecticides to gain their neuronal targets. However, the resistance mechanism in mosquito legs is unclear. Herein, we employed transcriptomic analyses and isobaric tags for relative and absolute quantitation techniques to investigate the resistance mechanism, focusing on Cx. pipiens legs. We discovered 2346 differentially expressed genes (DEGs) between deltamethrin-resistant (DR) and deltamethrin-sensitive (DS) mosquito legs, including 41 cytochrome P450 genes. In the same comparison, we identified 228 differentially expressed proteins (DEPs), including six cytochrome P450 proteins). Combined transcriptome and proteome analysis revealed only two upregulated P450 genes, CYP325G4 and CYP6AA9. The main clusters of DEGs and DEPs were associated with metabolic processes, such as cytochrome P450-mediated metabolism of drugs and xenobiotics. Transcription analysis revealed high CYP325G4 and CYP6AA9 expression in the DR strain at 72 hours post-eclosion compared with that in the DS strain, particularly in the legs. Mosquitoes knocked down for CYP325G4 were more sensitive to deltamethrin than the controls. CYP325G4 knockdown reduced the expression of several chlorinated hydrocarbon (CHC)-related genes, which altered the cuticle thickness and structure. Conversely, CYP6AA9 knockdown increased CHC gene expression without altering cuticle thickness and structure. P450 activity analysis demonstrated that CYP325G4 and CYP6AA9 contributed to metabolic resistance in the midgut and legs. This study identified CYP325G4 as a novel mosquito deltamethrin resistance factor, being involved in both metabolic and cuticular resistance mechanisms. The previously identified CYP6AA9 was investigated for its involvement in metabolic resistance and potential cuticular resistance in mosquito legs. These findings enhance our comprehension of resistance mechanisms, identifying P450s as promising targets for the future management of mosquito vector resistance, and laying a theoretical groundwork for mosquito resistance management. Author SummaryCulex pipiens mosquitoes are the primary vector of the filamentous nematode, Wuchereria bancrofti and also involved in the transmission of other pathogens, such as West Nile virus (WNV), avian malarias, and avian pox virus. Insecticides, particularly pyrethroids, continue to be the primary method to control these significant vectors. Worryingly, resistance to insecticides has become widespread and is rapidly intensifying in Culex mosquitoes throughout China, posing a threat to the efficacy of insecticides. Legs are the main sites of contact with ITNs and sprayed insecticides, and the insecticides have to penetrate the leg cuticle to reach their targets.Therefore, the resistance mechanisms in mosquito legs deserve further investigation. Several reports have found a certain amount of P450 in insect legs. Unfortunately, none of the above reports have conducted further functional studies on P450s in the legs. Here, we have identified two P450 enzymes, CYP325G4 and CYP6AA9, through the integrated analysis of transcriptomics and proteomics. CYP325G4 enriched in the cuticle of resistant mosquitoes might possess a dual resistance mechanism involving metabolic resistance and cuticle resistance. CYP6AA9 was slightly different, possibly exerting metabolic resistance as its main function and also being involved in cuticle synthesis. Understanding the dual resistance mechanism of P450s in the metabolism of pyrethroid insecticides will have an important role in optimizing vector control strategies.