No mechanoreceptor potential C (NompC) is a major mechanotransduction channel with an important role in sensing of external mechanical stimuli by insects, which help these organisms to avoid injury and adapt to environmental changes. To explore the biological functions of NompC in Bactrocera dorsalis, a notorious agricultural pest, we successfully generated NompC knockout strains using clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) technology. BdorNompC knockout led to an adult lethal phenotype, with approximately 100% mortality at 3 d after eclosion. Morphological observation revealed that the legs and wings of BdorNompC knockout insects were deformed, while behavioral assays showed that the locomotion was impaired in both adults and larvae, relative to that of the wild-type strain. Moreover, BdorNompC knockout reduced gentle-touch response in larvae. These results suggest that BdorNompC is critical for B. dorsalis survival, and that this mechanosensation channel represents a potential new target for pest control agents. Our findings also represent novel evidence indicating that insect NompC is involved in modulating adult wing and leg morphology.

In insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E) pathways jointly regulate fecundity, but only methyl farnesoate (MF) and ponasterone A exist in mites. Comparative transcriptomic analysis in Panonychus citri showed that E75B was significantly downregulated when exposed to lufenuron. Knockdown of E75B significantly affects the expression of vitellogenin (Vg), Fushi tarazu factor 1 (Ftz-f1) and juvenile hormone acid O-methyltransferase (JHAMT), reducing fecundity in mites. The knockdown of Ftz-f1 produced a more significant effect than the knockdown of E75B, indicating that the ponasterone A pathway positively regulates fecundity in P. citri. After the knockdown of JHAMT, the expression levels of both Vg and Ftz-f1 and fecundity were significantly increased, along with the inhibition of Kr-h1, suggesting that JHAMT was negatively correlated with fecundity in the regulatory network. Knockdown of Kr-h1 inhibited the expression of Vg and Ftz-f1 and fecundity, and whether the drop in fecundity is caused by Kr-h1 or Ftz-f1 is unclear. Subsequent feeding with MF induced Kr-h1 and Vg expression, whereas no significant effects were observed for JHAMT and Ftz-f1. Therefore, the MF pathway stimulates fecundity independently. RNA interference (RNAi) showed that JHAMT and Ftz-f1 inhibited each other, resulting in opposite effects of MF and ponasterone A pathways on steady-state fecundity when either factor changed. Meanwhile, JHAMT knockdown led to increased fecundity, indicating that the stimulating effect of the ponasterone A pathway was greater than the inhibiting effect of the MF pathway, and demonstrating the dominant role of the ponasterone A pathway. Therefore, the interaction between JHAMT and Ftz-f1 may be closely associated with the maintenance of MF-ponasterone A regulatory network homeostasis and is involved in the reduction of fecundity in P. citri induced by exposure to lufenuron.

Previous studies have shown oviposition deterring properties of 8 coconut free fatty acid (CFFA) compounds on fruit flies with different key deterrent components for different species. Here we evaluated oviposition deterrence of CFFA using laboratory 2-choice bioassays against Zeugodacus cucurbitae, determined key-bioactive deterrent compounds, and evaluated their behavioral mode. Unlike other reported fruit fly species, CFFA mixture increased Z. cucurbitae oviposition when directly applied on an oviposition substrate. When tested individually in subsequent tests, 4 compounds (caprylic, capric, oleic, and linoleic acids) significantly reduced the oviposition ("negative-compounds"), 1 compound (stearic acid) had no effect ("neutral-compound"), and 3 compounds (lauric, myristic, and palmitic acids) stimulated the oviposition ("positive-compounds"). The 4-component negative-compound blend was effective at reducing oviposition. However, adding stearic acid to the 4-component blend (5-component blend, 5c) further reduced oviposition. Adding any of the positive-compounds to the 5c resulted in loss of oviposition deterrence, suggesting the 5c as the key deterrent component blend. The blend was also effective in no-choice assays and when applied on cucumbers, a preferred host of Z. cucurbitae. When given a choice, Z. cucurbitae made 48.5% fewer visits, spent 39% less time, and oviposited 88.2% fewer eggs per min on 5c treated pumpkin agar than on control agar, suggesting that the 5c blend has both spatial repellency and contact deterrence. Given that all compounds are registered food additives and generally regarded as safe, this blend has potential application in behavioral control strategies, such as push-pull, to protect host fruit against Z. cucurbitae.

The gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect-specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single-nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV-infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single-nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single-cell level.

Insect lytic polysaccharide monooxygenases (LPMO15s) are newly discovered copper-dependent enzymes that promote chitin degradation in insect through oxidative cleavage of glycosidic bonds. They are potential pesticide targets due to their critical role for chitin turnover in the integument, trachea, and peritrophic matrix of the midgut during insect molting. However, the knowledge about whether and how LPMO15s participate in chitin turnover in other tissues is still insufficient. Here, using the orthopteran pest Locusta migratoria as a model, a novel alternative splicing site of LmLPMO15-1 was discovered and it produces 2 variants, LmLPMO15-1a and LmLPMO15-1b. The transcripts of LmLPMO15-1a and LmLPMO15-1b were specifically expressed in the trachea and foregut, respectively. RNA interference targeting LmLPMO15-1 (a common fragment shared by both LmLPMO15-1a and LmLPMO15-1b), a specific region of LmLPMO15-1a or LmLPMO15-1b all significantly reduced survival rate of nymphs and induced lethal phenotypes with developmental stasis or molt failure. Ultrastructure analysis demonstrated that LmLPMO15-1b was specifically involved in foregut old cuticle degradation, while LmLPMO15-1a was exclusively responsible for the degradation of the tracheal old cuticle. This study revealed LmLPMO15-1 achieved tissue-specific functional differentiation through alternative splicing, and proved the significance of the spliced variants during insect growth and development. It provides new strategies for pest control targeting LPMO15-1.

Antimicrobial peptides (AMPs) are critical components of innate immunity in diverse organisms, including plants, vertebrates, and insects. This study identified and characterized a novel Lepidoptera-specific AMP, named lepidoptin, from the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae). Lepidoptin is a 116-amino acid protein containing a signal peptide and a novel β-sandwich domain that is distinct from previously reported AMPs. Temporal and spatial expression analyses revealed a significant upregulation of the lepidoptin gene in vivo and in cultured SF9 cells in response to pathogens. Molecular docking analysis identified a specific binding cavity. Enzyme-linked immunosorbent assay and binding assays confirmed that lepidoptin can bind to pathogen-associated molecular patterns, bacteria, and fungi. Recombinant lepidoptin exhibited potent antibacterial activity by inducing bacterial agglutination, inhibiting bacterial growth, increasing bacterial membrane permeability, and preventing biofilm formation. Lepidoptin also showed antifungal activity against the entomopathogenic fungus Beauveria bassiana by inhibiting spore germination, increasing fungal cell permeability, and increasing reactive oxygen species. Injection of recombinant lepidoptin into S. frugiperda larvae increased survival after B. bassiana infection, whereas knockdown of lepidoptin by RNA interference decreased larval survival. In addition, lepidoptin showed antimicrobial activity against the plant pathogen Fusarium graminearum by inhibiting spore germination and alleviating disease symptoms in wheat seedlings and cherry tomatoes. This study demonstrates the remarkable dual functionality of lepidoptin in enhancing S. frugiperda immunity and controlling plant pathogens, making it a promising candidate for biocontrol strategies in both pest management and plant disease prevention.

Juvenile hormones (JHs) play a crucial role in regulating development and reproduction in insects. Most insects predominantly synthesize JH III, which typically involves esterification followed by epoxidation, lepidopteran insects use a pathway of epoxidation followed by esterification. Although hemipteran insects have JH III and JH skipped bisepoxide III (JH SB3), the synthesis pathway and key epoxidases remain unclear. This study was conducted on Aphis craccivora, and demonstrated that corpora allata, microsomes, Ac-CYP15C1, and Ac-JHAMT catalyze JH III production in vitro, establishing the pathway of epoxidation followed by esterification. These findings were further confirmed through RNA interference and molecular docking. The presence of JH III and JH SB3 in A. craccivora was identified, and their synthesis pathway was elucidated as follows: Ac-CYP15C1 oxidizes farnesic acid to JH A, followed by methylation to JH III by Ac-JHAMT, possibly providing an epoxidation site on the second carbon for JH SB3. This alteration may significantly contribute to the differentiation and functional diversification of JH types in insects.

Spiders are an abundant group of natural enemies preying on insect pests in agroecosystem. But their potential in biological control has not been fully realized due to difficult mass production. One hindrance is the intense intraspecific aggression in spiders. Neurotransmitters such as serotonin play important roles in modulating aggression. Here, we investigated the regulatory function of serotonin (5-hydroxytryptamine [5-HT]) signaling in the intraspecific aggression in a wandering spider Pardosa pseudoannulata (Araneae, Lycosidae). The aggression was quantified with 5 escalated aggression behaviors as approach, chasing, lunging, boxing, and biting. Virgin (VG) females exhibited higher aggression levels but less 5-HT content than post-reproductive (PR) females. Systemic increase of 5-HT via 5-HT injection decreased aggression, while decrease of 5-HT via RNA interference (RNAi) of the tryptophan hydroxylase gene, increased aggression. The involvement of the four 5-HT receptors were determined via individual or combined RNAi. Co-RNAi of the three 5-HT1 genes increased overall aggression with decreased incidents of approach, chasing, lunging, and increased biting. RNAi of 5-HT1B decreased approach and increased biting, whereas RNAi of 5-HT1A or 5-HT1C did not affect aggression. RNAi of 5-HT7 decreased approach only. Therefore, different 5-HT receptor types contribute to different aspects of the inhibitory effects of 5-HT on aggression and provide several pharmacological targets for manipulating spider aggression. 5-HT injection did not affect spiders' predation on their insect prey, the brown planthopper Nilaparvata lugens. The findings reveal 1 neuronal mechanism regulating intraspecific aggression in spiders and provide an insight in developing aggression suppression strategies for spider mass rearing.

Entomopathogenic fungi have been widely used as the main mycoinsecticide for controlling agricultural and forest pests. The effector molecules of these mycopathogens have evolved to adapt to their hosts. The role of fungal effectors in evading the host immune system in insects remains mainly unclear. We characterized the widely distributed fungal effector necrosis-inducing-like secreted protein 1 (NLS1) in the entomopathogenic fungus Metarhizium robertsii. Our findings revealed the presence of M. robertsii NLS1 (MrNLS1) in host hemocytes during the early stage of hemocoel infection. MrNLS1 knock down (ΔMrNLS1) reduced fungal pathogenicity during infection and altered the expression of host immune genes. The molecular docking results and the yeast 2-hybrid assay confirmed that MrNLS1 interacts with the host defense protein Hdd11. The phylogenetic analysis indicated that Hdd11 is conserved across a broad range of Lepidoptera species. Knock down of hdd11 in Helicoverpa armigera, Bombyx mori, and Galleria mellonella markedly suppressed their immune responses against M. robertsii. However, no significant difference was observed in the mean lethal time between hdd11-knockdown Lepidoptera species infected with ΔMrNLS1 and those infected with wild-type M. robertsii. Therefore, in Lepidoptera insects, Hdd11 is essential for fungal defense. In conclusion, M. robertsii infects Lepidoptera insects by targeting host Hdd11 through its protein MrNLS1, thereby suppressing the host immune response. Our findings clarify the molecular mechanisms underlying fungal infection pathogenesis.

In many animals, drastic changes are observed during sexual maturation characterized by the reproductive system development concomitantly to the sexual behavior ontogenesis. These modifications are under the control of internal and external factors such as food. Sexual maturation requires considerable energetic investment, and diet has been shown to affect reproductive activities in many taxonomic groups, especially in insects and vertebrates. By contrast, diet effects on sexual behavior development remain largely unexplored. To elucidate this aspect, we used the male moth Agrotis ipsilon which undergoes sexual maturation occurring between the third and the fifth day postemergence. During this period, males are sensitive to female sex pheromones and a stereotypical sexual behavior characterized by female-oriented flight takes place. In our study, we compared (1) sex pheromone detection by electroantennography recordings and (2) behavioral response in wind tunnel assays between males fed with different diets found in nature. Compared to standard sucrose diet, males fed with sucrose, fructose, and glucose supplemented with sodium (a mineral element necessary for the locomotor activity in several moths) did not respond better to female sex pheromones but clearly exhibited an earlier behavioral response. Thus, such a diet accelerates the development of sex pheromone-mediated oriented flight, probably by facilitating the central processing of sex pheromone information in male A. ipsilon moths. Our results provide new information on the influence of nutritional intake on the ontogenesis of male sexual behavior in animals.

In this paper, we provide an approach that can simulate the behavior of insects, and the aggressive behavior of fruit flies is shown as an example. The specific workflow is as follows. (1) We obtained high-speed camera video of the fly's aggressive behavior. (2) Based on the high-speed camera video, we generated the key action diagrams for each movement. (3, 4) We used micro-computed tomography imaging to segment the leg exoskeleton models using Amira 6.0. (5) With the Blender software, we optimized the OBJ model. (6) We gave motion properties to the 3-dimensional biomechanical model in Blender. (7) Based on high-speed camera videos and the key action diagrams, we generated a 4-dimensional precision adult Drosophila melanogaster biomechanical model. Our study provides a new approach to study rapid locomotion in insects. In addition, our study provides a new idea for establishment of a 4D database, the design and fabrication of bionic multipedal robots, and the linking of nerve signaling and muscle stretching processes.

The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for "NGG" or "CCN" target sequences in the BmNPV genome, excluding non-recurring and potential targets in the B. mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome-one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild-type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains.

In this review, we show that predatory ants have a wide range of foraging behavior, something expected given their phylogenetic distance and the great variation in their colony size, life histories, and nesting habitats as well as prey diversity. Most ants are central-place foragers that detect prey using vision and olfaction. Ground-dwelling species can forage solitarily, the ancestral form, but generally recruit nestmates to retrieve large prey or a group of prey. Typically, ants are omnivorous, but some species are strict predators preying on detritivorous invertebrates or arthropod eggs, while those specialized on termites or other ants often have scouts that localize their target and then trigger a raid. They can use compounds that ease this task, including chemical insignificance, mimicry, and venoms triggering submissive behavior. Army ants include 8 Dorylinae and some species from other subfamilies, all having wingless queens and forming raids. Dorylinae from the Old World migrate irregularly to new nesting sites. The foraging of most New World species that prey on the brood of other ants is regulated by their biological cycle that alternates between a "nomadic phase" when the colony relocates between different places and a "stationary phase" when the colony stays in a bivouac constituting a central place. Among arboreal ants, dominant species forage in groups, detecting prey visually, but can use vibrations, particularly when associated with myrmecophytes. Some species of the genera Allomerus and Azteca use fungi to build a gallery-shaped trap with small holes under which they hide to ambush prey.

Oviposition preferences of plant-feeding predators remain a complex topic, as such omnivores choose oviposition sites by assessing both plant characteristics and the quality and quantity of nearby animal food sources. Orius predators are omnivores that oviposit endophytically, thus plant characteristics play an important role in their oviposition choices. In this study, we assessed the oviposition and foraging preferences of O. laevigatus and O. majusculus on vegetative and flowering chrysanthemum plants, and assessed the survival of their offspring on differently aged tissues. Our results show a preference of O. laevigatus for young and tender chrysanthemum tissues, where the survival of the nymphs was longer on a plant diet. In contrast, O. majusculus selected older plant parts when laying its eggs, and nymphs did not survive long on any of the plant tissues offered. The foraging activity of Orius females for animal prey (Ephestia kuehniella eggs) did not reveal any specific pattern for either of the two predators. Furthermore, we tested the plasticity of the within-plant oviposition preferences of O. laevigatus, by offering sentinel prey (E. kuehniella eggs) on distinct plant parts. We found that more eggs were laid in older plant tissue when animal prey was offered lower on the plant. Overall, our findings show that oviposition choices of Orius predators are based on a dynamic interplay between plant characteristics, presence of animal and/or floral food sources among other factors, and that differences may well occur between closely related species based on the importance of plant resources in their diet.

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye-staining properties were used to characterize hemocyte types from T. castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2 h), then increased over time (at 4 and 6 h) and eventually returned to the naive state by 24 h post-injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T. castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph in vivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes in vitro, forming capsules to encapsulate and melanize nickel-nitrilotriacetic acid (Ni-NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T. castaneum larvae, offering valuable insights into cell-mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T. castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T. castaneum hemocytes in combating pathogens.

Insulin-like peptides (ILPs) act as crucial reproductive neuropeptides in insects, regulating insect reproduction through the insulin signaling pathway (ISP). Our previous studies have found that the sublethal concentrations (LC and LC) of lambda-cyhalothrin (λCy) could induce severe reproductive toxicity in the lacewing, Chrysoperla sinica (Tjeder), but the toxicological mechanism remains unclear. This study discovered that λCy could inhibit CsILP transcription, leading to a decrease in insulin content and downregulation of C. sinica insulin receptor (CsInR) and C. sinica forkhead box O (CsFOXO) expression in ISP. Interference with CsILP expression resulted in downregulation of C. sinica vitellogenin (CsVg) and decreasing fecundity, while exogenous injection of bovine insulin promoted upregulation of CsVg expression and facilitated reproduction in female adults of C. sinica. Meanwhile, interference with FOXO of ILP downstream transcription factor could lead to downregulation of CsVg, hindering ovarian development and resulting in a decrease in egg production. However, exogenous injection of bovine insulin could remedy the effects caused by FOXO interference. In addition, ILP mediates juvenile hormone and 20-hydroxyecdysone biosynthesis by acting on their synthetic regulatory enzymes and influences the signal transduction of the 2 reproductive endocrine hormones, thereby regulating the reproductive endocrine environment in C. sinica. In conclusion, λCy inhibits CsILP expression, leading to disorder of ISP, leading to the reduced fecundity of C. sinica.

The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles' progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed "direct parental" CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.

The rise of biological invasions threatens biodiversity and food security, with the vespid family, including Vespa soror, being of particular concern. Our study focused on the alarm pheromone components of V. soror. By using gas chromatography-mass spectrometry (GC-MS) chemical analyses, electroantennograms, and field bioassays, we identified 5 compounds-2-pentanol, 3-methyl-1-butanol, 2-heptanol, 2-nonanol (2-N), and isopentyl acetate (IPA)-in hornet sting venom that elicited defensive behavior from hornets. IPA and 2-N also serve as alarm pheromone components in multiple honey bee species that are important prey for V. soror. This shared chemical signaling may allow cross-detection by each species on the other's alarm cues. While it should be advantageous for bees to detect V. soror alarm pheromone, the benefits to V. soror of using IPA and 2-N are unclear. V. soror may manipulate bee behavior, potentially distracting defenders, because they mark victim bee colonies by rubbing their abdomens, which contain their sting glands, at bee hive entrances. Our findings pose new evolutionary questions about the role of manipulation in the arms races.

In the organisms with XX/XY sex chromosomes, Y chromosome is unique to males and plays an important role in male reproductive development. The study of Y chromosome genes will contribute to the development of pest genetic prevention and control technology. In this study, we identified 9 Y chromosome genes in Zeugodacus tau (Diptera: Tephritidae), including gene 16222. Protein structure analysis showed that 16222 was highly similar to odorant binding protein, and thus gene 16222 was named obp-Y. Obp-Y knockout (KO) significantly reduced hatching rate of offspring. Sperm detection results showed that obp-Y KO did not affect sperm number in the testes or sperm transfer during mating. We further examined the storage of sperms in females, and found that sperms in females mating with wild-type males began to transfer from spermathecal ducts to the spermathecae at hour 0 after the end of mating (AEM), and at 0-24 h AEM, the sperm count in the spermathecae gradually increased. However, no sperms were observed in spermathecae of females mating with mutant males at hours 0, 4, 8, 24 and 48 AEM. In summary, this study revealed that Y chromosome gene obp-Y was necessary for the storage of sperms in females. Our findings not only provide theoretical basis for elucidating the function of the Y chromosome, but also offer a molecular target for the genetic control over Z. tau.

Insect herbivores adapt and develop strategies to counteract plant chemical defenses. The aphid Uroleucon formosanum is a serious sap-sucking pest that infests lettuces containing toxic sesquiterpene lactones (STLs). Herein, we employed a combination of genome sequencing and RNA-seq transcriptome profiling to understand the mechanisms underlying phytotoxin tolerance in U. formosanum. We generated the first chromosome-level genome assembly for U. formosanum, with a total size of 453.26 Mb and a scaffold N50 of 33.22 Mb. Comparative genomic analyses revealed an enrichment of signals for positive selection and gene family expansion in immune-related pathways. Specifically, the expanded set of heat shock protein 70 (HSP70) genes showed upregulation after treatment with lactucin, suggesting that they may play a role in the immune response against STLs. The expression of takeout-like genes and cuticle-associated genes was also significantly increased in the lactucin-treated samples. Additionally, 53 cytochrome P450 monooxygenase, 30 carboxylesterase, 19 glutathione S-transferase, 32 uridine diphosphate glycosyltransferase and 63 ATP-binding cassette (ABC) transporter genes were identified in the U. formosanum genome. CYP4C1, CYP6A13 and 7 ABC genes were strongly upregulated in response to lactucin treatment, indicating the involvement of detoxifying enzymes in the tolerance of U. formosanum to STLs. Our findings suggest that the cuticle barrier, immune response and enzyme-mediated metabolic detoxification jointly enhance the tolerance of U. formosanum to phytotoxins and promote its adaptation to host plants. This study presents a valuable genomic resource and provides insights into insect adaptation to plant chemical challenges and future technological developments for pest management.

In addition to preventing precocious larval metamorphosis, juvenile hormone (JH), synthesized in corpora allata (CA), is known to stimulate female reproduction of insects. JH titer is extremely low or absent during metamorphosis, but thereafter rapidly increases in the previtellogenic stage and rises to a peak in the vitellogenic phase. However, the mechanisms underlying the biosynthesis of high levels of JH in adults remain unclear. We found in this study that 12 genes involved in JH synthesis pathway were highly expressed in the CA of adult locusts. By transcriptome analysis and quantitative real-time - polymerase chain reaction validation, a total of 106 evolutionary conserved micro RNAs (miRNAs) and 163 species-specific miRNAs were identified in locust CA. Dual-luciferase assay revealed that 17 miRNAs bound to 10 JH synthesis genes (JHSGs) and downregulated their expression. These miRNAs were expressed in low levels during vitellogenic stage, which was oppositive from that of targeting JHSGs. Six miRNAs including miR-971-3p, miR-31a, miR-9-5p, miR-1-3p, miR-315, and miR-282 were selected for function study. Co-application of agomiRs resulted in significantly decreased levels of targeting JHSGs, accompanied by significantly reduced vitellogenin expression as well as arrested ovarian development. The data suggest that multiple miRNAs expressed synchronously at low levels in the vitellogenic phase, thereby ensuring the high levels of JHSG expression to facilitate JH biosynthesis required for JH-dependent female reproduction. The findings provide important information for deciphering miRNA-messenger RNA modules for JH biosynthesis as well as JH regulation of insect metamorphosis and reproduction.