The complete sequence of the W chromosome, which carries feminization activity in the silkworm, is crucial for understanding the sex-determination system in Lepidoptera. However, extensive accumulation of transposons due to lack of recombination, the very rare protein-coding genes and almost no information about molecular markers has hindered full W sequencing. We report the first complete silkworm W sequence (T2T_W, 11683305 bp) obtained by combining sequencing-assembly technologies and newly developed error detection methods, evaluated with genetically mapped W-RAPD markers, W-mutants, and W-derived BAC clones. The T2T_W sequence showed that the W is composed of a massive 92% accumulation of transposons and repeat sequences, among which the main constituents are intact LTR/LINE retrotransposons indicating recent expansions. In addition to Fem clusters producing Fem piRNA (Feminizer-derived PIWI-interacting RNA), we found 26 protein-coding genes in the W sequence. These include four gene pairs encoding zinc-finger motifs designated z1:z20 and a gene encoding serine/arginine repetitive matrix protein 1-like (SRRM1-like). To identify candidate genes for female sex-determination and differentiation we also sequenced the shortest W (3.8 Mb) from a translocation mutant with feminizing activity, which harbored four conventional genes: a Fem cluster, a pair of z1:z20 isoforms, z20-S, and a SRRM1-like gene. Phylogenetic analysis revealed that z1:z20 originated from a copy of an autosomal zinc-finger gene pair, z2:z21, translocated onto the W around 2.43 Mya and subsequently amplified to yield 4 W-linked zinc-finger gene pairs. The complete W sequence revealed that large-scale deletions and amplifications played a significant role in W chromosome evolution.

Azadirachtin, a major botanical insecticide, is widely recognized as a sustainable alternative to synthetic pesticides, yet the molecular mechanisms underlying its effects on insect hosts remain poorly understood. Transcriptomic analysis, RT-PCR, and qPCR revealed significant upregulation of Sirt5 in the midgut following azadirachtin exposure. In the present study, we investigated the role of Sirt5, a key regulator of metabolic and stress responses, in mediating midgut damage in Spodoptera litura under azadirachtin stress. Functional assays showed that overexpression of Sirt5 in SpLi-221 cells suppressed lysosomal activity under stress, whereas CRISPR/Cas9-mediated knockout (KO) of Sirt5 led to elevated expression of autophagy-related genes (Atg6, Atg16, Atg101) and the apoptosis marker caspase-1. Compared with the wild-type control group, the mortality of the larvae lacking Sirt5 increased from 49% to 95% when fed with the median lethal dose of azadirachtin. These findings indicate that Sirt5 mitigates azadirachtin-induced midgut damage by modulating autophagy and apoptosis pathways. The present study provides the first experimental evidence linking Sirt5 to host responses against azadirachtin, offering new mechanistic insights into botanical insecticide action and identifying a potential target for enhancing sustainable pest management strategies.

BACKGROUND:Plants have evolved abundant defensive secondary metabolites to resist insect herbivores. Lycorine is an alkaloid with insecticidal activity from Amaryllidaceae plants, which the destructive pest Spodoptera litura naturally avoids. Cytochrome P450 enzymes are central to xenobiotic detoxification in insects, but the mechanism by which lycorine acts against S. litura remains unknown. This study aimed to reveal the toxic mechanism of lycorine focusing on P450-mediated detoxification. RESULTS:Lycorine exhibited substantial toxicity to first-instar S. litura larvae (LD50 = 0.55 μg larva-1). Subsequently, when fifth-instar larvae were exposed to a sublethal dose (LD30) of lycorine, Lyc disrupted metabolic pathways, damaged Malpighian tubules, and induced oxidative stress. Furthermore, lycorine strongly repressed a CYP6AE gene cluster (CYP6AE47, CYP6AE50, CYP6AE70, CYP6AE138 and CYP6AE139) and decreased total P450 activity to 45% in the Malpighian tubules. RNAi co-silencing of these cluster genes increased larval mortality (+30%) under lycorine treatment. Finally, molecular docking and microscale thermophoresis analyses further confirmed direct binding between Lyc and this CYP6AE gene cluster, with the strongest affinity observed for CYP6AE47 (Kd = 518.5 nM). A key residue, ARG170, may be vital for the interaction between Lyc and CYP6AE47. CONCLUSIONS:These results demonstrate that the insecticidal mechanism of Lyc involves suppressing the expression and function of a CYP6AE gene cluster, thereby impairing detoxification capacity, which leads to Lyc accumulation and larval mortality. Elucidation of the detoxification system-targeted mechanism for this plant-derived compound provides a foundation for developing novel, sustainable pest management strategies against S. litura and potentially other noctuid pests. © 2026 Society of Chemical Industry.

Neoseiulus barkeri Hughes, a widely used biological control agent for small pests, relies on its sophisticated olfactory system for key behaviors such as foraging, prey location, and mating. While herbivore-induced plant volatiles (HIPV) are well-studied in other insects and large natural enemies, the olfactory mechanisms of phytoseiid mites like N. barkeri remain largely uncharacterized, hindering advances in biological control. This study investigated the potential function of foreleg tarsal sensory organs of the phytoseiid mites in HIPV recognition. The results showed N. barkeri females and males were both attracted to the full blend and six individual volatile originated from cowpea leaves infested by Tetranychus urticae Koch. N. barkeri females no longer showed odor preferences once tarsi of leg I were excised, but attraction was not affected when tarsi of leg IV were excised. SEM analysis characterized the setae types in the distal part of tarsi in leg I as stout peg-shaped structures without sockets, identified as olfactory sensilla. Excising tarsi of legs did not affect predation or copulation in N. barkeri, but reduced its walking speed and mate-searching efficiency. Expression of three Niemann-Pick type C2 genes in excised tarsi of leg I was significantly down regulated when induced by ocimene. These results suggest that tarsi of forelegs of phytoseiids are involved in the perception of HIPV, as well as in discrimination of odor signals. Our study provides a foundation for further elucidation of effective control strategies against small insect and mite pests with predators and HIPV-based components as attractants or repellents.

Wolbachia is a genus of symbiotic bacteria prevalent in arthropods, with diverse effects on host reproduction and fecundity; however, it is unclear how Wolbachia modulates the host reproductive system. In this study, a novel Wolbachia strain, wSpic, was identified in the Noctuid moth Spodoptera picta and its effect on the reproduction of this host was investigated. We sequenced and annotated the 1,339,720 bp genome of wSpic. We identified a total of five WO phage regions in the genome and found no evidence of any plasmids associated with wSpic. Evolutionary analysis revealed that wSpic belongs to supergroup B and has undergone horizontal transmission between S. picta and Trichogramma pretiosum, a wasp parasitoid of insect eggs. The removal of Wolbachia by antibiotic treatment resulted in significantly decreased fecundity and abnormal development of S. picta ovaries, but no differences in egg hatching rate. An integrated transcriptome and proteome analysis indicated that major molecular pathways for Wolbachia-induced reproduction fitness benefits include its effects on insect juvenile hormone, vitellogenesis, choriogenesis, and nutritional metabolism. Our findings demonstrate that wSpic plays a critical role in promoting ovary development and sustaining fecundity in S. picta hosts.

Bt has been applied as a gene source for insect-resistant transgenic crops, which represents efficient control of insect pests. In this study, we evaluated the pesticidal specificity of one Bt maize strain, DBN9936, that expresses Cry1Ab protein in Spodoptera litura larvae. The results showed that this Bt maize is active against the younger larvae while causing a sublethal effect on older larvae. To further assess the biological responses of S. litura under sublethal Cry1Ab exposure, the relative concentrations of Cry1Ab in different tissues of fifth instar larvae were investigated, indicating that Cry1Ab is transported along the gut and out of the body via excretion or into the hemocoel and lead to sequestration by molting. Furthermore, the result of ultrastructural observation in sublethal Cry1Ab-treated midgut, namely an increase in lysosome number, with the lysosomal activity activation simultaneously provides a strong indication that lysosome plays an active role in response to sublethal Cry1Ab exposure.

BACKGROUND:Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS:After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION:Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

Genome sequences contain the fundamental genetic information that largely determines the biology of a species. Over the past 20 years, advancements in high-throughput sequencing technologies and bioinformatics tools have matured, facilitating genome assembly and ushering in the telomere-to-telomere (T2T) era. Bombyx mori is renowned as a silk-producing insect and serves as an important model organism extensively studied across various fields of biology. In this study, we present the first assembled T2T genome by integrating HiFi, ultra-long ONT, NGS, and Hi-C data. This assembly comprises 450,267,439 base pairs from 28 chromosomes and includes annotations for a total of 18,253 protein-coding genes. A completeness evaluation revealed that 99.1% of conserved single-copy genes were included, as determined by a BUSCO analysis. Furthermore, the consensus quality (QV) assessed through Merqury was recorded at 59.88. The proportion of repeat sequence achieved 60.77%, marking it as the highest reported value for B. mori to date. In comparison to previously published genomes, our assembly offers a more complete and higher quality representation, particularly concerning highly homologous tandem regions such as telomeres, rDNA clusters, and Gr family regions. Furthermore, our extensive experience in genome assembly, including sample preparation experience and assembly strategies to reduce complexity, will provide valuable references for other species aiming to achieve their own T2T genome assemblies.

In arthropods, a host of studies focused on insect olfactory systems, where most functional proteins including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) have been identified. However, in other arthropods such as ticks and mites, genes encoding for OBPs and CSPs are absent. The Niemann-Pick type C2 (NPC2), as a third class of binding proteins, was indicated as potential carriers for semiochemicals and participants in chemical communication. Here, we cloned three full-length NPC2 genes (NbNPC2-1, NbNPC2-2 and NbNPC2-3) from a predatory mite Neoseiulus barkeri Hughes (Acari: Phytoseiidae) and investigated the expression levels of these genes by quantitative real-time PCR. These NPC2 proteins possessed a conserved motif of six cysteines paired in three disulfide bridges with a coincidence to insect OBPs. The 3D molecular modeling of NbNPC2 showed ligand-binding pocket of NbNPC2-1 was composed of a flexible β-structure that contributed to binding to a wide range of potential semiochemicals. Three NPC2 genes all possessed one signal peptide revealed their specific character as secretory proteins. Three NPC2 genes in N. barkeri displayed the highest expression levels in adult males, suggesting a putative role in detecting of the female sex pheromones and highly specific binding to female sex pheromone. The expression levels were lowest in the stage of eggs and increased with developmental stage growing, indicating that NPC2 genes were continuously expressed in post-embryonic stages. NbNPC2-1 in the stage of adult females was expressed 6 and 4 times higher than that of larva and nymph, suggesting NbNPC2-1 might contribute an important role in odor recognition of adult females to hunt for suitable areas for the oviposition and predation. These results showed that NPC2 genes might be involved in chemical communications of adult males, and that NbNPC2-1 preformed specified olfactory functions in adult females. This study provided a foundation for research to determine the roles of NPC2 proteins acting as odorant carrier and involved in chemical communication in phytoseiid mites.

Biological control of spider mites using phytoseiid mites is frequently being disrupted by high temperatures, which results in an important and urgent research topic. We hypothesized that this scenario could be ameliorated if the thermal tolerance of a certain phytoseiid mite is enhanced. In this study, a high temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes was selected from its conventional strain (CS) via a long-term heat acclimation (maintained at 35 degrees C) and frequent heat hardenings (exposed at 45 degrees C for 2 h every 15-25 days) over multiple generations. As expected, heat acclimation greatly improved the survival probabilities of N. barkeri when a single heat stress event occurred, with a median lethal time (e.g., at 45 degrees C) calculated as 15.2 h for HTAS females and 1.9 h for CS ones, respectively. After the heat acclimatory, HTAS showed a faster growth and developmental rate, and a higher immature survival rate than CS did; a significant reduced total fecundity rather than longevity was observed at 35 degrees C; a shifting from larger females to smaller males was observed (about 21% reduced in female ratio). Meanwhile, single heat stress event (e.g., exposed at 42 degrees C for 4 h) had a detrimental effect on the reproductive traits of the newly-emerged females, particularly obvious for mites from CS colony, by delaying the onset of oviposition and reducing reproductive output. This is the first experimental demonstration that the artificially heat acclimatory can shape the intraspecific variations on thermal susceptibility in a phytoseiid mite. It is suggested that HTAS N. barkeri might be a promising strain for the biological control of the high temperature favored spider mites, and more field and greenhouse trials should be evaluated in the future.

To explore the effects of heat shock proteins in the conventional strain (CS) and high temperature-adapted strain (HTAS) of Neoseiulus barkeri, we cloned and characterized the cDNA of the NbHsp40, NbHsp60, NbHsp70, and NbHsp90 genes and examined changes in expression levels with short-term exposure to heat or cold stress. All four of the genes showed highly conserved amino acid sequences and high homology to Galendromus occidentalis and N. cucumeris. Phylogenetic analysis of NbHsps indicated a high consistency with the known genes of model insects. These results demonstrate variations in the sensitivity of NbHsps to heat or cold stress; i.e., the expression of NbHsp60 did not change with the application of heat and cold stress, NbHsp90 and NbHsp40 were upregulated at high temperature compared to that at low temperature responses, and the expression of NbHsp70 increased with both heat and cold stress. Hsp expression also differed between the two strains: The application of a 40°C heat shock resulted in a decrease in the expression of NbHsp40/70/90 in the CS within 1–2 h, but increased in the HTAS strain in 2 h. However, exposure to cold stress (0 °C and 5 °C) did not result in distinct changes in the expression of NbHsps in the CS, but a significant decrease in the HTAS. The expression of NbHsps further demonstrates that the HTAS has been acclimated to high temperatures. The results also determine that heat acclimation decreases cold tolerance in HTAS mites. This study reveals differences in the characteristics of heat and cold tolerance between the two strains of N. barkeri and provides useful information for future commercialization of N. barkeri for pest biological control, particularly the HTAS. Furthermore, it also describes high heat tolerance in HTAS at the molecular level.