摘要: MicroRNAs (miRNAs) have started to play an important role in pest control, and novel miRNA-based transgenic insect-resistant plants are now emerging. However, an environmental risk assessment of these novel transgenic plants expressing insect miRNAs must be undertaken before promoting their application. Here, transgenic miR-14 rice, which has high resistance to the rice stem borer Chilo suppressalis, was used as an example for evaluation in this study. Taking the tier 1 risk assessment method in Bacillus thuringiensis (Bt) crops as a reference, the effects of the direct exposure of a non-target parasitoid, Cotesia chilonis, to a high concentration of miRNA were evaluated. The results showed that direct feeding with miR-14 at high concentration had no significant effects on the biological parameters of Co. chilonis, whereas when miR-14 was injected into Ch. suppressalis-parasitized larvae, the development duration of Co. chilonis was significantly affected. In combination with the real conditions of the rice paddy field, it could be inferred that transgenic miR-14 rice has no significant negative effects on the important non-target parasitoid, Co. chilonis. These results will provide a foundation for the establishment of a new safety evaluation system for novel RNAi-based transgenic plants.

摘要: We made separate experiments to examine life-history traits and activities of protective enzymes as affected by carbon dioxide (CO2) elevation to 780 mu L/L as compared to 390 mu L/L in imidacloprid- or buprofezin-resistant strains of the brown planthopper (BPH) Nilaparvata lugens. We found an interaction effect between resistance and the CO2 level on the nymphal survival and duration in both resistant strains. Nymphal durations in both resistant strains were much shorter in the resistant than susceptible BPH at 780 mu L/L but similar between them or slightly shorter in the resistant than susceptible BPH at 390 mu L/L. Nymphal survival was lower for imidacloprid-resistant than its susceptible BPH at 390 mu L/L but higher at 780 mu L/L; it stayed unaffected by the CO2 elevation in buprofezin-resistant BPH. We did not observe an interaction effect between resistance and the CO2 level on major reproductive parameters in both resistant strains. But the 2 strains were not consistent across CO2 levels in all parameters. Our measurements of protective enzyme activities of superoxide dismutase, catalase, and peroxidase showed an interaction between resistance and the CO2 level. Overall, these enzymes became similar in activity between resistant and susceptible BPH at 780 mu L/L compared to 390 mu L/L and the change was more distinct in the imidacloprid- than buprofezin-resistant BPH strains. Our findings suggest that CO2 elevation can affect life-history traits of insecticide-resistant BPH, while the effect may vary depending on the kind of insecticides it is resistant to.

摘要: Kings and queens of termites are endowed with an extraordinary longevity coupled with lifelong fecundity. We recently reported that termite kings and queens display a dramatically increased enzymatic activity and abundance of telomerase in their somatic organs when compared to short-lived workers and soldiers. We hypothesized that this telomerase activation may represent a noncanonical pro-longevity function, independent of its canonical role in telomere maintenance. Here, we explore this avenue and investigate whether the presumed noncanonical role of telomerase may be due to alternative splicing of the catalytic telomerase subunit TERT and whether the subcellular localization of TERT isoforms differs among organs and castes in the termite Prorhinotermes simplex. We empirically confirm the expression of four in silico predicted splice variants (psTERT1-A, psTERT1-B, psTERT2-A, psTERT2-B), defined by N-terminal splicing implicating differential localizations, and C-terminal splicing giving rise to full-length and truncated isoforms. We show that the transcript proportions of the psTERT are caste- and tissue-specific and that the extranuclear full-length isoform TERT1-A is relatively enriched in the soma of neotenic kings and queens compared to their gonads and to the soma of workers. We also show that extranuclear TERT protein quantities are significantly higher in the soma of kings and queens compared to workers, namely due to the cytosolic TERT. Independently, we confirm by microscopy the extranuclear TERT localization in somatic organs. We conclude that the presumed pleiotropic action of telomerase combining the canonical nuclear role in telomere maintenance with extranuclear functions is driven by complex TERT splicing. We recently reported that long-lived termite kings and queens display an increased enzymatic activity and abundance of telomerase in their soma when compared to short-lived workers. We hypothesized that this telomerase activation may represent its noncanonical pro-longevity function, independent of its canonical role in telomere maintenance.Here, we show that the catalytic telomerase subunit TERT occurs in multiple splice variants in the termite Prorhinotermes simplex, and that their expression patterns differ between workers a and kings and queens. Extranuclear full-length isoform was found enriched in the soma of reproductives, supporting once again an unknown cytosolic function of telomerase in the soma of long-lived termites castes. image

摘要: Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata, using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Escherichia coli and Serratia marcescens) into larvae triggered a time-specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+-dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies.

摘要: Short-chain dehydrogenases/reductases (SDRs) are ubiquitously distributed across diverse organisms and play pivotal roles in the growth, as well as endogenous and exogenous metabolism of various substances, including drugs. The expression levels of SDR genes are reportedly upregulated in the fenpropathrin (FEN)-resistant (FeR) strain of Tetranychus cinnabarinus. However, the functions of these SDR genes in acaricide tolerance remain elusive. In this study, the activity of SDRs was found to be significantly higher (2.26-fold) in the FeR strain compared to the susceptible strain (SS) of T. cinnabarinus. A specific upregulated SDR gene, named SDR112C1, exhibited significant overexpression (3.13-fold) in the FeR population compared with that in the SS population. Furthermore, the expression of SDR112C1 showed a significant increase in the response to FEN induction. Additionally, knockdown of the SDR112C1 gene resulted in decreased SDR activity and reduced mite viability against FEN. Importantly, heterologous expression and in vitro incubation assays confirmed that recombinant SDR112C1 could effectively deplete FEN. Moreover, the overexpression of the SDR112C1 gene in Drosophila melanogaster significantly decreased the toxicity of FEN to transgenic fruit flies. These findings suggest that the overexpression of SDR SDR112C1 is a crucial factor contributing to FEN tolerance in T. cinnabarinus. This discovery not only enhances our understanding of SDR-mediated acaricide tolerance but also introduces a new family of detoxification enzymes to consider in practice, beyond cytochrome P450s, carboxyl/choline esterases and glutathione S-transferases.

摘要: The sweet potato weevil (Cylas formicarius) is a critical pest producing enormous global losses in sweet potato crops. Traditional pest management approaches for sweet potato weevil, primarily using chemical pesticides, causes pollution, food safety issues, and harming natural enemies. While RNA interference (RNAi) is a promising environmentally friendly approach to pest control, its efficacy in controlling the sweet potato weevil has not been extensively studied. In this study, we selected a potential target for controlling C. formicarius, the Troponin I gene (wupA), which is essential for musculature composition and crucial for fundamental life activities. We determined that wupA is abundantly expressed throughout all developmental stages of the sweet potato weevil. We evaluated the efficiency of double-stranded RNAs in silencing the wupA gene via microinjection and oral feeding of sweet potato weevil larvae at different ages. Our findings demonstrate that both approaches significantly reduced the expression of wupA and produced high mortality. Moreover, the 1st instar larvae administered dswupA exhibited significant growth inhibition. We assessed the toxicity of dswupA on the no-target insect silkworm and assessed its safety. Our study indicates that wupA knockdown can inhibit the growth and development of C. formicarius and offer a potential target gene for environmentally friendly control.

摘要: Syntaxin 1A (Syx1A) has diverse and indispensable functions in animals. Previous studies have mainly focused on the roles of Syx1A in Drosophila, and so how Syx1A operates during the development of other insects remains poorly understood. This study investigated whether disrupting LmSyx1A using RNA interference (RNAi) affects the growth and development of Locusta migratoria. LmSyx1A was expressed in all tissues tested, with the highest expression observed in the fat body. After 5th-instar nymphs were injected with double-stranded LmSyx1A (dsLmSyx1A), none of the nymphs were able to molt normally and all eventually died. The silencing of LmSyx1A resulted in the cessation of feeding, body weight loss, and atrophy of the midgut and gastric cecum in locusts. Hematoxylin and eosin (H&E) staining showed that the columnar cells in the midgut were severely damaged, with microvilli defects visible in dsLmSyx1A-injected nymphs. Secretory vesicles were observed with transmission electron microscopy (TEM). In addition, reverse transcription quantitative polymerase chain reaction (RT-qPCR) further indicates that silencing LmSyx1A repressed the expression of genes involved in the insulin/mammalian target of rapamycin (mTOR)-associated nutritional pathway. Taken together, these results suggest that LmSyx1A significantly affects the midgut cell layer of locust nymphs, which was partially associated with the downregulation of the insulin/mTOR-associated nutritional pathway. Thus, we argue that LmSyx1A is a suitable target for developing dsRNA-based biological pesticides for managing L. migratoria.

摘要: The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with omega values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.

摘要: The escalating use of nanodiamonds (NDs) has raised concerns about their ecotoxicological impact, prompting exploration of therapeutic interventions. This paper pioneers the examination of Vitamin B12-conjugated sericin (VB12-SER) as a potential therapeutic approach against ND-induced toxicity in darkling beetles (Blaps polychresta). The study analyzes mortality rates and organ-specific effects, covering the testis, ovary, and midgut, before and after treatments. Following exposure to 10 mg NDs/g body weight, within a subgroup of individuals termed ND2 with a mortality rate below 50%, two therapeutic treatments were administered, including pure sericin (SER) at 10 mg/mL and VB12-SER at 10.12 mg/mL. Consequently, five experimental groups (control, SER, ND2, ND2+SER, ND2+SER+VB12) were considered. Kaplan-Meier survival analysis was performed to assess the lifespan distribution of the insects in these groups over a 30-d period. Analyses revealed increased mortality and significant abnormalities induced by NDs within the examined organs, including cell death, DNA damage, enzyme dysregulation, antioxidant imbalances, protein depletion, lipid peroxidation, and morphological deformities. In contrast, the proposed treatments, especially (ND2+SER+VB12), demonstrated remarkable recovery, highlighting VB12-conjugated SER's potential in mitigating ND-triggered adverse effects. Molecular docking simulations affirmed binding stability and favorable interactions of the VB12-SER complex with target proteins. This research enhances understanding of NDs' effects on B. polychresta, proposing it as an effective bioindicator, and introduces VB12-conjugated SER as a promising therapeutic strategy in nanotoxicological studies.

摘要: Wing dimorphism in Nilaparvata lugens is controlled by the insulin-like growth factor 1 (IGF-1) signaling - Forkhead transcription factors (IIS-FoxO) pathway. However, the role of this signal in the wing development program remains largely unclear. Here, we identified 2 R-SMAD proteins, NlMAD1 and NlMAD2, in the brown planthopper (BPH) transcriptome, derived from the intrinsic transforming growth factor-beta pathway of insect wing development. Both proteins share high sequence similarity and conserved domains. Phylogenetic analysis placed them in the R-SMAD group and revealed related insect orthologs. The expression of Nlmad1 was elevated in the late instar stages of the macropterous BPH strain. Nlmad1 knockdown in nymphs results in malformed wings and reduced wing size in adults, which affects the forewing membrane. By contrast, Nlmad2 expression was relatively consistent across BPH strains and different developmental stages. Nlmad2 knockdown had a milder effect on wing morphology and mainly affected forewing veins and cuticle thickness in the brachypterous strain. NlMAD1 functions downstream of the IIS-FoxO pathway by mediating the FoxO-regulated vestigial transcription and wing morph switching. Inhibiting Nlmad1 partially reversed the long-winged phenotype caused by NlFoxO knockdown. These findings indicate that NlMAD1 and NlMAD2 play distinct roles in regulating wing development and morph differentiation in BPH. Generally, NlMAD1 is a key mediator of the IIS-FoxO pathway in wing morph switching.