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Arylalkylamine N‐acetyltransferase (aaNAT) is a crucial enzyme that catalyses the transfer of acetyl groups from acetyl coenzyme A to arylalkylamines and arylamines. Evolutionary studies have identified a distinct class of aaNATs specific to mosquitoes, yet their functions remain elusive. This study focuses on Ae‐aaNAT7, a mosquito‐unique gene in Aedes aegypti (Diptera:Culicidae), to explore its functionality. Temporal and spatial expression analysis of Ae‐aaNAT7 mRNA revealed high expression during embryonic development and in first‐instar larvae, with notable expression in the limbs of adult mosquitoes based on tissue expression profiling. By further employing CRISPR/Cas9 technology for loss‐of‐function studies, our investigation revealed a reduction in the area of white spotting in the limbs of Ae‐aaNAT7 mutant adult mosquitoes. Further investigation revealed a significant decrease in the fecundity and hatchability of the mutants. Dissection of the ovaries from Ae‐aaNAT7 heterozygous mutants showed a noticeable reduction in the oocyte area compared with wild type. Dissection of the exochorion of the eggs from Ae‐aaNAT7 homozygous mutants consistently revealed a striking absence of mature embryos. In addition, RNA interference experiments targeting Ae‐aaNAT7 in males resulted in a reduction in fecundity, but no effect on hatchability was observed. These collective insights underscore the substantial impact of Ae‐aaNAT7 on reproduction and its pivotal contribution to adult limb pigmentation in Ae. aegypti. These revelations offer insights pivotal for the strategic design of future insecticide targets. [ABSTRACT FROM AUTHOR]

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi‐derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo‐siRNA) pathway, with minimal engagement from the PIWI‐interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus‐derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo‐siRNA pathway. [ABSTRACT FROM AUTHOR]

Akirin is a nuclear protein that controls development in vertebrates and invertebrates. The function of Akirin has not been assessed in any Coleopteran insects. We found that high levels of akirin transcripts in Henosepilachna vigintioctopunctata, a serious Coleopteran potato defoliator (hereafter Hvakirin), were present at prepupal, pupal and adult stages, especially in larval foregut and fat body. RNA interference (RNAi) targeting Hvakirin impaired larval development. The Hvakirin RNAi larvae arrested development at the final larval instar stage. They remained as stunted larvae, gradually blackened and finally died. Moreover, the remodelling of gut and fat body was inhibited in the Hvakirin depleted larvae. Two layers of cuticles, old and newly formed, were noted in the dsegfp‐injected animals. In contrast, only a layer of cuticle was found in the dsakirin‐injected beetles, indicating the arrest of larval development. Furthermore, the expression of three transforming growth factor‐β cascade genes (Hvsmox, Hvmyo and Hvbabo), a 20‐hydroxyecdysone (20E) receptor gene (HvEcR) and six 20E response genes (HvHR3, HvHR4, HvE75, HvBrC, HvE93 and Hvftz‐f1) was significantly repressed, consistent with decreased 20E signalling. Conversely, the transcription of a juvenile hormone (JH) biosynthesis gene (Hvjhamt), a JH receptor gene (HvMet) and two JH response genes (HvKr‐h1 and HvHairy) was greatly enhanced. Our findings suggest a critical role of Akirin in larval development in H. vigintioctopunctata. [ABSTRACT FROM AUTHOR]

The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance‐related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post‐feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests. [ABSTRACT FROM AUTHOR]

The primary function of odorant‐binding proteins (OBPs) is thought to be binding and transporting odorants molecules. The research on the molecular mechanisms of SfruOBPs binding with sex pheromones is beneficial to the prevention and control of Spodoptera frugiperda (S. frugiperda). Herein, two screened SfruOBPs (Sfru10933 and Sfru21187) were knocked down by RNA interference, and then the changes in electroantennogram (EAG) and behavioral responses of male mutants to their main sex pheromone components were recorded. Upon interfering with Sfru10933, the EAG response and behavioural preferences of males to sex pheromones were significantly reduced or eliminated. The interference with Sfru21187 resulted in a weakening of the behavioural preference, while the EAG response remained unchanged. However, the co‐interference of Sfru10933/Sfru21187 led to a significant reduction in the EAG activity of male to sex pheromones. These results reveal that Sfru10933 and Sfru21187 play a crucial role in sex pheromone recognition, and there may be a synergistic effect between them. Furthermore, these results will help clarify the perception of sex pheromones by S. frugiperda, which may provide new targets for the development of mating inhibitors in the future. [ABSTRACT FROM AUTHOR]

Background: DEAD‐box protein (DDX) is a member of the DDX RNA helicase family that exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility, particularly in mammals. Nevertheless, the biological functions of DDXs in insects have not been fully resolved and attracted increasing attention these years. Laodelphax striatellus (Hemiptera) is a notorious rice pest through feeding on rice sap and transmitting plant viruses. In this study, we aim to elucidate the functional characterization of DDXs in L. striatellus, and to exploit potential target genes for the development of pest control strategies. Results: In this study, we characterized the expression patterns of LsDDX6, LsDDX47, and LsDDX51 in planthoppers and analyzed their conserved motifs. These genes were found to be expressed in all tissues and developmental stages examined, with significantly higher transcript levels observed in the ovary. Knockdown of LsDDX6, LsDDX47, and LsDDX51 resulted in an obvious lethal phenotype in nymphs and abnormal ovarian development in adults. Furthermore, a total of 27 DDXs were identified in L. striatellus, and most DDXs were highly expressed in ovary and structure analysis result revealed that all of the DDXs possessed nine motifs that were unique to the DDX family. Conclusion: The three DDX RNA helicases (LsDDX6, LsDDX47, and LsDDX51) are essential for both survivorship and reproduction in L. striatellus. Considering a total number of 27 DDXs identified in L. striatellus, they might serve as promising candidates for application in RNAi‐based control of this destructive pest. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cuticular proteins (CPs) play essential roles in forming cuticular structures in insects. However, the specific functions and regulatory mechanisms of CPs remain largely unexplored. In this study, the Larval cuticular protein 17 (HcLCP‐17) gene was identified from Hyphantria cunea, a highly destructive and polyphagous forest pest. To investigate the role of HcLCP‐17 in cuticular function and transcriptional regulation mediated by 20E‐responsive transcription factors (ERTFs), we employed RNA interference (RNAi) and yeast one‐hybrid assay techniques. Additionally, we examined the molecular mechanism by which chlorbenzuron, a type of benzoylphenylurea (BPU) that functions as a chitin synthesis inhibitor (CSI), affects the 20E signaling pathway and ultimately regulates HcLCP‐17 expression. RESULTS: HcLCP‐17 encodes a polypeptide consisting of 393 amino acids, which includes a chitin‐binding domain. Silencing HcLCP‐17 resulted in a disturbance in the structural organization of the larval cuticle and a notable reduction in chitin levels. HcLCP‐17 expression was controlled by the interaction between Broad‐Complex (Br‐C) and beta Fushi Tarazu Factor‐1 (βFTZ‐F1) with its promoter fragment. Furthermore, the inhibitory effect of chlorbenzuron on HcLCP‐17 expression was found to be potentially mediated by Br‐C and βFTZ‐F1. CONCLUSION: The study presents a novel mode of action for the 20E signaling pathway in regulating the expression of CPs and reveals the potential mode‐of‐action of BPUs in insect cuticles. These findings provide a theoretical basis for future utilization of LCP‐17 as a pesticide target making a significant contribution to the development of effective pest management strategies. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: RNA interference (RNAi) is an endogenous eukaryote viral defence mechanism representing a unique form of post‐transcriptional gene silencing. Owing to its high specificity, this technology is being developed for use in dsRNA‐based biopesticides for control of pest insects. Whilst many lepidopteran species are recalcitrant to RNAi, Tuta absoluta, a polyphagous insect responsible for extensive crop damage, is sensitive. Ryanodine receptors (RyRs) are intracellular calcium channels regulating calcium ion (Ca2+) release. The chemical pesticide class of diamides functions agonistically against lepidopteran RyR, resulting in uncontrolled Ca2+ release, feeding cessation and death. Resistance to diamides has emerged in T. absoluta, derived from RyR point mutations. RESULTS: RNAi was used to target RyR transcripts of T. absoluta. Data presented here demonstrate the systemic use of exogenous T. absoluta RyR‐specific (TaRy) dsRNA in tomato plants (Solanum lycopersicum) to significantly downregulate expression of the target gene, resulting in significant insect mortality and reduced leaf damage. Using a leaflet delivery system, daily dosing of 3 μg TaRy dsRNA for 72 h resulted in 50% downregulation of the target gene and 50% reduction in tomato leaf damage. Corrected larval mortality and adult emergence were reduced by 38% and 33%, respectively. TaRy dsRNA demonstrated stability in tomato leaves ≤72 h after dosing. CONCLUSIONS: This work identifies TaRy as a promising target for RNAi control of this widespread crop pest. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The insect cuticle consists of chitin fibers and a protein matrix, which plays an important role in protecting the body from invasion of various pathogens and prevents water loss. Periodic synthesis and degradation of the cuticle is required for the growth and development of insects. Key genes involved in cuticle formation have long been considered a potential target for pest control. RESULTS: In this study, a member of the RR‐2 subfamily of cuticular protein 8 (DcCP8) was identified from the Diaphorina citri genome database. Immunofluorescence analysis suggested that DcCP8 was mainly located in the Diaphorina citri exocuticle and can be induced to up‐regulate 12 h following 20‐hydroxyecdysone (20E) treatment. Silencing of DcCP8 by RNA interference (RNAi) significantly disrupted the metamorphosis to the adult stage, and improved the permeability of the cuticle. Transmission electron microscopy (TEM) analysis revealed that the synthesis of the exocuticle was impressed after silencing of DcCP8. Furthermore, the recombinant DcCP8 protein exhibited chitin‐binding properties in vitro, down‐regulation of DcCP8 significantly inhibited expression levels of chitin metabolism‐related genes. Additionally, a sprayable RNAi method targeting DcCP8 based on star polycation (SPc) nanoparticles‐wrapped double‐stranded RNA (dsRNA) significantly increased Diaphorina citri mortality. Transcriptome sequencing further confirmed that genes associated with the endocytic pathway and immune response were up‐regulated in Diaphorina citri after SPc treatment. CONCLUSIONS: The current study indicated that DcCP8 is critical for the formation of Diaphorina citri exocuticles, and lays a foundation for Diaphorina citri control based on large‐scale dsRNA nanoparticles. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Protein kinases are a type of transferase enzyme that catalyze the phosphorylation of protein substrates, including receptor proteins. Testis‐specific serine/threonine kinases (TSSKs) are a highly conserved group of protein kinases found in various organisms. They play an essential role in male reproduction by influencing sperm development and function. RESULTS: In this study, we report on the characterization of BmTSSK3, a TSSK from the silkworm, Bombyx mori. We found that BmTSSK3 is specifically expressed in the testis and localized to the sperm flagella, particularly in the sperm tail cyst. Furthermore, we developed BmTSSK3 inhibitors through molecular docking and binding assays. Small molecules 5‐(4‐Fluorophenyl)‐2‐ureidothiophene‐3‐carboxamide (TPCA‐1) and Imidurea were identified to bind to BmTSSK3. Using site‐specific mutation technology, we identified amino acid residues R134 and S184 as crucial binding sites for small molecules. RNA interference assay and Western blot analysis showed that knockdown of BmTSSK3 significantly decreased histone 3 phosphorylation. To confirm the inhibitory effect of these small molecules, we treated silkworm testes with TPCA‐1 and observed a strong inhibitory effect. CONCLUSION: TPCA‐1 is an inhibitor of BmTSSK3, which raises its potential as a future candidate for male sterility of the silkworm. Thus, this study may offer a novel strategy for sterile silkworms as well as insects. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Summary Cross‐kingdom RNA interference (ckRNAi) is a mechanism of interspecies communication where small RNAs (sRNAs) are transported from one organism to another; these sRNAs silence target genes in trans by loading into host AGO proteins. In this work, we investigated the occurrence of ckRNAi in Arbuscular Mycorrhizal Symbiosis (AMS). We used an in silico prediction analysis to identify a sRNA (Rir2216) from the AM fungus Rhizophagus irregularis and its putative plant gene target, the Medicago truncatula MtWRKY69 transcription factor. Heterologous co‐expression assays in Nicotiana benthamiana, 5′ RACE reactions and AGO1‐immunoprecipitation assays from mycorrhizal roots were used to characterize the Rir2216–MtWRKY69 interaction. We further analyzed MtWRKY69 expression profile and the contribution of constitutive and conditional MtWRKY69 expression to AMS. We show that Rir2216 is loaded into an AGO1 silencing complex from the host plant M. truncatula, leading to cleavage of a host target transcript encoding for the MtWRKY69 transcription factor. MtWRKY69 is specifically downregulated in arbusculated cells in mycorrhizal roots and increased levels of MtWRKY69 expression led to a reduced AM colonization level. Our results indicate that MtWRKY69 silencing, mediated by a fungal sRNA, is relevant for AMS; we thus present the first experimental evidence of fungus to plant ckRNAi in AMS. [ABSTRACT FROM AUTHOR]

Sex pheromones emitted by female moths play important roles in mate attraction. The molecular mechanism underlying pheromone biosynthesis activating neuropeptide (PBAN)‐regulated sex pheromone biosynthesis has been well elucidated in many moth species, although this mechanism is species‐dependent. Spodoptera litura, an important pest, has caused serious economic losses to agricultural production, yet the mechanism for its sex pheromone biosynthesis has not been fully identified. The present study investigates in detail mechanism underlying PBAN‐regulated sex pheromone biosynthesis in S. litura. The transcriptome sequencing of S. litura pheromone glands (PGs) was analysed to identify a serial of candidate genes potentially involved in sex pheromone biosynthesis. Further investigation revealed a bimodal pattern in both sex pheromone release and mating frequency. PBAN was found to regulate sex pheromone biosynthesis via its receptor by using Ca2+ as a secondary messenger, as demonstrated by RNA interference and the application of pharmacological inhibitors. Furthermore, PBAN/Ca2+ signalling activated calcineurin (CaN) and acetyl‐CoA carboxylase (ACC), which mediated sex pheromone biosynthesis in response to PBAN stimulation. Mostly importantly, hexokinase 2 (HK2) was confirmed to be activated by PBAN/PBANR /Ca2+/PKC signalling via phosphorylation at two specific sites (ser423 and ser434 sites of HK2). Overall, our findings shed light on the intricate processes involved in sex pheromone production in S. litura, in which PBAN regulates sex pheromone biosynthesis through PBAN/PBANR/Ca2+/CaN/ACC and PBAN/PBANR/Ca2+/PKC/HK2 signalling pathways. These insights significantly contribute to our comprehension of the specific mechanisms underlying sex pheromone biosynthesis in this moth species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SUMMARY: Pustule formation is pivotal for the development of the Xanthomonas citri subsp. citri (Xcc)‐induced citrus canker disease (CCD). Although our previous study demonstrated that the exogenous application of abscisic acid (ABA) facilitated pustule formation induced by Xcc, the precise mechanism remains elusive. The 9‐cis‐epoxycarotenoid dioxygenase (NCED) is a crucial enzyme in ABA biosynthesis. This study explored the role of citrus CsNCED1‐1 in CCD resistance through overexpression and RNA interference of CsNCED1‐1 in Wanjincheng orange (Citrus sinensis). Our findings indicated that CsNCED1‐1 negatively modulated CCD resistance by fostering ABA accumulation, concomitant with an increase in jasmonic acid (JA) and a decrease in salicylic acid (SA). Plants overexpressing CsNCED1‐1 displayed shortened leaves with smaller and denser stomata along with irregular and increased palisade cells. CsLOB1 is a known susceptibility gene for CCD, and CsbZIP40 positively influences resistance to this disease. We further confirmed that CsLOB1 promoted and CsbZIP40 suppressed the transcription of CsNCED1‐1 by directly binding to the CsNCED1‐1 promoter. Notably, CsbZIP40 and CsLOB1 showed a competitive relationship in the regulation of CsNCED1‐1 expression, with CsbZIP40 exhibiting greater competitiveness. Overall, our findings highlight that CsNCED1‐1 promotes susceptibility to citrus canker by disrupting JA‐ and SA‐mediated defense mechanisms and triggering the proliferation and remodeling of palisade cells, thereby facilitating pathogen colonization and pustule formation. This study offers novel insights into the regulatory mechanisms underlying citrus canker resistance and the role of CsNCED1‐1 in citrus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The role of epigenetics in regulating caste polyphenism in social insects has been debated. Here, we tested the importance of histone de/acetylation processes for the maintenance of queen hallmarks like a high fecundity and a long lifespan. To this end, we performed RNA interference experiments against histone deacetylase 3 (HDAC3) in the termite Cryptotermes secundus. Fat body transcriptomes and chemical communication profiles revealed that silencing of HDAC3 leads to signals indicative of queen hallmarks. This includes fostering of queen signalling, defence against ageing and a reduction of life‐shortening IIS (insulin/insulin‐like growth factor signalling) and endocrine JH (juvenile hormone) signalling via Kr‐h1 (Krüppel‐homologue 1). These observed patterns were similar to those of a protein‐enriched diet, which might imply that histone acetylation conveys nutritional effects. Strikingly, in contrast to solitary insects, reduced endocrine JH signalling had no negative effect on fecundity‐related vitellogenesis in the fat bodies. This suggests an uncoupling of longevity pathways from fecundity in fat bodies, which can help explain queens' extraordinary lifespans combined with high fecundity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Natural indole alkaloids provide important medicinal resources and defences to environmental stresses. The Uncaria genus is a recorded traditional medicinal woody plant with high alkaloids. Genomic insights into alkaloid variation remain elusive. Here, we have dissected the haploid‐resolved chromosomal T2T genome assembly of Uncaria rhynchophylla with a size of ~634 Mb and contig N50 of 27 Mb using PacBio HiFi long‐reads plus Hi‐C reads and anchored the contigs on 22 pairs of confirmed chromosomes. This genome contains 56% repeat sequences and ~29 000 protein‐encoding genes. U. rhynchophylla diverged from a common ancestor shared with Coffea around 20 million years ago and contains expanded and contracted gene families associated with secondary metabolites and defences/resistance to stresses. We constructed the pathway and mined genes for rhynchophylline alkaloid biosynthesis. Fifty‐three alkaloids in this pathway and eight differentially expressed genes are the keys to alkaloid accumulation. Elevated alkaloid levels are driven by high copy numbers of critical genes STRs and SGRs involved in strictosidine synthesis and hydrolysis as evidenced by phylogenetic, expression and RNA interference analyses. These results advance our genetic understanding and guide further breeding improvements, stress adaptation studies and pharmaceutical development. [ABSTRACT FROM AUTHOR]

Neuropeptide F (NPF) and short neuropeptide F (sNPF) are important neuropeptides and mainly affect feeding behaviour of insects. However, the regulation of insect feeding behaviour by NPF and sNPF appears to differ between species, and it is not clear how NPF and sNPF regulate the food intake of the brown planthopper (Nilaparvata lugens). Therefore, the functions of NPF and sNPF in regulating food intake and affecting the growth and antioxidant levels of N. lugens fed on host rice plants were investigated by knocking down NPF and sNPF respectively and simultaneously knocking down both of them by RNA interference. The results showed that NPF and sNPF were mainly expressed in the head of N. lugens, and N. lugens increased food intake after NPF and sNPF were knocked down, which was reflected in the prolonged duration of N4a and N4b waves in the electrical penetration graph (EPG) experiment after knocking down NPF and sNPF. In addition, knocking down NPF and sNPF led to the increase of body weight and mortality of N. lugens, and also led to the increase of antioxidant level of N. lugens. So it was concluded that NPF and sNPF could regulate food intake, maintain body weight stability and oxidative balance in N. lugens. Our study clarified the molecular mechanism of NPF and sNPF regulating feeding behaviour and affect the growth and antioxidant level of N. lugens. [ABSTRACT FROM AUTHOR]

Germline regulates the expression of life‐history traits and mediates the trade‐off between reproduction and somatic maintenance. However, germline maintenance in itself can be costly, and the costs can vary between the sexes depending on the number of gametes produced across the lifetime. We tested this directly by germline ablation using glp‐1 RNA interference (RNAi) in a dioecious nematode Caenorhabditis remanei. Germline removal strongly increased heat‐shock resistance in both sexes, thus confirming the role of the germline in regulating somatic maintenance. However, germline removal resulted in increased lifespan only in males. High costs of mating strongly reduced lifespan in both sexes and obliterated the survival benefit of germline‐less males even though neither sex produced any offspring. Furthermore, germline removal reduced male growth before maturation but not in adulthood, while female growth rate was reduced both before and especially after maturation. Thus, germline removal improves male lifespan without major growth costs, while germline‐less females grow slower and do not live longer than reproductively functional counterparts in the absence of environmental stress. Overall, these results suggest that germline maintenance is costlier for males than for females in C. remanei. [ABSTRACT FROM AUTHOR]

SUMMARY: Alfalfa is one of the most widely cultivated forage crops worldwide. However, soil salinization restricts alfalfa growth and development and affects global productivity. The plant cell wall is the first barrier against various stresses. Therefore, elucidating the alterations in cell wall architecture is crucial for stress adaptation. This study aimed to clarify the impact of myo‐inositol oxygenase 2 (MsMIOX2) on cell wall pectin and hemicellulose biosynthesis under saline–alkali stress and identify the upstream transcription factors that govern MsMIOX2. MsMIOX2 activation induced cell wall pectin and hemicellulose accumulation under saline–alkali stress. The effects of MsMIOX2 in saline–alkali tolerance were investigated by characterizing its overexpression and RNA interference lines. MsMIOX2 overexpression positively regulated the antioxidant system and photosynthesis in alfalfa under saline–alkali stress. MsMIOX2 exhibited myo‐inositol oxygenase activity, which increased polysaccharide contents, facilitated pectin and hemicellulose biosynthesis, and extended the cell wall thickness. However, MsMIOX2 RNA interference decreased cell wall thickness and alleviated alfalfa saline–alkali stress tolerance. In addition, MsbZIP53 was identified as an upstream transcriptional MsMIOX2 regulator by yeast one‐hybrid, electrophoretic mobility shift assay, dual‐luciferase, and beta‐glucuronidase assays. MsbZIP53 overexpression increased MsMIOX2 expression, elevated MIOX activity, reinforced the antioxidant system and photosynthesis, and increased saline–alkali stress tolerance in alfalfa. In conclusion, this study presents a novel perspective for elucidating the molecular mechanisms of saline–alkali stress tolerance in alfalfa and emphasizes the potential use of MsMIOX2 in alfalfa breeding. [ABSTRACT FROM AUTHOR]

Objective: Visceral adiposity is associated with increased proinflammatory activity, insulin resistance, diabetes risk, and mortality rate. Numerous individual genes have been associated with obesity, but studies investigating gene regulatory networks in human visceral obesity have been lacking. Methods: We analyzed gene regulatory networks in human visceral adipose tissue (VAT) from 48 and 11 Chinese patients with and without obesity, respectively, using gene coexpression and gene regulatory network construction from RNA‐sequencing data. We also conducted RNA interference‐based functional tests on selected genes for effects on adipocyte differentiation. Results: A scale‐free gene coexpression network was constructed from 360 differentially expressed genes between VAT samples from patients with and without obesity (absolute log fold change > 1, false discovery rate [FDR] < 0.05), with edge probability > 0.8. Gene regulatory network analysis identified candidate transcription factors associated with differentially expressed genes. A total of 15 subnetworks (communities) displayed altered connectivity patterns between obesity and nonobesity networks. Genes in proinflammatory pathways showed increased network connectivity in VAT samples with obesity, whereas the oxidative phosphorylation pathway displayed reduced connectivity (enrichment FDR < 0.05). Functional screening via RNA interference identified genes such as SOX30, SIRPB1, and OSBPL3 as potential network‐derived candidates influencing adipocyte differentiation. Conclusions: This approach highlights the network architecture in human obesity, identifies novel candidate genes, and generates new hypotheses regarding network‐assisted gene regulation in VAT. [ABSTRACT FROM AUTHOR]

BACKGROUND: Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip tolerance poses a significant threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress, and it may also regulate fall armyworm (FAW, Spodoptera frugiperda) Vip3Aa tolerance. RESULTS: In this study, a DNA methylation map was developed for FAW, and its function in regulating FAW Vip3Aa tolerance was explored. The FAW genome‐wide DNA methylation map showed that exons were preferred regions for DNA methylation and housekeeping genes were highly methylated. FAW was screened using Vip3Aa for ten generations, and bioassays indicated that Vip3Aa tolerance increased trans‐generationally. A comparison of DNA methylation maps between Vip3Aa‐tolerant and ‐susceptible strains showed that gene body methylation was positively correlated with gene expression level. FAW exhibits significant variation in DNA methylation among individuals, and Vip3Aa screening induces epigenetic variation based on DNA methylation. Moreover, the study demonstrated that a reduction in methylation density within the gene body of a 3′5′‐cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased tolerance of FAW to Vip3Aa, which was validated through RNA interference experiments. CONCLUSION: The DNA methylation map and mechanism of Vip3Aa tolerance improve our understanding of DNA methylation and its function in Lepidoptera and provide a new perspective for developing pest management strategies. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Calliptamus italicus L. is a major pest in Xinjiang grassland. The diapause overwintering strategy is one of the important reasons for the large population of this pest. This study investigated the function of the genes associated with the release of diapause (DIB, JHE and CAM) in Calliptamus italicus by RNA interference (RNAi) technology to aid in its biological control. RESULTS: The expression levels of DIB and its downstream‐associated genes (EcR and FTZ‐F1) in the eggs injected with dsDIB for 12 h decreased by 96.6%, 55.8% and 81.8%, respectively. Diapause began to terminate on day 3, and development was almost complete on day 6. However, the head was significantly smaller. The expression levels of JHE and its downstream‐associated genes (JHEH and VgR) at 48 h after dsJHE treatment decreased by 76.5%, 85.6% and 85.9%, respectively. The termination of diapause occured on day 3 of incubation. The development was basically complete on day 6, but the yolk had been incompletely absorbed. The expression of CAM and its downstream‐associated genes (CAMK4 and MYL) at 24 h after dsCAM treatment decreased by 42.4%, 95.3% and 82.7%, respectively. Diapause termination was completed on day 4 for incubation, and development was abnormal on day 6. The absorption of yolk was incomplete. CONCLUSION: DIB, JHE and CAM can delay the diapause termination of Calliptamus italicus eggs to different degrees and can be developed as potential target genes for its biological control. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The RNA interference (RNAi) efficiency of double‐stranded RNA (dsRNA) delivery to insects by various methods is different and the reduced efficacy of feeding dsRNA is partly due to the presence of DNA/RNA non‐specific endonuclease in the insect gut. However, the mechanism leading to the low RNAi efficiency of Nilaparvata lugens by feeding remains elusive. RESULTS: In this study, we identified a putatively DNA/RNA non‐specific endonuclease gene in the N. lugens genome database that was highly expressed in the first nymphal instar and the midgut. Different expression levels of NldsRNase after feeding and injection suggested that NldsRNase might interfere with oral RNAi in N. lugens. A co‐delivery RNAi strategy further revealed that the presence of NldsRNase reduces RNAi efficiency. In vitro dsRNA degradation experiments also showed that the stability of dsRNA was higher in a gut mixture from nymphs injected with dsNldsRNase. These results support the idea that the low oral RNAi response observed in N. lugens is likely due to the presence of NldsRNase. CONCLUSIONS: Our study provides insight into the differences in RNAi response between the injection and feeding of dsRNA in N. lugens and sheds light on the mechanisms underlying the reduced efficacy of RNAi via feeding. These findings may help to inform the development of more‐effective RNAi‐based strategies controlling N. lugens and other insect pests. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin‐encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta. RESULTS: Gut content (GC) and RNase A degraded double‐stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion–cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin‐encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial‐mediated RNA interference with 0.05 μg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8‐day‐old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged. CONCLUSION: The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin‐targeting pesticides. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In insects, tyrosine hydroxylase (TH) plays essential roles in cuticle tanning and cuticle pigmentation. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a leaf‐eating forest pest in salicaceous trees worldwide. However, the function of PverTH in P. versicolora is still unknown. In this study, we obtained a PverTH gene from transcriptome analysis. The expression analysis of PverTH showed that the highest expression was found in epidermis of larvae. In this study, we used RNA interference (RNAi) technology to knockdown the PverTH gene. The results showed that ingestion of dsTH led to cuticle coloration became lighter in larvae, pupae and adults. Knockdown of PverTH gene inhibited larval growth, and consequently caused higher mortality. In addition, RNAi of TH disrupted the cuticle tanning, caused lower pupation rate, lower eclosion rate and higher deformity rate. This study indicates that PverTH is vital for the cuticular pigments and cuticle tanning. Moreover, this research suggested that the development of PverTH gene as a potential target gene to control P. versicolora. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) has emerged as an eco‐friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi‐mediated pest control. We argue that the knowledge‐based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome‐wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large‐scale RNAi screenings are typically challenging. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Persistent plant viruses are widespread in natural ecosystems. However, little is known about why persistent infection with these viruses may cause little or no harm to their host. Here, we discovered a new polerovirus that persistently infected wild rice plants by deep sequencing and assembly of virus‐derived small‐interfering RNAs (siRNAs). The new virus was named Rice tiller inhibition virus 2 (RTIV2) based on the symptoms developed in cultivated rice varieties following Agrobacterium‐mediated inoculation with an infectious RTIV2 clone. We showed that RTIV2 infection induced antiviral RNA interference (RNAi) in both the wild and cultivated rice plants as well as Nicotiana benthamiana. It is known that virulent virus infection in plants depends on effective suppression of antiviral RNAi by viral suppressors of RNAi (VSRs). Notably, the P0 protein of RTIV2 exhibited weak VSR activity and carries alanine substitutions of two amino acids broadly conserved among diverse poleroviruses. Mixed infection with umbraviruses enhanced RTIV2 accumulation and/or enabled its mechanical transmission in N. benthamiana. Moreover, replacing the alanine at either one or both positions of RTIV2 P0 enhanced the VSR activity in a co‐infiltration assay, and RTIV2 mutants carrying the corresponding substitutions replicated to significantly higher levels in both rice and N. benthamiana plants. Together, our findings show that as a persistent plant virus, RTIV2 carries specific mutations in its VSR gene to weaken viral suppression of antiviral RNAi. Our work reveals a new strategy for persistent viruses to maintain long‐term infection by weak suppression of the host defence response. [ABSTRACT FROM AUTHOR]

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium‐induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi‐based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi‐based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation. [ABSTRACT FROM AUTHOR]

The foregut, located at the front of the digestive tract, serves a vital role in insects by storing and grinding food into small particles. The innermost layer of the foregut known as the chitinous intima, comes into direct contact with the food and acts as a protective barrier against abrasive particles. Knickkopf (Knk) is required for chitin organization in the chitinous exoskeleton, tracheae and wings. Despite its significance, little is known about the biological function of Knk in the foregut. In this study, we found that LmKnk was stably expressed in the foregut, and highly expressed before molting in Locusta migratoria. To ascertain the biological function of LmKnk in the foregut, we synthesized specific double‐stranded LmKnk (dsLmKnk) and injected it into locusts. Our findings showed a significant decrease in the foregut size, along with reduced food intake and accumulation of residues in the foregut after dsLmKnk injection. Morphological observations revealed that newly formed intima became thinner and lacked chitin lamella. Furthermore, fluorescence immunohistochemistry revealed that LmKnk was located in the apical region of new intima and epithelial cells. Taken together, this study provides insights into the biological function of LmKnk in the foregut, and identifies the potential target gene for exploring biological pest management strategies. [ABSTRACT FROM AUTHOR]

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)—a destructive invasive pest—can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real‐time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full‐length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node‐conserved structural features and six conserved DNA‐binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double‐stranded RNA and exposing Tuta absoluta to 35°C heat shock or −12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1‐mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi‐driven regulation of Tuta absoluta. [ABSTRACT FROM AUTHOR]

The function of DNA methylation in insects and the DNA methyltransferase (Dnmt) genes that influence methylation remains uncertain. We used RNA interference to reduce the gene expression of Dnmt1 within the whitefly Bemisia tabaci (Hemiptera:Aleyrodidae; Gennadius), a hemipteran species that relies on Dnmt1 for proper gametogenesis. We then used RNA‐seq to test an a priori hypothesis that meiosis‐related genetic pathways would be perturbed. We generally did not find an overall effect on meiosis‐related pathways. However, we found that genes in the Wnt pathway, genes associated with the entry into meiosis in vertebrates, were differentially expressed. Our results are consistent with Dnmt1 knockdown influencing specific pathways and not causing general transcriptional response. This is a finding that is also seen with other insect species. We also characterised the methylome of B. tabaci and assessed the influence of Dnmt1 knockdown on cytosine methylation. This species has methylome characteristics comparable to other hemipterans regarding overall level, enrichment within gene bodies, and a bimodal distribution of methylated/non‐methylated genes. Very little differential methylation was observed, and difference in methylation were not associated with differences in gene expression. The effect on Wnt presents an interesting new candidate pathway for future studies. [ABSTRACT FROM AUTHOR]

BACKGROUND: A GPCR (G protein‐coupled receptor) target‐based approach was applied to identify antagonists of the arthropod‐specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty‐three structural analogs of previously identified tick kinin antagonists were screened in a 'dual‐addition' calcium fluorescence assay using a CHO‐K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non‐cytotoxic antagonists, four of which were partial (SACC‐0428764, SACC‐0428780, SACC‐0428800, and SACC‐0428803), and three were full antagonists (SACC‐0428799, SACC‐0428801, and SACC‐0428815). Four of these antagonists (SACC‐0428764, SACC‐0428780, SACC‐0428799, and SACC‐0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC‐0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 μm). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Pyriproxyfen is an insect growth regulator (IGR) that is effective against various types of insect pests. However, the molecular mechanism underlying pyriproxyfen effects on insect reproduction remains unclear. Thus, in this study, we attempted to uncover the mechanisms underlying the impact of pyriproxyfen on the reproductive system of the model organism Drosophila melanogaster. RESULTS: A significant decrease in Drosophila reproduction was observed after pyriproxyfen treatment. The juvenile hormone (JH) titer was significantly increased (120.4%) in the ovary samples of pyriproxyfen‐treated flies. Likewise, the concentrations of key enzymes and the expression of key genes related to the JH signaling pathway were also increased in the pyriproxyfen‐treated group compared with the control group. Furthermore, pyriproxyfen treatment significantly increased (15.6%) the number of germline stem cells (GSCs) and significantly decreased (17%) the number of cystoblasts (CBs). However, no significant differences were observed in the number of somatic cells. We performed RNA interference (RNAi) on five key genes (Met, Tai, gce, ftz‐f1, and hairy) related to the JH signaling pathway in germ cells using the germ cell‐specific Gal4 driver. Interestingly, RNAi of the selected genes significantly decreased the number of both GSCs and CBs in pyriproxyfen‐treated transgenic flies. These results further validate that pyriproxyfen enhances GSC proliferation by up‐regulating JH signaling. CONCLUSION: Our results indicate that pyriproxyfen significantly decreases reproduction by affecting germ cells in female adult ovaries. The effect of pyriproxyfen on germ cell proliferation and differentiation is mediated by an increase in JH signaling. This study has significant implications for optimizing pest control strategies, developing sustainable agriculture practices, and understanding the mechanism of insecticide action. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap‐sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV‐tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. RESULTS: Insecticide susceptible D. citri reared on citrus infected with CTV‐tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non‐infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV‐tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field‐collected psyllids from areas with known broad‐spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV‐tCYP450 citrus trees as compared with those reared on controls. CONCLUSION: The integration of citrus‐mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Summary: Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5′untranslated region (5′UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG).Here, we report an efficient gene silencing method by introducing a tailor‐designed uATG‐containing element (ATGE) into the 5′UTR of genes in plants, occupying the original start site to act as a new pATG.Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding.Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement. See also the Commentary on this article by Li & Wei, 243: 2052–2054. [ABSTRACT FROM AUTHOR]

ABSTRACT Plants have developed various resistance mechanisms against herbivorous insects through prolonged coevolution. Plant defence responses can be triggered by specific compounds present in insect saliva. Apyrase, a known enzyme that catalyzes the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and inorganic phosphorus, has recently been identified in some herbivorous insects. However, whether insect salivary apyrase induces or inhibits plant responses remains poorly understood. In this study, we identified an apyrase‐like protein in the salivary proteome of the fall armyworm, Spodoptera frugiperda, named Sfapyrase. Sfapyrase was primarily expressed in the salivary gland and secreted into plants during insect feeding. Transient expression of Sfapyrase in tobacco and maize enhanced plant resistance and resulted in decreased insect feeding. Knockdown of Sfapyrase through RNA interference led to increased growth and feeding of S. frugiperda. Furthermore, we showed that Sfapyrase activates the jasmonic acid signalling pathway and promotes the synthesis of secondary metabolites, especially benzoxazinoids, thereby enhancing resistance to S. frugiperda. In summary, our findings demonstrated that Sfapyrase acts as a salivary elicitor, inducing maize jasmonic acid defence responses and the production of insect‐resistant benzoxazinoids. This study provides valuable insights into plant–insect interactions and offers potential targets for developing innovative insect pest management strategies. [ABSTRACT FROM AUTHOR]

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post‐ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC‐layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post‐ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)‐cAMP‐PKA‐CREB signaling cascade, is necessary for mGC‐layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC‐layer stiffening", shedding light on the mechanism that prevents mGC escape from the post‐ovulatory follicle. [ABSTRACT FROM AUTHOR]

Antiviral responses induced by double‐stranded RNA (dsRNA) include RNA interference (RNAi) and pattern‐triggered immunity (PTI), but their relative contributions to antiviral defence are not well understood. We aimed at testing the impact of exogenous applied dsRNA on both layers of defence against potato virus X expressing GFP (PVX‐GFP) in Nicotiana benthamiana. Co‐inoculation of PVX‐GFP with either sequence‐specific (RNAi) or nonspecific dsRNA (PTI) showed that nonspecific dsRNA reduced virus accumulation in both inoculated and systemic leaves. However, nonspecific dsRNA was a poor inducer of antiviral immunity compared to a sequence‐specific dsRNA capable of triggering the RNAi response, and plants became susceptible to systemic infection. Studies with a PVX mutant unable to move from cell to cell indicated that the interference with PVX‐GFP triggered by nonspecific dsRNA operated at the single‐cell level. Next, we performed RNA‐seq analysis to examine similarities and differences in the transcriptome triggered by dsRNA alone or in combination with viruses harbouring sequences targeted or not by dsRNA. Enrichment analysis showed an over‐representation of plant‐pathogen signalling pathways, such as calcium, ethylene and MAPK signalling, which are typical of antimicrobial PTI. Moreover, the transcriptomic response to the virus targeted by dsRNA had a greater impact on defence than the non‐targeted virus, highlighting qualitative differences between sequence‐specific RNAi and nonspecific PTI responses. Together, these results further our understanding of plant antiviral defence, particularly the contribution of nonspecific dsRNA‐mediated PTI. We envisage that both sequence‐specific RNAi and nonspecific PTI pathways may be triggered via topical application of dsRNA, contributing cumulatively to plant protection against viruses. [ABSTRACT FROM AUTHOR]

Background: Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. Methods: The mouse model of IRI‐AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation‐mass spectrometry was performed to identify potential ANKRD1‐interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. Results: First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno‐associated virus (rAAV9)‐mitigated ischaemia/reperfusion‐induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti‐ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post‐translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63‐linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. Conclusions: Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI‐AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. Key Points/Highlights: Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro.ANKRD1 knockdown mitigates kidney damage and preserves renal function.Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI.ANKRD1 promotes acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway.The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1‐mediated ubiquitination of ACSL3. [ABSTRACT FROM AUTHOR]

To present the expression of calsyntenin‐1 (Clstn1) in the brain and investigate the potential mechanism of Clstn1 in lithium–pilocarpine rat seizure models. Thirty‐five male SD adult rats were induced to have seizures by intraperitoneal injection of lithium chloride pilocarpine. Rats exhibiting spontaneous seizures were divided into the epilepsy (EP) group (n = 15), whereas those without seizures were divided into the control group (n = 14). Evaluate the expression of Clstn1 in the temporal lobe of two groups using Western blotting, immunohistochemistry, and immunofluorescence. Additionally, 55 male SD rats were subjected to status epilepticus (SE) using the same induction method. Rats experiencing seizures exceeding Racine's level 4 (n = 48) were randomly divided into three groups: SE, SE + control lentivirus (lentiviral vector expressing green fluorescent protein [LV‐GFP]), and SE + Clstn1‐targeted RNA interference lentivirus (LV‐Clstn1‐RNAi). The LV‐GFP group served as a control for the lentiviral vector, whereas the LV‐Clstn1‐RNAi group received a lentivirus designed to silence Clstn1 expression. These lentiviral treatments were administered via hippocampal stereotactic injection 2 days after SE induction. Seven days after SE, Western blot analysis was performed to evaluate the expression of Clstn1 in the hippocampus and temporal lobe. Meanwhile, we observed the latency of spontaneous seizures and the frequency of spontaneous seizures within 8 weeks among the three groups. The expression of Clstn1 in the cortex and hippocampus of the EP group was significantly increased compared to the control group (p <.05). Immunohistochemistry and immunofluorescence showed that Clstn1 was widely distributed in the cerebral cortex and hippocampus of rats, and colocalization analysis revealed that it was mainly co expressed with neurons in the cytoplasm. Compared with the SE group (11.80 ± 2.17 days) and the SE + GFP group (12.40 ± 1.67 days), there was a statistically significant difference (p <.05) in the latency period of spontaneous seizures (15.14 ± 2.41 days) in the SE + Clstn1 + RNAi group rats. Compared with the SE group (4.60 ± 1.67 times) and the SE + GFP group (4.80 ± 2.05 times), the SE + Clstn1 + RNAi group (2.0 ±.89 times) showed a significant reduction in the frequency of spontaneous seizures within 2 weeks of chronic phase in rats (p <.05). Elevated Clstn1 expression in EP group suggests its role in EP onset. Targeting Clstn1 may be a potential therapeutic approach for EP management. [ABSTRACT FROM AUTHOR]

Summary: The foundation of most food production systems underpinning global food security is the careful management of soil resources. Embedded in the concept of soil health is the impact of diverse soil‐borne pests and pathogens, and phytoparasitic nematodes represent a particular challenge. Root‐knot nematodes and cyst nematodes are severe threats to agriculture, accounting for annual yield losses of US$157 billion. The control of soil‐borne phytoparasitic nematodes conventionally relies on the use of chemical nematicides, which can have adverse effects on the environment and human health due to their persistence in soil, plants, and water. Nematode‐resistant plants offer a promising alternative, but genetic resistance is species‐dependent, limited to a few crops, and breeding and deploying resistant cultivars often takes years. Novel approaches for the control of phytoparasitic nematodes are therefore required, those that specifically target these parasites in the ground whilst minimizing the impact on the environment, agricultural ecosystems, and human health. In addition to the development of next‐generation, environmentally safer nematicides, promising biochemical strategies include the combination of RNA interference (RNAi) with nanomaterials that ensure the targeted delivery and controlled release of double‐stranded RNA. Genome sequencing has identified more than 75 genes in root knot and cyst nematodes that have been targeted with RNAi so far. But despite encouraging results, the delivery of dsRNA to nematodes in the soil remains inefficient. In this review article, we describe the state‐of‐the‐art RNAi approaches targeting phytoparasitic nematodes and consider the potential benefits of nanotechnology to improve dsRNA delivery. [ABSTRACT FROM AUTHOR]

Porphyrias are rare, mostly inherited disorders resulting from altered activity of specific enzymes in the haem synthesis pathway that lead to accumulation of pathway intermediates. Photocutaneous symptoms occur when excess amounts of photoreactive porphyrins circulate in the blood to the skin, whereas increases in potentially neurotoxic porphyrin precursors are associated with neurovisceral symptoms. Current therapies are suboptimal and their mechanisms are not well established. As described here, emerging therapies address underlying disease mechanisms by introducing a gene, RNA or other specific molecule with the potential to cure or slow progression of the disease. Recent progress in nanotechnology and nanoscience, particularly regarding particle design and formulation, is expanding disease targets. More secure and efficient drug delivery systems have extended our toolbox for transferring specific molecules, especially into hepatocytes, and led to proof‐of‐concept studies in animal models. Repurposing existing drugs as molecular chaperones or haem synthesis inhibitors is also promising. This review summarizes key examples of these emerging therapeutic approaches and their application for hepatic and erythropoietic porphyrias. [ABSTRACT FROM AUTHOR]

In the wake of global population increases and stagnating capture fishery production, there exists a requirement for novel biotechnologies to increase aquaculture productivity to offset demand. Through recognition of limitations imposed by aquaculture—increased incidence of disease, stress, cannibalism, and mortality in captivity—researchers continue to investigate RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR) technologies for their potential to alleviate these at a genetic level. In decapods, with the exception of generating desirable all‐male monosex populations in Macrobrachium rosenbergii, RNAi and CRISPR technologies have yet to provide a significant commercial use case. This literature review provides a comprehensive examination of RNAi and CRISPR in decapods, along with popular delivery methods (injection/microinjection; electroporation; receptor‐mediated ovary transduction of cargo [ReMOT]; and nanoparticles). It identifies a number of limitations including scalability bottlenecks imposed by RNAi delivery methods, and deferred gene editing progress as a result of CRISPR's infancy. Finally, it recognises gene editing induced gene silencing (GEiGS®) as a novel method for silencing gene expression, and suggests an interim examination of transgenerational gene silencing (TGS) ('parental RNAi') in decapods to enhance aquaculture productivity. [ABSTRACT FROM AUTHOR]

The review encapsulates the comprehensive exploration of RNA interference technology's application in shrimp aquaculture, covering molecular intricacies, production methods, and practical applications. The emphasis is on exploring the stability and delivery of gene‐specific double‐stranded RNA, particularly through nanoencapsulation. This is because the susceptibility of dsRNA to degradation and limited cellular penetration, emphasising the need for effective delivery mechanisms. The review presents an in‐depth exploration of nanoparticles for the encapsulation and delivery of dsRNA, including virus‐like particles (VLPs), and non‐viral based nanoparticles such as liposomes, chitosan, and beta‐glucan. VLPs, derived from non‐infectious shrimp virus structures, exhibit biocompatibility and natural functionality, making them suitable carriers for dsRNA. The application of chitosan and its derivatives are explored for their efficacy in reducing viral infections in shrimp. Beta‐glucan particles are examined for their immunostimulant properties in shrimp aquaculture. The text emphasises the use of yeast‐based glucan particles for encapsulating dsRNA, showcasing their potential in preventing viral diseases in shrimp. The review introduces cautionary considerations for nanoparticle formulation, highlighting factors such as pH, organic solvents, metal ions, and environmental ribonuclease that can impact dsRNA stability during synthesis. In conclusion, the text discusses the potential economic worthiness and environmental risk assessment of dsRNA technology in shrimp aquaculture. While acknowledging successful applications in other environments, it underscores the need for regulatory approval and risk assessment for dsRNA‐based products in the aquaculture industry. The evaluation of dsRNA in real‐world shrimp farms is deemed necessary for commercial utilisation, taking into account income, expenses, safety, and environmental considerations. [ABSTRACT FROM AUTHOR]

BACKGROUND: Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical‐based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double‐stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. RESULTS: A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid‐based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi‐mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. CONCLUSION: This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA‐mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]