Developmental Tissues Research Articles

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae)

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

EpOME mediates the immune resolution and its alkoxide analog enhances the virulence of microbial insecticides against the legume pod borer, Maruca vitrata

Excessive and unnecessary immune responses cause serious adverse effects due to self-tissue damage and energy consumption, particularly at the late stage of infection to terminate the induced immunity. Unlike mammals, which use long-chain fatty acid oxylipins, C18 oxygenated polyunsaturated fatty acids are suggested to act as immune resolvins in insects, including two epoxyoctadecamonoenoic acids (9,10-EpOME and 12,13-EpOME). This study investigated the physiological roles of EpOMEs in immune resolution in the lepidopteran insect, Maruca vitrata. The levels of two EpOMEs in the larvae increased during the late infection stage upon immune challenge. At their peak concentrations at 96 h post-infection (pi), both EpOMEs were found at similar levels: 323.18 pg/mg body weight for 9,10-EpOME and 322.07 pg/mg body weight for 12,13-EpOME. Both EpOMEs inhibited cellular and humoral immune responses, with 12,13-EpOME being more potent than 9,10-EpOME. Genes associated with EpOME synthase and degradation, identified as Mv-CYP1 and Mv-sEH, were detected in various developmental stages and tissues of M. vitrata. RNA interference (RNAi) targeting Mv-CYP1 failed to inhibit the immune response, whereas RNAi targeting Mv-sEH enhanced the immunosuppression. In contrast to the acute (< 12 h pi) immune response involving eicosanoid biosynthesis, the expression of these two genes linked to EpOME metabolism increased significantly at the late infection stage (> 12 h pi). Several alkoxide analogs of EpOME, with the epoxide group replaced by an alkoxide group, were synthesized; one such derivative demonstrated substantially greater efficacy than the natural EpOMEs in inhibiting the immune response. Additionally, using EpOME alkoxide significantly increased the effectiveness of microbial insecticides. Moreover, exposing young larvae to sublethal doses of EpOME alkoxide or sEH inhibitor induced severe developmental delays. These results suggest a novel strategy for insect pest control using insect immune resolvin analogs.

Genome-Wide Identification and Expression Analysis of the Cys2His2 Zinc Finger Protein Gene Family in Flammulina filiformis.

Zinc finger proteins (ZFPs) are essential transcription factors in eukaryotes, particularly the extensively studied C2H2 family, which is known for its involvement in various biological processes. This research provides a thorough examination and analysis of the C2H2-ZFP gene family in Flammulina filiformis. Using bioinformatics tools, 58 FfC2H2-ZFP genes spread across 11 chromosomes were identified and scrutinized in detail for their gene structures, protein characteristics, and phylogenetic relationships. The study of phylogenetics and synteny sheds light on the evolutionary relationships among C2H2-ZFPs in F. filiformis and other fungi, revealing a complex evolutionary past. The identification of conserved cis-regulatory elements in the gene promoter regions suggests intricate functionalities, particularly in the developmental and stress response pathways. By utilizing RNA-seq and qRT-PCR techniques, the expression patterns of these genes were explored across different developmental stages and tissues of F. filiformis, unveiling distinct expression profiles. Notably, significant expression variations were observed in the stipe elongation region and pilei of various sizes, indicating potential roles in fruiting body morphogenesis. This study enhances our knowledge of the C2H2-ZFP gene family in F. filiformis and lays the groundwork for future investigations into their regulatory mechanisms and applications in fungal biology and biotechnology.

Conservation and Divergence of PEPC Gene Family in Different Ploidy Bamboos.

Phosphoenolpyruvate carboxylase (PEPC), as a necessary enzyme for higher plants to participate in photosynthesis, plays a key role in photosynthetic carbon metabolism and the stress response. However, the molecular biology of the PEPC family of Bambusoideae has been poorly studied, and the function of its members in the growth and development of Bambusoideae is still unclear. Here, we identified a total of 62 PEPC family members in bamboo. All the PEPC genes in the bamboo subfamily were divided into twelve groups, each group typically containing significantly fewer PEPC members in Olyra latifolia than in Phyllostachys edulis, Dendrocalamus latiflorus and Dendrocalamus brandisii. The results of an intraspecific and interspecies collinearity analysis showed that fragment replication and whole genome replication were the main driving forces of bamboo PEPC members. Furthermore, the Ka/Ks values of collinear genes showed that bamboo PEPC experienced purification selection. In addition, the promoter region of PEPC genes contains cis-acting elements related to light response, plant hormone response and response to stress. An analysis of the expression levels of the PEPC family in different developmental stages and tissues of bamboo shoots has shown that PhePEPC7, PhePEPC9 and PhePEPC10 were highly expressed in the leaves of non-flowering plants and culms. Furthermore, PhePEPC6 was significantly upregulated in leaves after GA treatment. Further research has shown that PhePEPC6 was mainly localized in the cell membrane. This provides a solid bioinformatics foundation for further understanding the biological functions of the bamboo PEPC family.

Identification and expression patterns of voltage-gated sodium channel genes with intron retentions in different strains of Bactrocera dorsalis

Pyrethroid are the primary insecticides used for controlling of Bactricera dorsalis, a highly destructive and invasive fruit pest. Field populations have developed serious resistance, especially to β-cypermethrin. While mutations in the voltage-gated sodium channel (Vgsc) are a common mechanism of pyrethroid resistance, variations in BdVgsc associated with β-cypermethrin resistance remain unclear. Here, we reported the resistance levels of five field populations from China, with resistance ratio ranging from 1.54 to 21.34-fold. Cloning the full length of BdVgsc revealed no specific or known amino acid mutations between the most resistant population and the susceptible strain. However, three types of partial intron retention (IRE4–5, IRE19-f and IREL-24) were identified in BdVgsc transcripts, with these intron retentions containing stop codons. The expression of IRE4–5 transcripts and total BdVgsc showed different trends across developmental stages and tissues. Exposure to β-cypermethrin led to increased expression of IRE4–5. Comparison of genomic and transcriptional sequences reveled that IRE4–5 transcripts had two types (IRE4–5.5 T and IRE4–5.6 T) caused by genomic variations. Both field and congenic strains indicated that homozygotes for IRE4–5.5 T had lower IRE4–5 transcript levels than homozygotes for IRE4–5.6 T. However, congenic and field strains exhibited inconsistent results about the association of expression levels of IRE4–5 transcripts with sensitivity to β-cypermethrin. In summary, this study is the first to identify intron retention transcripts in the Vgsc gene from B. dorsalis and to examine their expression patterns across different developmental stages, tissues, and strains with varying sensitivities to β-cypermethrin. The potential role of the intron retentions of BdVgsc in insecticide toxicity is also discussed.

Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four LdKnk-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing LdKnk and LdKnk3-5' led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.

Dynamic patterns of gene expressional and regulatory variations in cotton heterosis.

Although the application of heterosis has significantly increased crop yield over the past century, the mechanisms underlying this phenomenon still remain obscure. Here, we applied transcriptome sequencing to unravel the impacts of parental expression differences and transcriptomic reprogramming in cotton heterosis. A high-quality transcriptomic atlas covering 15 developmental stages and tissues was constructed for XZM2, an elite hybrid of upland cotton (Gossypium hirsutum L.), and its parental lines, CRI12 and J8891. This atlas allowed us to identify gene expression differences between the parents and to characterize the transcriptomic reprogramming that occurs in the hybrid. Our analysis revealed abundant gene expression differences between the parents, with pronounced tissue specificity; a total of 1,112 genes exhibited single-parent expression in at least one tissue. It also illuminated transcriptomic reprogramming in the hybrid XZM2, which included both additive and non-additive expression patterns. Coexpression networks between parents and hybrid constructed via weighted gene coexpression network analysis identified modules closely associated with fiber development. In particular, key regulatory hub genes involved in fiber development showed high-parent dominant or over dominant patterns in the hybrid, potentially driving the emergence of heterosis. Finally, high-depth resequencing data was generated and allele-specific expression patterns examined in the hybrid, enabling the dissection of cis- and trans-regulation contributions to the observed expression differences. Parental transcriptional differences and transcriptomic reprogramming in the hybrid, especially the non-additive upregulation of key genes, play an important role in shaping heterosis. Collectively, these findings provide new insights into the molecular basis of heterosis in cotton.

Regulation of nitro-oxidative homeostasis: an effective approach to enhance salinity tolerance in plants.

Soil salinity is a major constraint for sustainable agricultural productivity, which together with the incessant climate change may be transformed into a severe threat to the global food security. It is, therefore, a serious concern that needs to be addressed expeditiously. The overproduction and accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the key events occurring during salt stress, consequently employing nitro-oxidative stress and programmed cell death in plants. However, very sporadic studies have been performed concerning different aspects of nitro-oxidative stress in plants under salinity stress. The ability of plants to tolerate salinity is associated with their ability to maintain the cellular redox equilibrium mediated by both non-enzymatic and enzymatic antioxidant defense mechanisms. The present review emphasizes the mechanisms of ROS and RNS generation in plants, providing a detailed evaluation of how redox homeostasis is conserved through their effective removal. The uniqueness of this article stems from its incorporation of expression analyses of candidate genes for different antioxidant enzymes involved in ROS and RNS detoxification across various developmental stages and tissues of rice, utilizing publicly available microarray data. It underscores the utilization of modern biotechnological methods to improve salinity tolerance in crops, employing different antioxidants as markers. The review also explores how various transcription factors contribute to plants' ability to tolerate salinity by either activating or repressing the expression of stress-responsive genes. In summary, the review offers a thorough insight into the nitro-oxidative homeostasis strategy for extenuating salinity stress in plants.

Accelerated evolution in the human lineage led to gain and loss of transcriptional enhancers in the RBFOX1 locus.

A long-standing goal of evolutionary biology is to decode how changes in gene regulatory networks contribute to human-specific traits. Human accelerated regions (HARs) are prime candidates for driving gene regulatory modifications in human development. The RBFOX1 locus is densely populated with HARs, providing a set of potential regulatory elements that could have changed its expression in the human lineage. Here, we examined the role of RBFOX1-HARs using transgenic zebrafish reporter assays and identified 15 transcriptional enhancers that are active in the developing nervous system, 9 of which displayed differential activity between the human and chimpanzee sequences. The engineered loss of two selected RBFOX1-HARs in knockout mouse models modified Rbfox1 expression at specific developmental stages and tissues in the brain, influencing the expression and splicing of a high number of Rbfox1 target genes. Our results provided insight into the spatial and temporal changes in gene expression driven by RBFOX1-HARs.

Genome-wide identification and phylogenetic analysis of the tetraspanin gene family in lepidopteran insects and expression profiling analysis in Helicoverpa armigera.

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 ω 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.

Comparative transcriptome analysis reveals genes involved in trichome development and metabolism in tobacco

BackgroundThe glandular trichomes of tobacco (Nicotiana tabacum) can efficiently produce secondary metabolites. They act as natural bioreactors, and their natural products function to protect plants against insect-pests and pathogens and are also components of industrial chemicals. To clarify the molecular mechanisms of tobacco glandular trichome development and secondary metabolic regulation, glandular trichomes and glandless trichomes, as well as other different developmental tissues, were used for RNA sequencing and analysis.ResultsBy comparing glandless and glandular trichomes with other tissues, we obtained differentially expressed genes. They were obviously enriched in KEGG pathways, such as cutin, suberine, and wax biosynthesis, flavonoid and isoflavonoid biosynthesis, terpenoid biosynthesis, and plant–pathogen interaction. In particular, the expression levels of genes related to the terpenoid, flavonoid, and wax biosynthesis pathway mainly showed down-regulation in glandless trichomes, implying that they lack the capability to synthesize certain exudate compounds. Among the differentially expressed genes, 234 transcription factors were found, including AP2-ERFs, MYBs, bHLHs, WRKYs, Homeoboxes (HD-ZIP), and C2H2-ZFs. These transcription factor and genes that highly expressed in trichomes or specially expressed in GT or GLT. Following the overexpression of R2R3-MYB transcription factor Nitab4.5_0011760g0030.1 in tobacco, an increase in the number of branched glandular trichomes was observed.ConclusionsOur data provide comprehensive gene expression information at the transcriptional level and an understanding of the regulatory pathways involved in glandular trichome development and secondary metabolism.

In silico identification and expression analysis of superoxide dismutases in Tenebrio molitor.

Insects encounter various environmental stresses, in response to which they generate reactive oxygen species (ROS). Superoxide dismutase (SOD) is an antioxidant metalloenzyme that scavenges superoxide radicals to prevent oxidative damage. To investigate expressions of SODs under oxidative stress in Tenebrio molitor. Here, we investigated the transcriptional expression of SODs by pesticide and heavy metals in Tenebrio moltior. First, we searched an RNA-Seq database for T. molitor SOD (TmSOD) genes and identified two SOD isoforms (TmSOD1-iso1 and iso2). We examined their activities under developmental stage, tissue-specific, and various types (pesticide and heavy metal) of oxidative stress by using qPCR. Our results revealed two novel forms of TmSODs. These TmSODs had a copper/zinc superoxide dismutase domain, active site, Cu2+ binding site, Zn2+ binding site, E-class dimer interface, and P-class dimer interface. TmSODs (TmSOD1-iso1 and iso2) were expressed in diverse developmental phases and tissues. Pesticides and heavy metals caused an upregulation of these TmSODs. Our findings suggest that the two TmSODs have different functions in T. molitor, providing insights into the detoxification ability of T. molitor.

ChCaspase-1 cleaves ChGasdermin-E, inducing pyroptosis, and participates in the bacterial defence of the oyster Crassostrea hongkongensis

Oysters have evolved an effective and powerful innate immune system to recognize foreign substances and eliminate invaders through orchestrated immune responses such as programmed cell death (PCD), including apoptosis, autophagy, necroptosis, and pyroptosis. Both apoptosis and autophagy have been considered to be crucial mechanisms for oyster defence. However, the role of pyroptosis, a gasdermin (GSDM)-dependent programmed cell death pathway that is critical for defence against infection, in oysters remains unclear. In this study, we investigated the activity of Crassostrea hongkongensis GSDME (ChGSDME), which is cleaved by inflammatory C.hongkongensis caspase-1 (ChCASP1). qRT–PCR analysis showed that ChCASP1 and ChGSDME were widely expressed in oyster developmental stages and tissues, and responded to Vibrio coralliilyticus challenge. Meanwhile, hemocyte death was reduced after knockdown of ChCASP1 or ChGSDME by RNA interference (RNAi). Following injection of V. coralliilyticus, the cell death rate of the knockdown ChCASP1 or ChGSDME group was significantly lower than that of the control group. In vitro, coexpressing ChCASP1 and ChGSDME in HEK293T cells resulted in pyroptotic morphology, a bubble-like membrane, and a truncated protein (ChGSDME N-terminal). Subsequently, immunofluorescence revealed that ChGSDME-NT oligomerized at the cell membrane, inducing bubble-like membrane formation, thereby causing low cytotoxicity. Luciferase reporter gene analysis revealed that ChCASP1 significantly upregulated Nuclear factor kappa-B (NF-κB) and modestly upregulated the p53 signalling pathway. Taken together, our study demonstrated that the pyroptosis-related genes ChCASP1 and ChGSDME mediate pyroptosis in HEK293T cells and play important roles in cell death during V. coralliilyticus infection in C. hongkongensis.

DNA Methylation Machinery in Gastropod Mollusks.

The role of DNA methylation in mollusks is just beginning to be understood. This review synthesizes current knowledge on this potent molecular hallmark of epigenetic control in gastropods-the largest class of mollusks and ubiquitous inhabitants of diverse habitats. Their DNA methylation machinery shows a high degree of conservation in CG maintenance methylation mechanisms, driven mainly by DNMT1 homologues, and the presence of MBD2 and MBD2/3 proteins as DNA methylation readers. The mosaic-like DNA methylation landscape occurs mainly in a CG context and is primarily confined to gene bodies and housekeeping genes. DNA methylation emerges as a critical regulator of reproduction, development, and adaptation, with tissue-specific patterns being observed in gonadal structures. Its dynamics also serve as an important regulatory mechanism underlying learning and memory processes. DNA methylation can be affected by various environmental stimuli, including as pathogens and abiotic stresses, potentially impacting phenotypic variation and population diversity. Overall, the features of DNA methylation in gastropods are complex, being an essential part of their epigenome. However, comprehensive studies integrating developmental stages, tissues, and environmental conditions, functional annotation of methylated regions, and integrated genomic-epigenomic analyses are lacking. Addressing these knowledge gaps will advance our understanding of gastropod biology, ecology, and evolution.

The Expression and Function of Notch Involved in Ovarian Development and Fecundity in Basilepta melanopus.

Basilepta melanopus is a pest that severely affects oil tea plants, and the Notch signaling pathway plays a significant role in the early development of insect ovaries. In this study, we explored the function of the notch gene within the Notch signaling pathway in the reproductive system of B. melanopus. The functional domains and expression patterns of Bmnotch were analyzed. Bmnotch contains 45 epidermal growth factor-like (EGF-like) domains, one negative regulatory region, one NODP domain and one repeat-containing domain superfamily. The qPCR reveals heightened expression in early developmental stages and specific tissues like the head and ovaries. The RNA interference (RNAi)-based suppression of notch decreased its expression by 52.1%, exhibiting heightened sensitivity to dsNotch at lower concentrations. Phenotypic and mating experiments have demonstrated that dsNotch significantly impairs ovarian development, leading to reduced mating frequencies and egg production. This decline underscores the Notch pathway's crucial role in fecundity. The findings advocate for RNAi-based, Notch-targeted pest control as an effective and sustainable strategy for managing B. melanopus populations, signifying a significant advancement in forest pest control endeavors.

Functional analysis of adipokinetic hormone signaling in reproductive diapause of Coccinella septempunctata.

The ladybeetle, Coccinella septempunctata, an important predator, is widely used to control aphids, whiteflies, mites, thrips, and lepidopteran pests. Diapause control technology is key to extending C. septempunctata shelf-life and commercialization. Lipid accumulation is a major feature of reproductive diapause, but the function of AKH signaling as a regulator of lipid mobilization in reproductive diapause remains unclear. This study aimed to identify and characterize AKH and AKHR genes, and clarify their functions in reproductive diapause. The relative expression levels of CsAKH and CsAKHR were the highest in the head and fat body, respectively, and were significantly decreased under diapause conditions, both in developmental stages and tissues (head, midgut, fat body, and ovary). Furthermore, CsAKH and CsAKHR expression was increased significantly after juvenile hormone (JH) injection, but CsMet silencing significantly inhibited CsAKH and CsAKHR expression, whereas CsMet knockdown blocked the induction effect of JH. CsAKH and CsAKHR knockdown significantly reduced water content, increased lipid storage, and promoted the expression of genes related to lipid synthesis, but significantly blocked ovarian development, and induced forkhead box O (FOXO) gene expression in C. septempunctata under reproduction conditions. By contrast, injection of AKH peptide significantly inhibited FOXO expression, reduced lipid storage, and increased water content in C. septempunctata under diapause conditions. These results indicate that CsAKH and CsAKHR are involved in the regulation of lipid accumulation and ovarian development during diapause in C. septempunctata, and provide a promising target for manipulating C. septempunctata diapause. © 2024 Society of Chemical Industry.

RNAi efficiency is enhanced through knockdown of double-stranded RNA-degrading enzymes in butterfly Papilio xuthus.

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double-stranded RNA-degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1-6) were identified in P. xuthus genome, and their dsRNA-degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA-degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi-of-RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

Antimicrobial peptide cecropin B functions in pathogen resistance of Mythimna separata.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Identification, expression analysis, and functional verification of three opsin genes related to the phototactic behaviour of Ostrinia furnacalis.

Ostrinia furnacalis is a disreputable herbivorous pest that poses a serious threat to corn crops. Phototaxis in nocturnal moths plays a crucial role in pest prediction and control. Insect opsins are the main component of insect visual system. However, the inherent molecular relationship between phototactic behaviour and vision of insects remains a mystery. Herein, three opsin genes were identified and cloned from O. furnacalis (OfLW, OfBL, and OfUV). Bioinformatics analysis revealed that all opsin genes had visual pigment (opsin) retinal binding sites and seven transmembrane domains. Opsin genes were distributed across different developmental stages and tissues, with the highest expression in adults and compound eyes. The photoperiod-induced assay elucidated that the expression of three opsin genes in females were higher during daytime, while their expression in males tended to increase at night. Under the sustained darkness, the expression of opsin genes increased circularly, although the increasing amplitude in males was lower when compared with females. Furthermore, the expression of OfLW, OfBL, and OfUV was upregulated under green, blue, and ultraviolet light, respectively. The results of RNA interference showed that the knockout of opsin genes decreased the phototaxis efficiency of female and male moths to green, blue, and ultraviolet light. Our results reveal that opsin genes are involved in the phototactic behaviour of moths, providing a potential target gene for pest control and a basis for further investigation on the phototactic behaviour of Lepidoptera insects.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.