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The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway play a pivotal role in innate immunity. Among invertebrates, Domeless receptors serve as the key upstream regulators of this pathway. In our study on Bactrocera dorsalis, we identified three cytokine receptors: BdDomeless1, BdDomeless2, and BdDomeless3. Each receptor encompasses five fibronectin-type-III-like (FN III) extracellular domains and a transmembrane domain. Furthermore, these receptors exhibit the increased responsiveness to diverse pathogenic challenges. Notably, only BdDomeless3 is upregulated during symbiont-like viral infections. Moreover, silencing BdDomeless3 enhanced the infectivity of Bactrocera dorsalis cripavirus (BdCV) and B. dorsalis picorna-like virus (BdPLV), underscoring BdDomeless3’s crucial role in antiviral defense of B. dorsalis. Following the suppression of Domeless3 expression, six antimicrobial peptide genes displayed decreased expression, potentially correlating with the rise in viral infectivity. To our knowledge, this is the first study identifying cytokine receptors associated with the JAK/STAT pathway in tephritid flies, shedding light on the immune mechanisms of B. dorsalis.

ABSTRACT Parasitoid wasps are a diverse group of parasitoid insects that play a crucial role in biological control and integrated pest management programs due to their wide range of host species and complex behaviors. Previous studies have reported that select RNA viruses in parasitoid wasps can affect either the host-parasitoid wasp or the pest host parasitized by the wasps. Therefore, a study of the dynamics of RNA viruses in parasitoid wasps is essential for artificial breeding programs and improving their parasitic capabilities. In this study, we detected the presence of nine novel and three known RNA viruses with complete genomes in three parasitoid wasp species: Diachasmimorpha longicaudata, Fopius arisanus, and Spalangia endius. These wasps are parasitoids that can parasitel tephritid flies, one of the most important pest groups of fruits and vegetables. In D. longicaudata, the small RNAs derived from the viruses exhibited distinct peaks at 22 nt and were symmetrically distributed across the viral genome. These findings indicate that these viruses can activate the host’s RNAi immune response. PCR detection further revealed that DlNaLV efficiently infects female individuals of D. longicaudata with 100% efficiency, and ZcNLV is capable of infecting both D. longicaudata and its host, Zeugodacus cucurbitae. Our findings contribute to a better understanding of RNA viruses in parasitoid wasps and lay the foundation for future research on the triple-level trophic relationships between tephritid flies, parasitoid wasps, and RNA viruses. IMPORTANCE Parasitoid wasp populations have developed persistent beneficial symbiotic relationships with several viruses through repeated evolution. However, there have been limited reports on RNA viruses in parasitoid wasps of tephritid flies, a significant pest group affecting fruits and vegetables. This study explores the diversity of RNA viruses in three parasitoid wasps of tephritid flies and highlights the potential biological significance of specific viruses in Diachasmimorpha longicaudata. These findings have important implications for the development of sustainable pest management strategies and the enhancement of artificial rearing techniques for parasitoid wasps.

The Asian tramp snail, Bradybaena similaris (Ferusssac), is an invasive land snail species and has been a rising agricultural pest in south of China. As a pest, it also plays a role in transmission of Angiostrongylus cantonensis. However, present studies on this species are rare and the molecular information is limited. For this purpose, we sequenced the transcriptome and small RNA of B. similaris collected from citrus orchards. In total, 89,747 unigenes with an N50 size of 1287 bp and an average length of 817 bp were generated from ∼8.9 Gb transcriptome and 31 Mb clean reads were generated from ∼36 Mb small RNA library. To demonstrate the usefulness of these two datasets, we analyzed a series of genes associated with xenobiotic metabolism and core RNAi machinery. Analysis of the transcripts resulted in annotation of 126 putative genes encoding cytochrome P450 monooxygenases (CYP, 45), carboxyl/cholinesterases (CCE, 13), glutathione-S-transferases (GST, 24), and ATP-binding cassette transporters (ABC, 44). Analysis of the small RNA detected 42 miRNAs. In addition, four genes involved in small RNA pathways (miRNA, piRNA, and siRNA) were identified, and a total of 430 genes that can be targeted by miRNAs were predicted. Moreover, we found that a few miRNAs could target certain genes involved in xenobiotic metabolism. Therefore, we believe that these two datasets and the characterization of the identified/predicted genes will facilitate the molecular study of this species as well as other land snails with agricultural importance.

The tea aphid, Aphis aurantii, has become one of the destructive pests in tea plantations in the tropics and subtropics. Very few functional studies have so far focused on the developmental and reproductive biology at a molecular level, because of the lack of comprehensive genetic information. Full-length transcriptomes represent a very highly efficient approach to obtain reference gene sequences in non-model insects. In the present study, the transcriptome of A. aurantii was comprehensively sequenced using PacBio Iso-Seq technology. A total of 46.8 Gb nucleotides and 15,938 non-redundant full-length transcripts were obtained, 13,498 (84.69%) of which were annotated into seven databases. Of these transcripts, 2,029 alternative splicing events and 15,223 simple sequence repeats were detected. Among these transcripts, 4,571 (28.68%) and 11,367 (71.32%) were long non-coding RNAs (lncRNAs) and protein-coding genes, respectively. Five hundred and ninety transcription factors were detected. The first full-length transcriptome represents a significant increase in the known genetic information of A. aurantii. It will assist the future functional study of genes involved in its development and reproduction.

Carotenoids play many crucial roles in organisms. Recently, the de novo synthesis of carotenoids has been reported in pea aphid (Acyrthosiphon pisum) through horizontally transferred genes. However, their upstream pathway in the pea aphid is poorly understood. Geranylgeranyl diphosphate synthase (GGPPS) is the functional enzyme in the synthesis of geranylgeranyl diphosphate (GGPP) which is a precursor for the biosynthesis of many biological metabolites, including carotenoid synthesis. In this study, we performed a series of experiments to characterize GGPPS gene and its association with carotenoid biosynthesis. (1) determining the transcript abundance and carotenoid content in two geographical strain with red and green morphs, and (2) examining the abundance of carotenoid related genes and carotenoid levels after silencing of GGPPS in both red and green morphs. We observed that GGPPS was more highly expressed in the green morph than in the red morph of two strains of the pea aphid. The total level of carotenoids was also higher in green morphs than in red morphs in both strains. In addition to the total carotenoid difference, the carotenoids found in the two morphs also differed. There were α-carotene, β-carotene, and γ-carotene in the green morphs, but three additional carotenoids, including cis-torulene∗, trans-torulene∗, and 3,4-didehydrolycopene∗, were present in the red morphs. Silencing the GGPPS by RNAi in both the red and green morphs decreased the expression of some carotenoid biosynthesis-related genes, including carotenoid synthase/cyclase genes and carotenoid desaturase genes in green morphs. Carotenoid levels were decreased in both green and red morphs. However, the specific carotenoids present were not changed after silencing GGPPS. These results demonstrated that GGPPS may act as the upstream enzyme to influence the synthesis of the total amount of carotenoids. The present study provided important molecular evidence for the conserved roles of GGPPS associated with carotenoids biosynthesis and will enhance further investigation on the mechanisms of carotenoid biosynthesis in pea aphid.

Molting is essential for arthropods to grow. As one of the important arthropod pests in agriculture, key spider mite species (Tetranychus and Panonychus) can normally molt three times from the larva to adult stage within a week. This physiological strategy results in the short lifecycle of spider mites and difficulties in their control in the field. Long non-coding RNAs (lncRNAs) regulate transcriptional editing, cellular function, and biological processes. Thus, analysis of the lncRNAs in the spider mite molting process may provide new insights into their roles in the molting mechanism. For this purpose, we used high-throughput RNA-seq to examine the expression dynamics of lncRNAs and mRNAs in the molting process of different development stages in Panonychus citri. We identified 9199 lncRNAs from 18 transcriptomes. Analysis of the lncRNAs suggested that they were shorter and had fewer exons and transcripts than mRNAs. Among these, 356 lncRNAs were differentially expressed during three molting processes: late larva to early protonymph, late protonymph to early deutonymph, and late deutonymph to early adult. A time series profile analysis of differentially expressed lncRNAs showed that 77 lncRNAs were clustered into two dynamic expression profiles (Pattern a and Pattern c), implying that lncRNAs were involved in the molting process of spider mites. Furthermore, the lncRNA–mRNA co-expression networks showed that several differentially expressed hub lncRNAs were predicted to be functionally associated with typical molting-related proteins, such as cuticle protein and chitin biosynthesis. These data reveal the potential regulatory function of lncRNAs in the molting process and provide datasets for further analysis of lncRNAs and mRNAs in spider mites.

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and biological model organism, and RNA interference (RNAi) is an important tool for functional genomics and for insect pest management. However, the efficiency of RNAi in pea aphids is variable, limiting its application in aphids. In this study, we present optimized conditions for inducing and increasing the gene silencing efficiency of RNAi in pea aphids. The optimal gene silencing of the target Aphunchback gene was achieved by injecting 600 ng double-stranded (ds) RNA, and the highest mRNA depletion rate (74%) was detected at 36 h after injection. Moreover, the same gene silencing conditions were used to achieve transcript silencing for nine different genes in the pea aphid, although the silencing efficiencies for the different genes varied. Furthermore, the pre-exposure of aphids to dsRNA (600 ng dsGFP) led to significant hunchback silencing following a secondary exposure to 60 ng of dshunchback, a dose which did not lead to gene silencing when independently injected. The information presented here can be exploited to develop more efficient RNAi bioassays for pea aphids, both as gene functional study tools and an insect pest control strategy.

In this editorial for the Special Issue on ‘RNAi in insect pest control’, three important applications of RNA interference (RNAi) in insects are briefly discussed and linked to the different studies published in this Special Issue. The discovery of the RNAi mechanism revolutionized entomological research, as it presented researchers with a tool to knock down genes, which is easily applicable in a wide range of insect species. Furthermore, RNAi also provides crop protection with a novel and promising pest control mode-of-action. The sequence-dependent nature allows RNAi-based control strategies to be highly species selective and the active molecule, a natural biological molecule known as double-stranded RNA (dsRNA), has a short environmental persistence. However, more research is needed to investigate different cellular and physiological barriers, such as cellular uptake and dsRNA degradation in the digestive system in insects, in order to provide efficient control methods against a wide range of insect pest species. Finally, the RNAi pathway is an important part of the innate antiviral immune defence of insects, and could even lead to applications targeting viruses in beneficial insects such as honeybees in the future.

The pea aphid is an important pest of vegetables and causes serious losses worldwide. RNA interference (RNAi) is an effective pest control tool, and three sub-pathways have been described: The miRNA pathway, siRNA pathway, and piRNA pathway. A large number of genes in miRNA pathway and piRNA pathway are found to be expanded. To study the roles of these genes, the expression of 25 core RNAi genes was screened in spatiotemporal samples, artificially synthesized dsRNA and miRNA treated samples. The 25 genes were all expressed during different development stages and in different tissues. In dsRNA-treated samples and miRNA-treated samples, the expressions of genes in these three pathways were induced, especially the expanded genes. This suggests a complex network of RNAi core genes in the three sub-pathways. Treatment of miRNA seems to induce gene expression in a dosage-dependent manner. These results increase our knowledge of the siRNA pathway and related factors from RNAi pathway in aphids and promote the use of RNAi for the control of aphid pests.

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control. [ABSTRACT FROM AUTHOR]

The article focuses on the current knowledge and potential applications of RNAi-based pesticides for Integrated Pest Management. Topics discussed include the limitations of traditional insecticides, the advantages of RNA interference (RNAi) as a tool for crop protection with perspectives of using RNAi for pest control; and the importance of considering off-target effects and conducting safety assessments to ensure the compatibility of RNAi-based pest control with biological control methods.

Biopesticides based on RNA interference (RNAi) act in a sequence-dependent manner. Homology between target genes in insect pests and orthologs in non-target beneficial insects should therefore be investigated during the design of double-stranded RNA (dsRNA) constructs and by experimental testing to evaluate off-target effects. Here we investigated potential off-target effects against the ladybeetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae), an important biological control agent, in the context of RNAi-based aphid control. First, we sequenced the full-length P. japonica transcriptome and predicted potential off-target sites at the whole-transcriptome level in the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) as queries. We found off-target sites in almost every P. japonica genes. Second, we found that first-instar P. japonica larvae were more sensitive to dsRNA exposure than other developmental stages by comparing the silencing efficiency of three indicator genes (IAP, V-ATP and CHMP-4b). The expression of core siRNA pathway genes after dsRNA treatment confirmed that the response to dsRNA was more robust in first-instar larvae than adults. Finally, we used the A. pisum alpha-actinin gene as a case study to design dsRNA fragments with different degrees of efficacy against P. japonica, confirming that the safety of RNAi approaches can be enhanced by rational dsRNA design. [ABSTRACT FROM AUTHOR]

Wing dimorphism is a phenomenon of phenotypic plasticity in aphid dispersal. However, the signal transduction for perceiving environmental cues (e.g., crowding) and the regulation mechanism remain elusive. Here, we found that aci-miR-9b was the only down-regulated microRNA (miRNA) in both crowding-induced wing dimorphism and during wing development in the brown citrus aphid Aphis citricidus. We determined a targeted regulatory relationship between aci-miR-9b and an ABC transporter (AcABCG4). Inhibition of aci-miR-9b increased the proportion of winged offspring under normal conditions. Overexpression of aci-miR-9b resulted in decline of the proportion of winged offspring under crowding conditions. In addition, overexpression of aci-miR-9b also resulted in malformed wings during wing development. This role of aci-miR-9b mediating wing dimorphism and development was also confirmed in the pea aphid Acyrthosiphon pisum. The downstream action of aci-miR-9b-AcABCG4 was based on the interaction with the insulin and insulin-like signaling pathway. A model for aphid wing dimorphism and development was demonstrated as the following: maternal aphids experience crowding, which results in the decrease of aci-miR-9b. This is followed by the increase of ABCG4, which then activates the insulin and insulin-like signaling pathway, thereby causing a high proportion of winged offspring. Later, the same cascade, "miR-9b-ABCG4-insulin signaling," is again involved in wing development. Taken together, our results reveal that a signal transduction cascade mediates both wing dimorphism and development in aphids via miRNA. These findings would be useful in developing potential strategies for blocking the aphid dispersal and reducing viral transmission. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi), usually referring to the small interfering (si)RNA pathway, is promising in the development of a new generation of insect pest control products. However, prior to its exploitation for insect pest control, it is important to consider potential limiting factors, such as immune response and fitness cost, RNAi efficiency and dsRNA degradation, and virus-encoded suppressor of RNAi factors in the development of the RNAi-based pest control strategy. Additional challenges such as lack of feasible dsRNA delivery methods in practice, low efficiency in pest control capacity and evolution of resistance to RNAi have largely constrained the application of RNAi in practice. This review highlights how insect ecology and integrated pest management principles, taking into account RNAi, could be exploited to promote sustainable insect pest control. [ABSTRACT FROM AUTHOR]

Abamectin has been widely used as an insecticide/acaricide for more than 30 years because of its superior bioactivity. Recently, an interesting phenomenon was identified in the carmine spider mite, Tetranychus cinnabarinus, an important pest in agriculture. The gamma aminobutyric acid (GABA) contents in a laboratory abamectin resistant strain of T. cinnabarinus (AbR) were significantly increased. Decreases in activity and mRNA expression of GABA transaminase (GABA-T) were responsible for GABA accumulation in AbR mites. To clarify the mechanism of GABA accumulation mediated abamectin resistance, three artificial approaches were conducted to increase GABA contents in susceptible mites, including feeding of vigabatrin (a specific inhibitor of GABA-T), feeding of exogenous GABA, and inhibition of GABA-T gene expression. The results showed that susceptible mites developed resistance to abamectin when the GABA contents were artificially increased. We also observed that the mites with higher GABA contents moved more slowly, which is consistent with the fact that GABA is an inhibitory neurotransmitter in arthropods. Subsequently, functional response assays revealed that predation rates of predatory mites on GABA accumulated abamectin-resistant mites were much higher than control groups. The tolerance to abamectin, slow crawling speed, and vulnerability to predators were all resulted from GABA accumulation. This relationship between GABA and predation was also confirmed in a field-collected population. Our finding indicates that predatory mites might be used as a tool for biological control to circumvent the development of abamectin resistance in mites. [ABSTRACT FROM AUTHOR]