Cross-kingdom RNA interference mediated by insect salivary microRNAs may suppress plant immunity.

Communication between insects and plants relies on the exchange of bioactive molecules that traverse the species interface. Although proteinic effectors have been extensively studied, our knowledge of other molecules involved in this process remains limited. In this study, we investigate the role of salivary microRNAs (miRNAs) from the rice planthopper Nilaparvata lugens in suppressing plant immunity. A total of three miRNAs were confirmed to be secreted into host plants during insect feeding. Notably, the sequence-conserved miR-7-5P is specifically expressed in the salivary glands of N. lugens and is secreted into saliva, distinguishing it significantly from homologues found in other insects. Silencing miR-7-5P negatively affects N. lugens feeding on rice plants, but not on artificial diets. The impaired feeding performance of miR-7-5P-silenced insects can be rescued by transgenic plants overexpressing miR-7-5P. Through target prediction and experimental testing, we demonstrate that miR-7-5P targets multiple plant genes, including the immune-associated bZIP transcription factor 43 (OsbZIP43). Infestation of rice plants by miR-7-5P-silenced insects leads to the increased expression of OsbZIP43, while the presence of miR-7-5P counteracts this upregulation effect. Furthermore, overexpressing OsbZIP43 confers plant resistance against insects which can be subverted by miR-7-5P. Our findings suggest a mechanism by which herbivorous insects have evolved salivary miRNAs to suppress plant immunity, expanding our understanding of cross-kingdom RNA interference between interacting organisms.

Comparative transcriptomic analysis of salivary glands between the zoophytophagous Cyrtorhinus lividipennis and the phytozoophagous Apolygus lucorum.

BACKGROUND: Saliva plays a crucial role in shaping the feeding behavior of insects, involving processes such as food digestion and the regulation of interactions between insects and their hosts. Cyrtorhinus lividipennis serves as a predominant natural enemy of rice pests, while Apolygus lucorum, exhibiting phytozoophagous feeding behavior, is a destructive agricultural pest. In this study, a comparative transcriptome analysis, incorporating the published genomes of C.lividipennis and A.lucorum, was conducted to reveal the role of salivary secretion in host adaptation. RESULTS: In contrast to A.lucorum, C.lividipennis is a zoophytophagous insect. A de novo genome analysis of C.lividipennis yielded 19,706 unigenes, including 16,217 annotated ones. On the other hand, A.lucorum had altogether 20,111 annotated genes, as obtained from the published official gene set (20,353 unigenes). Functional analysis of the top 1,000 salivary gland (SG)-abundant genes in both insects revealed that the SG was a dynamically active tissue engaged in protein synthesis and secretion. Predictions of other tissues and signal peptides were compared. As a result, 94 and 157 salivary proteins were identified in C.lividipennis and A.lucorum, respectively, and were categorized into 68 and 81 orthogroups. Among them, 26 orthogroups were shared, potentially playing common roles in digestion and detoxification, including several venom serine proteases. Furthermore, 42 and 55 orthogroups were exclusive in C.lividipennis and A.lucorum, respectively, which were exemplified by a hyaluronidase in C.lividipennis that was associated with predation, while polygalacturonases in A.lucorum were involved in mesophyll-feeding patterns. CONCLUSIONS: Findings in this study provide a comprehensive insight into saliva secretions in C.lividipennis and A.lucorum via a transcriptome approach, reflecting the intricate connections between saliva secretions and feeding behaviors. It is found that conserved salivary secretions are involved in shaping the overlapping feeding patterns, while a plethora of unique salivary secretions may drive the evolution of specific feeding behaviors crucial for their survival. These results enhance our understanding of the feeding mechanisms in different insects from the perspective of saliva and contribute to future environmentally friendly pest control by utilizing predatory insects.

Horizontally Transferred Salivary Protein Promotes Insect Feeding by Suppressing Ferredoxin-Mediated Plant Defenses.

Herbivorous insects such as whiteflies, planthoppers, and aphids secrete abundant orphan proteins to facilitate feeding. Yet, how these genes are recruited and evolve to mediate plant-insect interaction remains unknown. In this study, we report a horizontal gene transfer (HGT) event from fungi to an ancestor of Aleyrodidae insects approximately 42 to 190 million years ago. BtFTSP1 is a salivary protein that is secreted into host plants during Bemisia tabaci feeding. It targets a defensive ferredoxin 1 in Nicotiana tabacum (NtFD1) and disrupts the NtFD1-NtFD1 interaction in plant cytosol, leading to the degradation of NtFD1 in a ubiquitin-dependent manner. Silencing BtFTSP1 has negative effects on B. tabaci feeding while overexpressing BtFTSP1 in N. tabacum benefits insects and rescues the adverse effect caused by NtFD1 overexpression. The association between BtFTSP1 and NtFD1 is newly evolved after HGT, with the homologous FTSP in its fungal donor failing to interact and destabilize NtFD1. Our study illustrates the important roles of horizontally transferred genes in plant-insect interactions and suggests the potential origin of orphan salivary genes.

Molecular and biological characterization of a bunyavirus infecting the brown planthopper (Nilaparvata lugens).

A negative-strand symbiotic RNA virus, tentatively named Nilaparvata lugens Bunyavirus (NLBV), was identified in the brown planthopper (BPH, Nilaparvata lugens). Phylogenetic analysis indicated that NLBV is a member of the genus Mobuvirus (family Phenuiviridae, order Bunyavirales). Analysis of virus-derived small interfering RNA suggested that antiviral immunity of BPH was successfully activated by NLBV infection. Tissue-specific investigation showed that NLBV was mainly accumulated in the fat-body of BPH adults. Moreover, NLBV was detected in eggs of viruliferous female BPHs, suggesting the possibility of vertical transmission of NLBV in BPH. Additionally, no significant differences were observed for the biological properties between NLBV-infected and NLBV-free BPHs. Finally, analysis of geographic distribution indicated that NLBV may be prevalent in Southeast Asia. This study provided a comprehensive characterization on the molecular and biological properties of a symbiotic virus in BPH, which will contribute to our understanding of the increasingly discovered RNA viruses in insects.

Complete genome sequence and genetic characterization of a novel segmented RNA virus infecting Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is a significant agricultural pest capable of long-distance migration and transmission of viruses that cause severe disease in rice. In this study, we identified a novel segmented RNA virus in a BPH, and this virus exhibited a close relationship to members of a recently discovered virus lineage known as "quenyaviruses" within the viral kingdom Orthornavirae. This newly identified virus was named "Nilaparvata lugens quenyavirus 1" (NLQV1). NLQV1 consists of five positive-sense, single-stranded RNAs, with each segment containing a single open reading frame (ORF). The genomic characteristics and phylogenetic analysis support the classification of NLQV1 as a novel quenyavirus. Notably, all of the genome segments of NLRV contained the 5'-terminal sequence AUCUG. The characteristic virus-derived small interfering RNA (vsiRNA) profile of NLQV1 suggests that the antiviral RNAi pathway of the host BPH was activated in response to virus infection. These findings represent the first documented report of quenyaviruses in planthoppers, contributing to our understanding of quenyaviruses and expanding our knowledge of insect-specific viruses in planthoppers.

Long-wave opsin involved in body color plastic development in Nilaparvata lugens.

BACKGROUND: As one of the components of visual photopigments in photoreceptor cells, opsin exhibits different spectral peaks and plays crucial roles in visual function. Besides, it is discovered to evolve other functions despite color vision. However, research on its unconventional function is limited nowadays. With the increase in genome database numbers, various numbers and types of opsins have been identified in insects due to gene duplications or losses. The Nilaparvata lugens (Hemiptera) is a rice pest known for its long-distance migration capability. In this study, opsins were identified in N. lugens and characterized by genome and transcriptome analyses. Meanwhile, RNA interference (RNAi) was carried out to investigate the functions of opsins, and then the Illumina Novaseq 6000 platform-based transcriptome sequencing was performed to reveal gene expression patterns. RESULTS: Four opsins belonging to G protein-coupled receptors were identified in the N. lugens genome, including one long-sensitive opsin (Nllw) together with two ultraviolet-sensitive opsins (NlUV1/2) and an additional new opsin with hypothesized UV peak sensitivity (NlUV3-like). A tandem array of NlUV1/2 on the chromosome suggested the presence of a gene duplication event, with similar exons distribution. Moreover, as revealed by spatiotemporal expression, the four opsins were highly expressed in eyes with age-different expression levels. Besides, RNAi targeting each of the four opsins did not significantly affect the survival of N. lugens in phytotron, but the silencing of Nllw resulted in the melanization of body color. Further transcriptome analysis revealed that silencing of Nllw resulted in up-regulation of a tyrosine hydroxylase gene (NlTH) and down-regulation of an arylalkylamine-N-acetyltransferases gene (NlaaNAT) in N. lugens, demonstrating that Nllw is involved in body color plastic development via the tyrosine-mediated melanism pathway. CONCLUSIONS: This study provides the first evidence in a Hemipteran insect that an opsin (Nllw) takes part in the regulation of cuticle melanization, confirming a cross-talk between the gene pathways underlying the visual system and the morphological differentiation in insects.

Molecular characterization of a novel benyvirus infecting wheat in China.

In this study, a novel positive single-stranded RNA (+ ssRNA) virus named wheat yellow stripe associated virus (WYSAV) was identified in wheat plants in China. Molecular characterization revealed that the complete genome of WYSAV is divided into two segments, RNA1 and RNA2, which are 6,460 and 4,935 nucleotides (nt) in length, excluding their respective poly(A) tails. RNA1 contains one large opening reading frame (ORF), encoding a replication-associated protein. RNA2 contains six ORFs, encoding a coat protein (CP), a coat protein readthrough domain protein (CP-RTD), triple gene block protein 1 (TGB1), triple gene block protein 2 (TGB2), triple gene block protein 3 (TGB3), and a cysteine-rich protein (CRP). Phylogenetic analysis showed that WYSAV is related to members of the genus Benyvirus in the family Benyviridae. Thus, WYSAV is proposed to be a new member of the genus Benyvirus. Wheat (Triticum aestivum L.) is one of the most important food crops and ranked third in the world in terms of production, only behind rice and maize [1]. During its growth cycle, wheat faces several biotic and abiotic stresses. Wheat soil-borne virus disease is an important disease that is difficult to control and causes severe yield loss in China each year [2]. The main pathogens causing wheat soil-borne virus disease are Chinese wheat mosaic virus (CWMV) and wheat yellow mosaic virus (WYMV), and their transmission vector is Polymyxa graminis [3-5]. Members of the viral family Benyviridae usually have two to five genomic RNA segments and are transmitted by root-infecting vectors belonging to the family "Plasmodiophoridae". Although few members of the family Benyviridae, of which beet necrotic yellow vein virus is the type member, have been identified [6], several recently identified viruses have been found to be phylogenetically related to benyviruses but are not classified as members of the family Benyviridae. These "unclassified benyviruses" include red clover RNA virus 1, Arceuthobium sichuanense virus 3, Dactylorhiza hatagirea beny-like virus, goji berry chlorosis virus [7], Guiyang benyvirus 1, Guiyang benyvirus 2, Mangifera indica latent virus [8], Rhizoctonia solani beny-like virus 1 [9], Sanya benyvirus 1 [10], and Sclerotium rolfsii beny-like virus 1 [11].In this study, we identified a novel + ssRNA virus in symptomatic leaf samples collected from cultivated wheat in the city of Zhumadian, Henan Province, China. We propose to name this virus "wheat yellow stripe associated virus" (WYSAV), and we have deposited its full-length sequence in the GenBank database under the accession numbers OQ547804 (RNA1) and OQ547805 (RNA2).

Complete nucleotide sequence of hackberry virus A, a tentative member of the genus Waikavirus.

The complete genomic sequence of a waikavirus from Chinese hackberry in Zhejiang province, China, named "hackberry virus A" (HVA), was determined using high-throughput sequencing (HTS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The bicistronic genomic RNA of HVA was found to consist of 12,691 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and to encode a large polyprotein of 3783 amino acids (aa) and an additional 10.3-kDa protein. The aa sequences of the Pro-Pol and the CP regions of this virus share 39.8-44.2% and 25.5-36.4% identity, respectively, with currently known waikaviruses. These values are significantly below the current species demarcation threshold (< 75% and < 80% aa identity for the CP and Pro-Pol region, respectively) for the family Secoviridae, indicating that HVA represents a new species in the genus Waikavirus. This is the first report of a virus infecting Chinese hackberry.

Molecular characterization of a novel virga-like virus associated with wheat.

In this work, we report the detection of a novel single-strand RNA virus from wheat, tentatively named "Triticum aestivum-associated virga-like virus 1" (TaAVLV1). Further characterization revealed that the complete genome of TaAVLV1 is divided into two segments, RNA1 and RNA2, which are 3530 and 3466 nt in length, excluding their respective polyA tails, and each contains only one open reading frame (ORF). The ORF of RNA1 encodes an RNA-dependent RNA polymerase (RdRp), while the ORF of RNA2 encodes a putative protein with methyltransferase and helicase domains. Phylogenetic analysis showed that the RdRp of TaAVLV1 is closely related to those of members of the unclassified virga-like virus group in the family Virgaviridae. Thus, we have identified TaAVLV1 as a putative novel virga-like virus belonging to the family Virgaviridae.

Complete genome analysis of a novel picorna-like virus from a ladybird beetle (Cheilomenes sexmaculata).

The ladybird beetle Cheilomenes sexmaculata (family Coccinellidae, order Coleoptera) is a common insect predator of agricultural pests. In this study, the full genome sequence of a novel picorna-like virus, tentatively named "Cheilomenes sexmaculata picorna-like virus 1" (CSPLV1), was identified in C. sexmaculata. The full-length sequence of CSPLV1 is 11,384 nucleotides (nt) in length (excluding the polyA tail), with one predicted open reading frame (ORF) encoding a polyprotein of 3727 amino acids, a 13-nt 5' untranslated region (UTR), and a 187-nt 3' UTR. The ORF of CSPLV1 consists of four distinct domains, including an RNA virus helicase domain (nt 3029-3319), a peptidase domain (nt 5555-6121), an RNA-dependent RNA polymerase domain (nt 7154-8101), and a picorna-like coat protein domain (nt 8606-9283). Phylogenetic analysis based on the conserved RdRP sequence showed that CSPLV1, together with Wuhan house centipede virus 3, Hypera postica associated virus 1, and Diabrotica undecimpunctata virus 1, forms an unclassified group that is closely related to members of the family Solinviviridae. To the best of our knowledge, CSPLV1 is the first picorna-like virus discovered in C. sexmaculata.

Complete genome analysis of a nege-like virus in aphids (Astegopteryx formosana).

Negeviruses are a group of insect-specific viruses that have a wide geographic distribution and broad host range. In recent years, nege-like viruses have been discovered in aphids of various genera of the family Aphididae, including Aphis, Rhopalosiphum, Sitobion, and Indomegoura. Here, we report the complete genome sequence of a nege-like virus isolated from Astegopteryx formosana aphids collected in Guangdong, China, which we have designated as "Astegopteryx formosana nege-like virus" (AFNLV). AFNLV has a genome length of 10,107 nt (excluding the polyA tail) and possesses the typical conserved domains of negeviruses. These include a viral methyltransferase, an S-adenosylmethionine-dependent methyltransferase, a viral helicase, and an RNA-dependent RNA polymerase (RdRP) domain in open reading frame 1 (ORF1), a DiSB-ORF2_chro domain in ORF2, and a SP24 domain in ORF3. The genome of AFNLV shares the highest nucleotide sequence identity (74.89%) with Wuhan house centipede virus, identified in a mixture of barley aphids. As clearly revealed by RdRP-based phylogenetic analysis, AFNLV, together with other negeviruses and nege-like viruses discovered in aphids, formed a distinct "unclassified clade" closely related to members of the proposed genus "Sandewavirus" and the family Kitaviridae. In addition, small interfering RNAs (siRNAs) derived from AFNLV did not exhibit typical characteristics of virus-derived siRNAs processed by the host RNAi-based antiviral pathway. However, the extremely high abundance of viral transcripts (average read coverage 73,403X) strongly suggested that AFNLV might actively replicate in the aphid host. AFNLV described in this study is the first nege-like virus discovered in aphids of the genus Astegopteryx, which will contribute to future study of the co-evolution of nege/nege-like viruses and their host aphids.

Complete genome sequence of a novel arlivirus from a yellow spotted stink bug (Erthesina fullo (Thunberg, 1783)).

Arlivirus is currently the only genus in the newly established viral family Lispiviridae. In this study, the complete genome sequence of a novel arlivirus, tentatively named "Nbu stink bug virus 1" (NbuSBV-1), was identified in an individual yellow spotted stink bug, Erthesina fullo (family Pentatomidae, order Hemiptera), which is a widely distributed phytophagous pest in Asia. NbuSBV-1 has a single negative-stranded RNA genome of 13,605 nucleotides in length, and it was predicted to contain six open reading frames (ORFs). Conserved domains of NbuSBV-1 were predicted in ORF1 (a nucleoprotein), ORF4 (a glycoprotein domain), ORF5 (a zinc-finger domain), and ORF6 (an RNA-directed RNA polymerase [RdRP] domain, an mRNA cap domain, and a methyltransferase domain). NbuSBV-1 shares 50.54% amino acid sequence identity in the RdRP region with its closest homolog, Lishì spider virus 2. In RdRP-based phylogenetic analysis, NbuSBV-1 was clearly clustered in a clade with other arliviruses. Furthermore, NbuSBV-1-derived small interfering RNAs (siRNAs) showed typical patterns of virus-derived siRNAs produced by the host antiviral RNA interference pathway. As far as we know, NbuSBV-1 is the first arlivirus identified in an insect of the family Pentatomidae.

Complete genome analysis of a novel iflavirus from the spotted lanternfly Lycorma delicatula.

The spotted lanternfly (Lycorma delicatula) is an invasive pest that causes serious economic losses in fruit and wood production. Here, we identified a novel iflavirus named "Lycorma delicatula iflavirus 1" (LDIV1), in a spotted lanternfly. The full genome sequence of LDIV1 is 10,222 nt in length and encodes a polyprotein containing a picornavirus capsid-protein-domain-like domain, a cricket paralysis virus capsid superfamily domain, an RNA helicase domain, a peptidase C3 superfamily domain, and an RNA-dependent RNA polymerase (RdRp) domain. LDIV1 replicates in the host insect and activates small interfering RNA (siRNA)-based host antiviral immunity. Phylogenetic analysis demonstrated that LDIV1 is most closely related to an unspecified member of the order Picornavirales, with 61.7% sequence identity in the RdRp region and 57.6% sequence identity in the coat protein region, and thus meets the demarcation criteria for new species in the genus Iflavirus. To the best of our knowledge, LDIV1 is the first virus discovered in L. delicatula.

Complete genome analysis of a novel chuvirus from a southern green stink bug (Nezara viridula).

A novel chuvirus from a southern green stink bug (Nezara viridula) was identified by RNA sequencing in this study and was tentatively named "Ningbo southern green stink bug chuvirus 1" (NBSGSBV-1). The complete genome sequence of NBSGSBV-1 consists of 11,375 nucleotides, and the genome was found to be circular by 'around-the-genome' reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Three open reading frames (ORFs) were predicted in the NBSGSBV-1 genome, encoding a large polymerase protein (L protein), a glycoprotein (G protein), and a nucleocapsid protein (N protein). A phylogenetic tree was constructed based on all of the currently available RNA-dependent RNA polymerase amino acid sequences of viruses of the family Chuviridae, and NBSGSBV-1 was found to cluster together with Sanya chuvirus 2 and Hubei odonate virus 11, indicating that NBSGSBV-1 might belong to the genus Odonatavirus. Five conserved sites were identified in the L proteins of NBSGSBV-1 and other chuviruses. The abundance and characteristics of the NBSGSBV-1-derived small interfering RNAs suggested that NBSGSBV-1 actively replicates in the host insect. To the best of our knowledge, this is the first report of a chuvirus identified in a member of the insect family Pentatomidae. The discovery and characterization of NBSGSBV-1 will help us to understand the diversity of chuviruses in insects.

Complete genome sequence of a novel nege-like virus in aphids (genus Indomegoura).

BACKGROUND: Aphids are important vectors of numerous plant viruses. Besides plant viruses, a number of insect specific viruses (ISVs), such as nege/nege-like viruses, have been recently discovered in aphids of the genera Aphis, Rhopalosiphum, and Sitobion. FINDINGS: In this study, the complete genome sequence of a novel nege-like virus, tentatively named "Indomegoura nege-like virus 1" (INLV1), was identified in aphids of the genus Indomegoura. INLV1 possessed a single positive-stranded RNA genome with 8945 nucleotides, which was predicted to contain three typical open reading frames (ORFs) of negeviruses (including ORF1, ORF2, and ORF3), a 44-nt 5' untranslated region (UTR) and a 98-nt 3' UTR. Five conserved domains were predicted for INLV1, including an Alphavirus-like methyltransferase domain, a RNA virus helicase core domain, and a RNA-dependent RNA polymerase domain (RdRP) in ORF1, a DISB-ORF2_chro domain in ORF2, and a SP24 domain in ORF3. According to the maximum likelihood phylogenetic tree based on RdRP, INLV1 was grouped with barley aphid RNA virus 1 and Hubei virga-like virus 4, together with another two invertebrate viruses, which formed a distinct clade in the proposed group Centivirus. The alignment of RdRP domains for INLV1 and other nege/kita-like viruses suggested that RdRP of INLV1 contained the permuted C (GDD)- A [DX(4-5)D] -B [GX(2-3)TX(3)N] motifs, which were conserved in the Centivirus and Sandewavirus groups. Furthermore, the high abundance and typical characteristics of INLV1 derived small interfering RNAs clearly showed the active replication of INLV1 in the aphid Indomegoura. CONCLUSION: INLV1 is the first nege-like virus infecting aphids of the genus Indomegoura. As far as we know, it is also the first ISV revealed in this aphid genus.

Complete genome analysis of a novel iflavirus from a leaf beetle, Aulacophora lewisii.

The leaf beetle Aulacophora lewisii (family Chrysomelidae, order Coleoptera) is a common insect pest of cucurbitaceous vegetables. In this study, the complete genome sequence of a novel virus from a single leaf beetle was determined using metagenomic sequencing and rapid amplification of cDNA ends. A homology search and phylogenetic analysis suggested that the new virus belongs to the genus Iflavirus, family Iflaviridae, and it was tentatively named "Aulacophora lewisii iflavirus 1" (ALIV1). ALIV1 has a single positive-stranded RNA genome of 9655 nucleotides in length (excluding the polyA tail) that is predicted to encode typical conserved domains of iflaviruses, including two picornavirus-like capsid protein domains, a helicase domain, and an RNA-dependent RNA polymerase (RdRp) domain. Sequence comparisons showed that the full genome sequence of ALIV1 is most similar to that of Brevicoryne brassicae picorna-like virus, with 42.4% sequence identity, and it shares 60% sequence identity in the coat protein region with its closest homolog, Watson virus. The average coverage of the ALIV1 sequence was approximately 5000X, suggesting that it might actively replicate in the host. Phylogenetic analysis based on deduced amino acid sequences suggested that ALIV1 is closely related to Dinocampus coccinellae paralysis virus. To the best of our knowledge, ALIV1 is the first virus discovered in A. lewisii and is also the first iflavirus identified in a member of the genus Aulacophora.

A pipeline contributes to efficient identification of salivary proteins in short-headed planthopper, Epeurysa nawaii.

Saliva, an oral secretion primarily originating from salivary glands (SGs), exert critical roles in the ongoing evolutionary interaction between insects and plants. However, identifying insect salivary components poses challenges due to the tiny size of insects, low secretion amounts, and the propensity for degradation after secretion. In this study, we developed a transcriptome-based approach to comprehensively analyze the salivary proteins of the short-headed planthopper, Epeurysa nawaii, a species with unique feeding habits on bamboo. A total of 165 salivary proteins were identified, with 114 secretory genes highly and specifically expressed in SGs. Consistent with most phloem-feeding insects, digestive enzymes, calcium-binding proteins, oxidoreductases, and a few previously reported salivary effectors were ubiquitously distributed in E. nawaii saliva. However, we also identified a substantial portion of salivary proteins exhibiting taxonomy specificity, including 60 E. nawaii-specific and 62 Delphacidae-specific proteins. These taxonomy-restricted proteins potentially play a role in insect adaptation to specific host plants. Our study provides an efficient pipeline for salivary protein identification and serves as a valuable resource for the functional characterization of effectors.

Discovery of Two Novel Viruses of the Willow-Carrot Aphid, Cavariella aegopodii.

The advancement of bioinformatics and sequencing technology has resulted in the identification of an increasing number of new RNA viruses. This study systematically identified the RNA virome of the willow-carrot aphid, Cavariella aegopodii (Hemiptera: Aphididae), using metagenomic sequencing and rapid amplification of cDNA ends (RACE) approaches. C. aegopodii is a sap-sucking insect widely distributed in Europe, Asia, North America, and Australia. The deleterious effects of C. aegopodii on crop growth primarily stem from its feeding activities and its role as a vector for transmitting plant viruses. The virome includes Cavariella aegopodii virga-like virus 1 (CAVLV1) and Cavariella aegopodii iflavirus 1 (CAIV1). Furthermore, the complete genome sequence of CAVLV1 was obtained. Phylogenetically, CAVLV1 is associated with an unclassified branch of the Virgaviridae family and is susceptible to host antiviral RNA interference (RNAi), resulting in the accumulation of a significant number of 22nt virus-derived small interfering RNAs (vsiRNAs). CAIV1, on the other hand, belongs to the Iflaviridae family, with vsiRNAs ranging from 18 to 22 nt. Our findings present a comprehensive analysis of the RNA virome of C. aegopodii for the first time, offering insights that could potentially aid in the future control of the willow-carrot aphid.

Identification and functional analysis of the female determiner gene in the bean bug, Riptortus pedestris.

BACKGROUND: Homing-based gene drives targeting sex-specific lethal genes have been used for genetic control. Additionally, understanding insect sex determination provides new targets for managing insect pests. While sex determination mechanisms in holometabolous insects have been thoroughly studied and employed in pest control, the study of the sex determination pathway in hemimetabolous insects is limited to only a few species. Riptortus pedestris (Fabricius; Hemiptera: Heteroptera), commonly known as the bean bug, is a significant pest for soybeans. Nonetheless, the mechanism of its sex determination and the target gene for genetic control are not well understood. RESULTS: We identified Rpfmd as the female determiner gene in the sex determination pathway of R. pedestris. Rpfmd encodes a female-specific serine/arginine-rich protein of 436 amino acids and one non-sex-specific short protein of 98 amino acids. Knockdown of Rpfmd in R. pedestris nymphs caused death of molting females with masculinized somatic morphology but did not affect male development. Knockdown of Rpfmd in newly emerged females inhibited ovary development, while maternal-mediated RNA interference (RNAi) knockdown of Rpfmd expression resulted in male-only offspring. Transcriptome sequencing revealed that Rpfmd regulates X chromosome dosage compensation and influences various biological processes in females but has no significant effect on males. Moreover, RNAi mediated knockdown of Rpfmd-C had no influence on the development of R. pedestris, suggesting that Rpfmd regulates sex determination through female-specific splicing isoforms. We also found that Rpfmd pre-mRNA alternative splicing regulation starts at the 24-h embryo stage, indicating the activation of sex differentiation. CONCLUSION: Our study confirms that Rpfmd, particularly its female-specific isoform (Rpfmd-F), is the female determiner gene that regulates sex differentiation in R. pedestris. Knockdown of Rpfmd results in female-specific lethality without affecting males, making it a promising target for genetic control of this soybean pest throughout its development stages. Additionally, our findings improve the understanding of the sex-determination mechanism in hemimetabolous insects. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification and Characterization of Three Novel Solemo-like Viruses in the White-Backed Planthopper, Sogatella furcifera.

Agricultural insects play a crucial role in transmitting plant viruses and host a considerable number of insect-specific viruses (ISVs). Among these insects, the white-backed planthoppers (WBPH; Sogatella furcifera, Hemiptera: Delphacidae) are noteworthy rice pests and are responsible for disseminating the southern rice black-streaked dwarf virus (SRBSDV), a significant rice virus. In this study, we analyzed WBPH transcriptome data from public sources and identified three novel viruses. These newly discovered viruses belong to the plant-associated viral family Solemoviridae and were tentatively named Sogatella furcifera solemo-like virus 1-3 (SFSolV1-3). Among them, SFSolV1 exhibited a prevalent existence in different laboratory populations, and its complete genome sequence was obtained using rapid amplification of cDNA ends (RACE) approaches. To investigate the antiviral RNA interference (RNAi) response in WBPH, we conducted an analysis of virus-derived small interfering RNAs (vsiRNAs). The vsiRNAs of SFSolV1 and -2 exhibited typical patterns associated with the host's siRNA-mediated antiviral immunity, with a preference for 21- and 22-nt vsiRNAs derived equally from both the sense and antisense genomic strands. Furthermore, we examined SFSolV1 infection and distribution in WBPH, revealing a significantly higher viral load of SFSolV1 in nymphs' hemolymph compared to other tissues. Additionally, in adult insects, SFSolV1 exhibited higher abundance in male adults than in female adults.