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Abstract Plant-sucking insects have intricate associations with a diverse array of microorganisms to facilitate their adaptation to specific ecological niches. The midgut of phytophagous true bugs is generally structured into four distinct compartments to accommodate their microbiota. Nevertheless, there is limited understanding regarding the origins of these gut microbiomes, the mechanisms behind microbial community assembly, and the interactions between gut microbiomes and their insect hosts. In this study, we conducted a comprehensive survey of microbial communities within the midgut compartments of a bean bug Riptortus pedestris, soybean plant, and bulk soil across 12 distinct geographical fields in China, utilizing high-throughput sequencing of the 16 S rRNA gene. Our findings illuminated that gut microbiota of the plant-sucking insects predominantly originated from the surrounding soil environment, and plants also play a subordinate role in mediating microbial acquisition for the insects. Furthermore, our investigation suggested that the composition of the insect gut microbiome was probably shaped by host selection and/or microbe-microbe interactions at the gut compartment level, with marginal influence from soil and geographical factors. Additionally, we had unveiled a noteworthy dynamic in the acquisition of core bacterial taxa, particularly Burkholderia, which were initially sourced from the environment and subsequently enriched within the insect midgut compartments. This bacterial enrichment played a significant role in enhancing insect host reproduction. These findings contribute to our evolving understanding of microbiomes within the insect-plant-soil ecosystem, shedding additional light on the intricate interactions between insects and their microbiomes that underpin the ecological significance of microbial partnerships in host adaptation.

Abstract Herbivorous insects employ an array of salivary proteins to aid feeding. However, the mechanisms behind the recruitment and evolution of these genes to mediate plant-insect interactions remain poorly understood. Here, we report a potential horizontal gene transfer (HGT) event from bacteria to an ancestral bug of Eutrichophora. The acquired genes subsequently underwent duplications and evolved through co-option. We annotated them as horizontal-transferred, Eutrichophora-specific salivary protein (HESPs) according to their origin and function. In Riptortus pedestris (Coreoidea), all nine HESPs are secreted into plants during feeding. The RpHESP4 to RpHESP8 are recently duplicated and found to be indispensable for salivary sheath formation. Silencing of RpHESP4-8 increases the difficulty of R. pedestris in probing the soybean, and the treated insects display a decreased survivability. Although silencing the other RpHESPs does not affect the salivary sheath formation, negative effects are also observed. In Pyrrhocoris apterus (Pyrrhocoroidea), five out of six PaHESPs are secretory salivary proteins, with PaHESP3 being critical for insect survival. The PaHESP5, while important for insects, no longer functions as a salivary protein. Our results provide insight into the potential origin of insect saliva and shed light on the evolution of salivary proteins.

Abstract Non-retroviral endogenous viral elements (nrEVEs) are widely dispersed throughout the genomes of eukaryotes. Although nrEVEs are known to be involved in host antiviral immunity, it remains an open question whether they can be domesticated as functional proteins to serve cellular innovations in arthropods. In this study, we found that endogenous toti-like viral elements (ToEVEs) are ubiquitously integrated into the genomes of three planthopper species, with highly variable distributions and polymorphism levels in planthopper populations. Three ToEVEs display exon‒intron structures and active transcription, suggesting that they might have been domesticated by planthoppers. CRISPR/Cas9 experiments revealed that one ToEVE in Nilaparvata lugens, NlToEVE14, has been co-opted by its host and plays essential roles in planthopper development and fecundity. Large-scale analysis of ToEVEs in arthropod genomes indicated that the number of arthropod nrEVEs is currently underestimated and that they may contribute to the functional diversity of arthropod genes.

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.

Salivary elicitors secreted by herbivorous insects can be perceived by host plants to trigger plant immunity. Here, the authors show that the small brown planthopper salivary sheath protein LsSP1 binds to salivary sheath proteins and contributes to insect feeding by manipulating rice plant defenses.

Abstract A large number of insect-specific viruses (ISVs) have recently been discovered, mostly from hematophagous insect vectors because of their medical importance, but little attention has been paid to important plant virus vectors such as the whitefly Bemisia tabaci, which exists as a complex of cryptic species. Public SRA datasets of B. tabaci and newly generated transcriptomes of three Chinese populations are here comprehensively investigated to characterize the whitefly viromes of different cryptic species. Twenty novel ISVs were confidently identified, mostly associated with a particular cryptic species while different cryptic species harbored one or more core ISVs. Microinjection experiments showed that some ISVs might cross-infect between the two invasive whitefly cryptic species, Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED), but others appeared to have a more restricted host range, reflecting the possibility of distinct long-term coevolution of these ISVs and whitefly hosts. Moreover, analysis of the profiles of virus-derived small-interfering RNAs indicated that some of the ISVs can successfully replicate in whitefly and the antiviral RNAi pathway of B. tabaci is actively involved in response to ISV infections. Our study provides a comprehensive analysis of the RNA virome, the distinct relationships and cross-cryptic species infectivity of ISVs in an agriculturally important insect vector.

The Toll pathway plays an important role in defense against infection of various pathogenic microorganisms, including viruses. However, current understanding of Toll pathway was mainly restricted in mammal and some model insects such as Drosophila and mosquitoes. Whether plant viruses can also activate the Toll signaling pathway in vector insects is still unknown. In this study, using rice stripe virus (RSV) and its insect vector (small brown planthopper, Laodelphax striatellus) as a model, we found that the Toll pathway was activated upon RSV infection. In comparison of viruliferous and non-viruliferous planthoppers, we found that four Toll pathway core genes (Toll, Tube, MyD88, and Dorsal) were upregulated in viruliferous planthoppers. When the planthoppers infected with RSV, the expressions of Toll and MyD88 were rapidly upregulated at the early stage (1 and 3 days post-infection), whereas Dorsal was upregulated at the late stage (9 days post-infection). Furthermore, induction of Toll pathway was initiated by interaction between a Toll receptor and RSV nucleocapsid protein (NP). Knockdown of Toll increased the proliferation of RSV in vector insect, and the dsToll-treated insects exhibited higher mortality than that of dsGFP-treated ones. Our results provide the first evidence that the Toll signaling pathway of an insect vector is potentially activated through the direct interaction between Toll receptor and a protein encoded by a plant virus, indicating that Toll immune pathway is an important strategy against plant virus infection in an insect vector.

The bean bug, Riptortus pedestris (Fabricius), is one of the most important soybean pests. It damages soybean leaves and pods with its piercing-sucking mouthparts, causing staygreen-like syndromes in the infested crops. During the feeding process, R. pedestris secretes a mixture of salivary proteins, which play critical roles in the insect–plant interactions and may be responsible for staygreen-like syndromes. The present study aimed to identify the major salivary proteins in R. pedestris saliva by transcriptomic and proteomic approaches, and to screen the proteins that potentially induced plant defense responses. Altogether, 136 salivary proteins were identified, and a majority of them were involved in hydrolase and binding. Additionally, R. pedestris saliva contained abundant bug-specific proteins with unknown function. Transient expression of salivary proteins in Nicotiana benthamiana leaves identified that RpSP10.3, RpSP13.4, RpSP13.8, RpSP17.8, and RpSP10.2 were capable of inducing cell death, reactive oxygen species (ROS) burst, and hormone signal changes, indicating the potential roles of these proteins in eliciting plant defenses. Our results will shed more light on the molecular mechanisms underlying the plant–insect interactions and are useful for pest management.

Negeviruses are a proposed group of insect-specific viruses that can be separated into two distinct phylogenetic clades, Nelorpivirus and Sandewavirus. Negeviruses are well-known for their wide geographic distribution and broad host range among hematophagous insects. In this study, the full genomes of two novel negeviruses from each of these clades were identified by RNA extraction and sequencing from a single dungfly (Scathophaga furcata) collected from the Arctic Yellow River Station, where these genomes are the first negeviruses from cold zone regions to be discovered. Nelorpivirus dungfly1 (NVD1) and Sandewavirus dungfly1 (SVD1) have the typical negevirus genome organization and there was a very high coverage of viral transcripts. Small interfering RNAs derived from both viruses were readily detected in S. furcata, clearly showing that negeviruses are targeted by the host antiviral RNA interference (RNAi) pathway. These results and subsequent in silico analysis (studies) of public database and published virome data showed that the hosts of nege-like viruses include insects belonging to many orders as well as various non-insects in addition to the hematophagous insects previously reported. Phylogenetic analysis reveals at least three further groups of negeviruses, as well as several poorly resolved solitary branches, filling in the gaps within the two sub-groups of negeviruses and plant-associated viruses in the Kitaviridae. The results of this study will contribute to a better understanding of the geographic distribution, host range, evolution and host antiviral immune responses of negeviruses.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in various biological processes. This study examined microRNA profiles of Laodelphax striatellus using the small RNA libraries derived from virus free (VF) and rice black-streaked dwarf virus (RBSDV) infected (RB) insects. A total of 59 mature miRNAs (46 miRNA families) were identified as conserved insect miRNAs in both VF and RB libraries. Among these conserved miRNAs, 24 were derived from the two arms of 12 miRNA precursors. Nine conserved L. striatellus miRNAs were up-regulated and 12 were down-regulated in response to RBSDV infection. In addition, a total of 20 potential novel miRNA candidates were predicted in the VF and RB libraries. The miRNA transcriptome profiles and the identification of L. striatellus miRNAs differentially expressed in response to RBSDV infection will contribute to future studies to elucidate the complex miRNA-mediated regulatory network activated by pathogen challenge in insect vectors.