Diverse RNA Viruses Discovered in Three Parasitoid Wasps of the Rice Weevil Sitophilus oryzae.

In this study, many virus-like fragments were obtained from transcriptomes of three wasp species, including Anisopteromalus calandrae (8), Lariophagus distinguendus (3), and Theocolax elegans (18), which can parasitize and control rice weevil Sitophilus oryzae, a serious insect pest of farm-stored grains. By further bioinformatic analysis and sequencing, we identified six novel RNA viruses with complete genomes and named them WWPSRV-1, WWPSRV-2, AcPSRV-1, AcNSRV-1, AcNSRV-2, and LdNSRV-1. PCR-based detection revealed that WWPSRV-1 and WWPSRV-2 had the possibility of interspecies virus transmission, especially WWPSRV-2, which was also present in the rice weevil adults. Phylogenetically, three out of these six viruses appeared to be members of order Picornavirales: WWPSRV-1 belonged to unassigned virus families of this order, whereas WWPSRV-2 and AcPSRV-1 belonged to families Iflaviridae and Dicistroviridae, respectively. The conserved picornavirus-typical domains helicase, protease, and RNA-dependent RNA polymerase could be found in the nonstructural protein encoded by the three viruses, whose genomes consisted of the different numbers of open reading frames (ORFs). The other three RNA viruses could be classified to order Mononegavirales: AcNSRV-1 and AcNSRV-2 belonged to family Lispiviridae, whereas LdNSRV-1 belonged to a big family Rhabdoviridae The genomes of the three viruses contained at least five ORFs, encoding deduced proteins in the following order: 3'-N-P-M-G-L-5'. All the ORFs were separated by conserved intergenic sequences which likely regulated the transcription termination and initiation. Our findings enhance the understanding of RNA viruses in weevil wasps and set the foundation for the future study of the association among weevils, weevil wasps, and RNA viruses.IMPORTANCE The enormous diversity of RNA viruses in insects is continuously validated. Parasitoid wasps, as biocontrol insects which are widely used against insect pests in agroecosystems, may also carry many "good" RNA viruses. Some RNA viruses in parasitoid wasps have been reported to affect the host wasps or the wasps' host. Here, six novel RNA viruses with complete genomes were identified in three parasitoid wasps of the rice weevil. One of these viruses was also detected in the rice weevil adults. Phylogenetically, WWPSRV-1 was the first unambiguous detection of Nora-like virus in insect parasitoids. WWPSRV-2 and AcPSRV-1 belong to families Iflaviridae and Dicistroviridae, some viruses of which can result in lethal infections in silkworms and honeybees. The other three RNA viruses belong to order Mononegavirales, which comprises many well-known insect-associated viruses.

Identification and characterization of a novel rhabdovirus in green rice leafhopper, Nephotettix cincticeps.

The family Rhabdoviridae is one of the most ecological diverse groups of RNA viruses, with remarkable genome complexity and wide host range. Rhabdoviruses are discovered in many insect species, and insects, such as midges, mosquitoes, aphids and leafhoppers, act as crucial vectors for rhabdovirus transmission. Here, a novel rhabdovirus was identified in green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), a common virus vector on rice. This virus was named as Nephotettix cincticeps negative-stranded RNA virus-1 (NcNSRV-1). The genome of NcNSRV-1 is 12,361 nucleotides in length, flanked by untranslated 3' leader and 5' trailer. The anti-sense viral genome consists of five major structural protein genes (N, P, M, G and L), which shares a typical architecture with the family Rhabdoviridae. An additional gene, P6, is interposed between G and L genes. NcNSRV-1 is phylogenetically clustered with the unclassified rhabdoviruses isolated from insects and exhibits low sequence identities with other viruses. The transcription regulatory sequences in NcNSRV-1 gene junctions were determined, revealing a unique transcription initiation sequence. In view of the phylogeny, sequence identity and genome organization, NcNSRV-1 is likely to be an insect rhabdovirus. Field surveys showed NcNSRV-1 was prevalent in the rice field populations of N. cincticeps in China.

Molecular and pharmacological characterization of a ß-adrenergic-like octopamine receptor from the green rice leafhopper Nephotettix cincticeps.

As the counterparts of noradrenaline and adrenaline in vertebrates, octopamine (OA) regulates multiple physiological and behavioral processes in invertebrate. OA mediates its effects via binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been reported in several insects. However, little work was documented in hemipteran insects. We cloned a ß-adrenergic-like OAR (NcOA2B2) from Nephotettix cincticeps. NcOA2B2 shares high similarity with members of the OA2B2 receptor class. Transcript level of NcOA2B2 varied in various tissues and was highly expressed in the leg. After heterologous expression in CHO-K1 cells, NcOA2B2 was dose-dependently activated by OA (EC50 = 2.56 nM) and tyramine (TA) (EC50 = 149 nM). Besides putative octopaminergic agonists, dopaminergic agonists and amitraz and DPMF potently activated NcOA2B2 in a dose-dependent manner. Receptor activity was blocked by potential antagonists and was most efficiently antagonized by asenapine. Phentolamine showed both antagonist and agonist effects on NcOA2B2. Our results offer the important information about molecular and pharmacological characterization of an OAR from N. cincticeps that will provide the basis for forthcoming studies on its roles in physiological processes and behaviors, and facilitate the design of novel insecticides for pest control.

A Novel Iflavirus Was Discovered in Green Rice Leafhopper Nephotettix cincticeps and Its Proliferation Was Inhibited by Infection of Rice Dwarf Virus.

The green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), is a key insect vector transmitting rice dwarf virus (RDV) that causes rice dwarf disease. We discovered a novel iflavirus from the transcriptomes of N. cincticeps and named it as Nephotettix cincticeps positive-stranded RNA virus-1 (NcPSRV-1). The viral genome consists of 10,524 nucleotides excluding the poly(A) tail and contains one predicted open reading frame encoding a polyprotein of 3,192 amino acids, flanked by 5' and 3' untranslated regions. NcPSRV-1 has a typical iflavirus genome arrangement and is clustered with the members of the family Iflaviridae in the phylogenetic analysis. NcPSRV-1 was detected in all tested tissues and life stages of N. cincticeps and could be transmitted horizontally and vertically. Moreover, NcPSRV-1 had high prevalence in the laboratory populations and was widely spread in field populations of N. cincticeps. NcPSRV-1 could also infect the two-striped leafhopper, Nephotettix apicalis, at a 3.33% infection rate, but was absent in the zigzag leafhopper, Recilia dorsalis, and rice Oryza sativa variety TN1. The infection of RDV altered the viral load and infection rate of NcPSRV-1 in N. cincticeps, for which it seems that RDV has an antagonistic effect on NcPSRV-1 infection in the host.