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.

The genomic history and global migration of a windborne pest.

Many insect pests, including the brown planthopper (BPH), undergo windborne migration that is challenging to observe and track. It remains controversial about their migration patterns and largely unknown regarding the underlying genetic basis. By analyzing 360 whole genomes from around the globe, we clarify the genetic sources of worldwide BPHs and illuminate a landscape of BPH migration showing that East Asian populations perform closed-circuit journeys between Indochina and the Far East, while populations of Malay Archipelago and South Asia undergo one-way migration to Indochina. We further find round-trip migration accelerates population differentiation, with highly diverged regions enriching in a gene desert chromosome that is simultaneously the speciation hotspot between BPH and related species. This study not only shows the power of applying genomic approaches to demystify the migration in windborne migrants but also enhances our understanding of how seasonal movements affect speciation and evolution in insects.

Co-option of a non-retroviral endogenous viral element in planthoppers.

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.

A chromosome-level genome assembly of Stenchaetothrips biformis and comparative genomic analysis highlights distinct host adaptations among thrips.

Insects have a limited host range due to genomic adaptation. Thysanoptera, commonly known as thrips, occupies distinct feeding habitats, but there is a lack of comparative genomic analyses and limited genomic resources available. In this study, the chromosome-level genome of Stenchaetothrips biformis, an oligophagous pest of rice, is assembled using multiple sequencing technologies, including PacBio, Illumina short-reads, and Hi-C technology. A 338.86 Mb genome is obtained, consisting of 1269 contigs with a contig N50 size of 381 kb and a scaffold N50 size of 18.21 Mb. Thereafter, 17,167 protein-coding genes and 36.25% repetitive elements are annotated. Comparative genomic analyses with two other polyphagous thrips, revealing contracted chemosensory-related and expanded stress response and detoxification gene families in S. biformis, potentially facilitating rice adaptation. In the polyphagous thrips species Frankliniella occidentalis and Thrips palmi, expanded gene families are enriched in metabolism of aromatic and anthocyanin-containing compounds, immunity against viruses, and detoxification enzymes. These expansion gene families play crucial roles not only in adapting to hosts but also in development of pesticide resistance, as evidenced by transcriptome results after insecticides treatment. This study provides a chromosome-level genome assembly and lays the foundation for further studies on thrips evolution and pest management.

High-throughput sequencing yields a complete mitochondrial genome of the rice thrips, Stenchaetothrips biformis (Thysanoptera: Thripidae).

Rice thrips, Stenchaetothrips biformis (Bagnall, 1913), are one of the destructive pests of rice. Here, the complete mitochondrial genome of S. biformis was sequenced using high-throughput sequencing. The mitogenome is 15,359 bp long with an A + T content of 76.94%, which contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), 2 ribosomal RNA genes (rRNAs) and 2 putative control regions (CRs). The phylogenetic analysis showed that S. biformis is closely related to Thrips imaginis and Thrips palmi. This new mitochondrial genome data can be better used to provide a basis for studies of the mitochondrial evolution of Thysanoptera.

Chromosome-level Assembly, Dosage Compensation and Sex-biased Gene Expression in the Small Brown Planthopper, Laodelphax striatellus.

In insects, sex chromosome differentiation often results in unequal gene dosages between sexes. Dosage compensation mechanisms evolve to balance gene expression, but the degree and mechanism of regulation often vary by insect species. In hemipteran species, the small brown planthopper (SBPH), Laodelphax striatellus, is an injurious crop pest, with a sex chromosome type XX in females and XO in males. This species offers the opportunity to study dosage compensation and sex-biased gene expression. In this study, we generated a chromosome-level genome of SBPH using Oxford Nanopore Technologies and high-throughput chromatin conformation capture (Hi-C) technology. We also sequenced RNA-seq data from 16 tissue samples to annotate the genome and analyze gene dosage compensation. We finally obtained a 510.2 megabases (Mb) genome with 99.12% of the scaffolds anchored on 15 chromosomes (14 autosomes and 1 X chromosome) and annotated 16,160 protein-coding genes based on full-length cDNA sequencing data. Furthermore, we found complete dosage compensation in all L. striatellus somatic tissues, but lack of dosage compensation in gonad tissue testis. We also found that female-biased genes were significantly enriched on the X chromosome in all tissues, whereas male-biased genes in gonad tissues were enriched on autosomes. This study not only provides a high-quality genome assembly but also lays a foundation for a better understanding of the sexual regulatory network in hemipteran insects.

A feminizing switch in a hemimetabolous insect.

The mechanism of sex determination remains poorly understood in hemimetabolous insects. Here, in the brown planthopper (BPH), Nilaparvata lugens, a hemipteran rice pest, we identified a feminizing switch or a female determiner (Nlfmd) that encodes a serine/arginine-rich protein. Knockdown of Nlfmd in female nymphs resulted in masculinization of both the somatic morphology and doublesex splicing. The female-specific isoform of Nlfmd, Nlfmd-F, is maternally deposited and zygotically transcribed. Depletion of Nlfmd by maternal RNAi or CRISPR-Cas9 resulted in female-specific embryonic lethality. Knockdown of an hnRNP40 family gene named female determiner 2 (Nlfmd2) also conferred masculinization. In vitro experiments showed that an Nlfmd2 isoform, NlFMD2340, bound the RAAGAA repeat motif in the Nldsx pre-mRNA and formed a protein complex with NlFMD-F to modulate Nldsx splicing, suggesting that NlFMD2 may function as an RNA binding partner of the feminizing switch NlFMD. Our results provide novel insights into the diverse mechanisms of insect sex determination.

Chromosome-level assembly of the brown planthopper genome with a characterized Y chromosome.

Hundreds of insect genome sequences have been reported; however, most sequencing projects have not determined the sex chromosomes, and no Y chromosomes from a heterometabolous insect have been identified and characterized to date. The brown planthopper (Nilaparvata lugens Stål) is the most economically damaging pest to rice and is also an ideal research subject for paddy ecology and functional genomics. We previously assembled a draft female genome mainly using second-generation sequencing technologies, with a contig N50 of only 24 kb, due to the large size and excessive repetitive regions in the N. lugens genome. Here, we utilize third-generation sequencing technologies and Hi-C data to generate a high-quality male N. lugens assembly with a contig N50 of 1.01 Mb, a scaffold N50 of 69.96 Mb and more than 95.6% of the assembled bases located on 16 chromosomes. Fourteen autosomes and two sex chromosomes (X + Y) were identified, filling in the gap related to the Y chromosome in heterometabolous insects. A total of 18,021 protein-coding genes and 6423 long-noncoding RNAs were predicted with full-length cDNA sequencing data. All 315 of the Y chromosome genes (Y-genes) were derived from autosomal and X-chromosome duplications. Large-scale RNA interference (RNAi) experiments were conducted against the N. lugens Y-genes, demonstrating that 7 Y-genes were essential for normal BPH development or male organ development, suggesting the importance of Y-genes. The first identified Y chromosome in heterometabolous insects will help gain more insight into sex determination, fertility and chromosome evolution.

Identification and functional analysis of the doublesex gene in the sexual development of a hemimetabolous insect, the brown planthopper.

In the sex determination cascade, the genes dsx (doublesex) in insects, mab-3 (male abnormal 3) in nematodes, and Dmrt1 (dsx/mab-3 related transcription factor-1) in vertebrates act as the base molecular switches and play important roles. Moreover, these genes share the same conserved feature domain-DNA-binding oligomerization domain (OD1), and female-specific dsx also has a conserved oligomerization domain 2 (OD2). Although sex determination and the functions of dsx in several holometabolous insects have been well documented, sex determination and the function of dsx in hemimetabolous insects remain a mystery. In this study, four dsx homologs were unexpectedly found in the Nilaparvata lugens (brown planthopper, BPH, order Hemiptera), which also showed a different evolutionary status. We found that only one of the four homologs, Nldsx, which has three alternative splicing variants (female-specific NldsxF, male-specific NldsxM, non-sex-specific NldsxC), was required in the sexual development of N. lugens. Compared with that of holometabolous species, the dsx of N. lugens contains a less conserved OD1, while the OD2 domain of BPH was not identifiable because the common region is poorly conserved, and the female-specific region is short. RNAi-mediated knockdown of Nldsx in female BPH resulted in a larger body size with a normal abdomen and reproductive system, while no changes in fertility were noted. However, adult males with RNA interference knockdown of NldsxM in nymphs became pseudofemales, were infertile, had abnormal copulatory organs, and had impassable deferent ducts with hyperplastic walls; additionally, the pseudofemales could not produce the normal courtship signals. Our results suggest that dsx plays a critical role in male BPH somatic development and mating behavior. This is the first study to show that dsx is essential for sexual development in a hemipteran species.