Molting-related proteases in the brown planthopper, Nilaparvata lugens.

Digestion and absorption of old cuticles during insect molting are necessary for new cuticle formation, during which complicated enzyme catalysis is essential. To date, a few carboxypeptidases, aminopeptidases and serine proteases (mostly trypsins) connected with cuticle digestion, zymogen activation and histological differentiation during the ecdysis of lepidopteran, dipteran and hymenopteran insects have been identified. However, little is known about these proteins in hemimetabolous insects. In this study, we identified 33 candidate trypsin and trypsin-like homologs, 14 metallocarboxypeptidase and 32 aminopeptidase genes in the brown planthopper Nilaparvata lugens, a hemipteran rice pest. Among the proteins encoded by these genes, 9 trypsin-like proteases, 3 metallocarboxypeptidases and 1 aminopeptidase were selected as potential procuticle hydrolases by bioinformatics analysis and in vivo validation. RNA interference targeting these genes demonstrated that 3 trypsin-like proteases (NlTrypsin-8, NlTrypsin-29 and NlTrypsin-32) genes and 1 metallocarboxypeptidase (NlCpB) gene were found to be essential for ecdysis in N. lugens; specifically, gene silencing led to incomplete cuticle degradation and arrested ecdysis, causing lethal morphological phenotype acquisition. Spatiotemporal expression profiling by quantitative PCR and western blotting revealed their specific expression in the integument and their periodic expression during each stadium, with a peak before ecdysis and eclosion. Transmission electron microscopy demonstrated corresponding ultrastructural defects after RNAi targeting, with NlCpB-silenced specimens having the most undigested old procuticles. Immunohistochemical staining revealed that NlTrypsin-8, NlTrypsin-29 and NlCpB were predominantly located in the exuvial space. This research further adds to our understanding of proteases and its potential role in insect ecdysis.

Threonyl-tRNA synthetase gene, a potential target for RNAi-based control of three rice planthoppers.

BACKGROUND: RNA interference (RNAi) has potential as a new strategy for pest control. However, the current overemphasis on the control of a single pest increased control costs. The aim of this study was to find a green method of controlling several pests without affecting the natural enemies with a single target gene. One possible RNAi target is the threonyl-tRNA synthetase (ThrRS), which is conserved and plays a significant role in protein biosynthesis. RESULTS: In this study, one threonyl-tRNA synthetase gene (NlthrS) was identified from the brown planthopper (Nilaparvata lugens). Spatio-temporal expression pattern analysis showed that NlthrS was highly expressed in the ovary, late embryogenesis, nymphs and female adults. In addition, RNAi-mediated knockdown of NlthrS caused 85.6% nymph mortality, 100% female infertility, molting disorder, extended nymph duration and shortened adult longevity. Target-specific results were obtained when dsNlthrS was used to interfere with the whiteback planthopper (Sogatella furcifera), small brown planthopper (Laodelphax striatellus), zig-zag winged leafhopper (Inazuma dorsalis) and their natural enemy (green mirid bug, Cyrtorhinus lividipennis). In addition, dsNlthrS could cause high mortalities of three species of planthoppers (85.6-100%), while only dsNlthrS-1 led to the death (97.3%) of I. dorsalis that was not affected by dsNlthrS-2. Furthermore, neither dsNlthrS-1 nor dsNlthrS-2 could influence the survival of C. lividipennis. CONCLUSION: The results reveal the biological functions of ThrRS in N. lugens in addtion to its protein synthesis, deepening our understanding of tRNA synthase in insects and providing a new method for the control of several rice pests via one dsRNA design. © 2022 Society of Chemical Industry.

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.

The Genetic Network of Forkhead Gene Family in Development of Brown Planthoppers.

We identified 18 distinct Fox genes in the genome of the brown planthopper, Nilaparvata lugens, and further found a novel insect-specific subfamily that we temporarily named FoxT. A total of 16 genes were highly expressed in the eggs, while NlFoxL2 and NlFoxT are female- and male-specific genes, respectively. Large scale RNAi and RNA-seq analyses were used to reveal the functions and potential targets of NlFoxs. In the eggs, NlFoxA, NlFoxN1 and NlFoxN2 are indispensable to early embryogenesis by regulating different target genes; NlFoxG and NlFoxQ co-regulate NlSix3 for brain development; and NlFoxC, NlFoxJ1 and NlFoxP have complementary effects on late embryogenesis. Moreover, NlFoxA, NlFoxNl and NlFoxQ have pleiotropism. NlFoxA and NlFoxQ regulate the expression of NlCHS1 and cuticular proteins, respectively, thereby participating in the formation of cuticles. NlFoxN1, which regulates the expression of NlKrt9 is involved in the formation of intermediate filament frameworks. Our previous studies have revealed that NlFoxL2 and NlFoxO play important roles in chorion formation and wing polyphenism. Altogether, N. lugens Fox genes exhibit functional diversity in embryonic development and organogenesis. This comprehensive study combines genomics, transcriptomics and phenomics, thereby constructing a complex genetic network that spans the entire life cycle of the brown planthopper.

Intersex Plays a Role in Microbial Homeostasis in the Brown Planthopper.

Insects harbor a wide variety of symbiotic microorganisms that are capable of regulating host health and promoting host adaptation to their environment and food sources. However, there is little knowledge concerning the mechanisms that maintain the microbial community homeostasis within insects. In this study, we found that the intersex (ix) gene played an essential role in maintaining microbial homeostasis in the brown planthopper (BPH), Nilaparvata lugens. Injection of the double-strand RNA targeting N. lugens ix (Nlix) into the newly emerged females resulted in abnormal expansion of the copulatory bursa of BPH after mating. Further observation by transmission electron microscopy (TEM) revealed that the abnormally enlarged copulatory bursa resulting from dsNlix treatment was full of microorganisms, while in contrast, the copulatory bursa of dsGFP-treated individuals stored a large number of sperm accompanied by a few bacteria. Moreover, RNA-seq analysis showed that the gene responses to bacteria were remarkably enriched in differentially expressed genes (DEGs). In addition, 16s rRNA sequencing indicated that, compared with control samples, changes in the composition of microbes presented in dsNlix-treated copulatory bursa. Together, our results revealed the immune functions of the Nlix gene in maintaining microbial homeostasis and combating infection in BPH.

Pleiotropic Roles of the Orthologue of the Drosophila melanogaster Intersex Gene in the Brown Planthopper.

Intersex(ix), a gene involved in the sex-determining cascade of Drosophila melanogaster, works in concert with the female-specific product of doublesex (dsx) at the end of the hierarchy to implement the sex-specific differentiation of sexually dimorphic characters in female individuals. In this study, the ix homolog was identified in the brown planthopper (BPH), Nilaparvata lugens, which contained two splice variants expressed in both female and male insects. We found that Nlix played a vital role in the early nymphal development of BPH, showing an accumulated effect. RNAi-mediated knockdown of Nlix at 4th instar led to the external genital defects in both sexes, consequently resulting in the loss of reproductive ability in female and male individuals. After dsRNA injection, the males were normal on testes, while the females had defective ovarian development. Nlix was also required for early embryogenesis. Notably, when the dsNlix microinjection was performed in newly emerged females, the copulatory bursas were abnormally enlarged while the other tissues of the reproductive system developed normally. Our results demonstrated the pleiotropic roles of Nlix in embryogenesis and development of the reproductive system in a hemimetabolous insect species.

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.

Tra-2 Mediates Cross-Talk Between Sex Determination and Wing Polyphenism in Female Nilaparvata lugens.

Sexual dimorphism and wing polyphenism are important and evolutionarily conserved features of many insect species. In this article, we found a cross-talk linking sexual differentiation with wing polyphenism in the brown planthopper (BPH) Nilaparvata lugens (order: Hemiptera). Knockdown of the sex determination gene Transformer-2 in N. lugens (NlTra-2) in nymph caused females to develop into infertile pseudomales containing undeveloped ovaries. Whereas males treated with dsNlTra-2 exhibited normal morphology, but lost fertility. Knockdown of NlTra-2 in adult females (maternal RNAi) resulted in long-winged female offspring, indicating that maternal RNAi changed the wing morphs in female offspring. In addition, silencing of NlTra-2 down-regulated the expression of the forkhead transcription factor FoxO (NlFoxO), and simultaneously up-regulated the expression of phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (NlAkt), the two critical genes in the insulin signaling pathway. Furthermore, the long-winged effect caused by maternal dsNlTra-2 RNAi could be reversed by silencing of NlInR1 and NlAkt, leading to short-winged morphs. We propose that there is a cross-talk between the sexual differentiation and wing polyphenism pathways mediated by NlTra-2 during embryonic stages.