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.

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.

[Comparison of Clinical Effects of Electroacupuncture of Abdominal and Limb Acupoints in the Treatment of Acute Pancreatitis].

OBJECTIVE: To compare the difference of therapeutic efficacy of electroacupuncture (EA) stimulation of abdominal acupoints and limb acupoints in the treatment of acute pancreatitis (AP), so as to provide a reference for EA treatment of AP. METHODS: A total of 60 patients with AP were equally and randomly divided into abdominal acupoint group and limb acupoint group. On the basis of retention enema of Chaiqin Chengqi Decoction (containing Radix Bupleurum, Radix Scutellaria, etc.), and abdominal external application of Liuhe Powder ointment (containing Cinnabaris, pearl powder, etc.), patients of the two groups were also treated with acupuncture stimulation of acupoints at the abdomen or limbs. The abdominal acupoints were Shangwan (CV 13), Zhongwan (CV 12), Xiawan(CV 10), Liangmen (ST 21, left), Taiyi(ST 23, left), Chengman (ST 20, left), Fu'ai (SP 16, left), Yindu(KI 19, right), and the limb acupoints were Hegu(LI 4), Neiguan(PC 6), Zusanli(ST 36), Shangjuxu(ST 37), Xiajuxu(ST 39), and Yinlingquan(SP 9) which were punctured with filiform needles by retaining the needles for 30 min after twirling for a while. CV 12 - ST 21, CV 10- SP 16 of the abdomen group, and bilateral PC 6 and ST 36 of the limb group were administered EA (2 Hz/15 Hz, 1 mA and duration of 30 min). The treatment was conducted once daily for 5 days. The abdominal pain severity and distension severity were assessed using the visual analogue scale (VAS), and the abdominal girth was measured by using a soft ruler. RESULTS: Following the treatment, the abdominal pain and distension symptom scores in the lightest and most severe phases of AP and the abdominal girth levels were considerably decreased on the 5th day in the two groups in comparison with their own pre-treatment in the same one group (P < 0.05); and the abdominal pain scores on the 2nd, 3rd and 4th day in the lightest phase, and those on the 2nd, 3rd, 4th and 5th day in the most severe phase were markedly lower in the abdomen acupoint group than in the limb acupoint group (P<0.05). No significant differences were found between two groups in the abdominal distension symptom scores and abdominal circumference levels in the lightest and most severe phases of AP (P>0.05). In addition, no adverse events were found in both groups. CONCLUSION: EA has a good curative effect in the treatment of AP patients. The curative effect of acupuncture of the abdominal acupoints is significantly superior to that of limb acupoints in relieving abdominal pain.