Characterization of light-dependent rhythm of courtship vibrational signals in Nilaparvata lugens: essential involvement of cryptochrome genes.

BACKGROUND: Vibrational signal plays a crucial role in courtship communication in many insects. However, it remains unclear whether insect vibrational signals exhibit daily rhythmicity in response to changes in environmental cues. RESULTS: In this study, we observed daily rhythms of both female vibrational signals (FVS) and male vibrational signals (MVS) in the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most notorious rice pests across Asia. Notably, oscillations of FVS and MVS in paired BPHs were synchronized as part of male-female duetting interactions, displaying significant day-night rhythmicity. Furthermore, we observed light dependency of FVS emissions under different photoperiodic regimes (18 L:6 D and 6 L:18 D) and illumination intensity levels (>300 lx, 50 lx, and 25 lx). Subsequently, the potential role of circadian clock genes cryptochromes (Nlcry1 and Nlcry2) in regulating FVS daily oscillations was examined using gene knockdown via RNA interference. We observed sharp declines and disrupted rhythms in FVS frequencies when either of the Nlcrys was downregulated, with Nlcry2 knockdown showing a more prominent effect. Moreover, we recorded a novel FVS variant (with a dominant frequency of 361.76 ± 4.31 Hz) emitted by dsNlcry1-treated BPH females, which significantly diminished the impact of courtship stimuli on receptive males. CONCLUSION: We observed light-dependent daily rhythms of substrate-borne vibrational signals (SBVS) in BPH and demonstrated essential yet distinct roles of the two Nlcrys. These findings enhanced our understanding of insect SBVS and illustrated the potential of novel precision physical control strategies for disrupting mating behaviors in this rice pest. © 2023 Society of Chemical Industry.

Genetic diversity of RNA viruses infecting invertebrate pests of rice.

Invertebrate species are a natural reservoir of viral genetic diversity, and invertebrate pests are widely distributed in crop fields. However, information on viruses infecting invertebrate pests of crops is limited. In this report, we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields. We identified 296 new RNA viruses and 13 known RNA viruses. These viruses clustered within 31 families, with many highly divergent viruses constituting potentially new families and genera. Of the identified viruses, 13 RNA viruses clustered within the Fiersviridae family of bacteriophages, and 48 RNA viruses clustered within families and genera of mycoviruses. We detected known rice viruses in novel invertebrate hosts at high abundances. Furthermore, some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species. Forty-five potential insect pathogenic RNA viruses were detected in invertebrate species. Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity. Cross-species transmission of RNA viruses was detected between invertebrate hosts. Newly identified viral genomes showed extensive variation for invertebrate viral families or genera. Together, the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species, the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

Resistance to Planthoppers and Southern Rice Black-Streaked Dwarf Virus in Rice Germplasms.

The damage caused by the white-back planthopper (WBPH, Sogatella furcifera) and brown planthopper (BPH, Nilaparvata lugens), as well as southern rice black-streaked dwarf virus (SRBSDV), considerably decreases the grain yield of rice. Identification of rice germplasms with sufficient resistance to planthoppers and SRBSDV is essential to the breeding and deployment of resistant varieties and, hence, the control of the pests and disease. In this study, 318 rice accessions were evaluated for their reactions to the infestation of both BPH and WBPH at the seedling stage using the standard seed-box screening test method; insect quantification was further conducted at the end of the tillering and grain-filling stages in field trials. Accessions HN12-239 and HN12-328 were resistant to both BPH and WBPH at all tested stages. Field trials were conducted to identify resistance in the collection to SRBSDV based on the virus infection rate under artificial inoculation. Rathu Heenati (RHT) and HN12-239 were moderately resistant to SRBSDV. In addition, we found that WBPH did not penetrate stems with stylets but did do more probing bouts and xylem sap ingestion when feeding on HN12-239 than the susceptible control rice Taichung Native 1. The resistance of rice accessions HN12-239, HN12-328, and RHT to BPH, WBPH, and/or SRBSDV should be valuable to the development of resistant rice varieties.

Effect of Paclobutrazol Application on Enhancing the Efficacy of Nitenpyram against the Brown Planthopper, Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive rice pests in Asia. It has already developed a high level of resistance to many commonly used insecticides including nitenpyram (NIT), which is a main synthetic insecticide that is used to control BPH with a much shorter persistence compared to other neonicotinoid insecticides. Recently, we found that an exogenous supplement of paclobutrazol (PZ) could significantly enhance the efficacy of NIT against BPH, and the molecular mechanism underlying this synergistic effect was explored. The results showed that the addition of a range of 150-300 mg/L PZ increased the toxicity of NIT against BPH with the highest mortalities of 78.0-87.0% on the 16th day after treatments, and PZ could also significantly prolong the persistence of the NIT efficacies. Further investigation suggested that PZ directly increased the content of flavonoids and H2O2 in rice and increased the activity of polyphenol oxidase, which might be involved in the constitutive defense of rice in advance. Additionally, there was an interaction between PZ and BPH infestation, indicating that PZ might activate the host defense responses. Therefore, PZ increased the efficacy of NIT against the brown planthoppers by enhancing the constitutive and inducible defense responses of rice. Our study showed for the first time that PZ could contribute to improving the control effects of insecticides via inducing the defense responses in rice plants against BPH, which provided an important theoretical basis for developing novel pest management strategies in the field.

SPARC plays an important role in the oviposition and nymphal development in Nilaparvata lugens Stål.

BACKGROUND: The brown planthopper (Nilaparvata lugens Stål)is a notorious rice pest in many areas of Asia. Study on the molecular mechanisms underlying its development and reproduction will provide scientific basis for effective control. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is one of structural component of the extracellular matrix, which influences a diverse array of biological functions. In this study, the gene for SPARC was identified and functionally analysed from N.lugens. RESULTS: The result showed that the NlSPARC mRNA was highly expressed in fat body, hemolymph and early embryo. The mortality increased significantly when NlSPARC was downregulated after RNA interference (RNAi) in 3 ~ 4th instar nymphs. Downregulation of NlSPARC in adults significantly reduced the number of eggs and offspring, as well as the transcription level of NlSPARC in newly hatched nymphs and survival rate in progeny. The observation with microanatomy on individuals after NlSPARC RNAi showed smaller and less abundant fat body than that in control. No obvious morphological abnormalities in the nymphal development and no differences in development of internal reproductive organ were observed when compared with control. CONCLUSION: NlSPARC is required for oviposition and nymphal development mainly through regulating the tissue of fat body in N.lugens. NlSPARC could be a new candidate target for controlling the rapid propagation of N.lugens population. Our results also demonstrated that the effect of NlSPARC RNAi can transfer to the next generation in N.lugens.

Knockdown of the chromatin remodeling ATPase gene Brahma impairs the reproductive potential of the brown planthopper, Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests in rice-growing regions of Asia. Extensive studies have suggested that SWI/SNF chromatin remodeling ATPase Brahma (BRM) plays multiple roles in the insect model Drosophila. Yet much less is known about the physiological properties for NlBRM. In the present study, the cloned full-length cDNA of NlBRM was 5637 bp and contained an ORF of 5292 bp encoding a 194.53 kD protein. The spatiotemporal dynamics of NlBRM was investigated by qPCR, which showed that it was abundantly expressed in the egg and ovary. Then significant downregulation of NlBRM by dsRNA injection had a relatively greater impact on female survival than male. Moreover, the number of oviposition marks of the NlBRM-RNAi females were declined by 61.11% - 73.33% compared with the controls during the subsequent 5 days after dsRNA injection. Meanwhile, the number of newly hatched BPH nymphs also decreased correspondingly by 93.56% - 100%. Phenotypic analysis revealed that none of normally banana-shaped eggs were discernable in the ovaries of NlBRM-deficient females, where mRNA expression of N. lugens vitellogenin gene was also reduced. Our results demonstrated that NlBRM played a crucial role in ovarian development and fecundity of BPH, likely by regulating the vitellogenin gene in vivo, which could be as a promising target for parental RNAi-based control of this serious rice pest.

Biological Traits of the Pincer Wasp Gonatopus Flavifemur (Esaki & Hashimoto) Associated with Different Stages of Its Host, the Brown Planthopper, Nilaparvata Lugens (Stål).

Gonatopus flavifemur (Esaki & Hashimoto) is a common parasitoid of the most important rice pest, the brown planthopper (BPH) Nilaparvata lugens (Stål), in eastern and southeastern Asia. We investigated the parasitism rates, feeding rates, and offspring development of G. flavifemur in association with five instars of BPH nymphs and male and female adults under laboratory conditions (27 ± 1 °C and 70 ± 5% Relative Humidity). The results showed that the life stage of the host significantly affects parasitism, host feeding, and offspring development by G. flavifemur. The parasitism rate was highest on 4th instar nymphs, and the feeding rate was highest on 1st instar nymphs. The cocooning rate on male adult BPHs was significantly lower than that on other stages; however, emergence rates did not significantly differ among the BPH stages. The rate of female offspring upon emergence from 5th instars was higher than other stages. Both the parasitism and host-feeding functional responses of G. flavifemur to different BPH stages fit well with Holling type II models, supporting the results of parasitism and feeding rates and indicating that G. flavifemur would be a good agent for BPH control. In conclusion, G. flavifemur prefers to feed on young nymphs but prefers to parasitize older nymphs. In addition, 5th instar nymphs are favorable to female offspring of the pincer wasp.

Phenotypic and transcriptomic responses of two Nilaparvata lugens populations to the Mudgo rice containing Bph1.

The Bph1 gene was the first reported brown planthopper (BPH, Nilaparvata lugens) resistance gene in Mudgo rice and was widely used as a commercial cultivar for controlling BPH infestations. However, rapid adaptations of BPH on the Mudgo rice resulted in its resistance breakdown and the emergence of virulent BPH populations. Thus, specific BPH populations and rice varieties can serve as good model systems for studying the roles of different bio-compounds and proteins in the insect-plant interactions. Although our understandings have been improved on the complexity of BPH and rice interactions, the underlying molecular mechanisms remain largely unknown. Here we analyzed the feeding performances and the transcriptomic responses of two BPH populations (Mugdo-BPH and TN1-BPH) during compatible (Mudog-BPH feeding on Mudgo rice) and incompatible (TN1-BPH feeding on Mudgo rice) interactions. The electrical penetration graph (EPG) results indicated that the BPH feeding and performances during the incompatible interaction are significantly affected in terms of decreased honeydew, loss of weight, decreased phloem sap ingestion (N4 waveform), but increased non-penetration (NP waveform) phase. Abundance of glucose and trehalose was reduced in BPH during the incompatible interaction. Transcriptomic surveys of insects in both interactions revealed that genes involved in cuticle formation, detoxification, metabolite transport, digestion, RNA processing, lipid or fatty acid metabolism, and proteolysis were significantly down-regulated during the incompatible interaction, whereas genes involved in insulin signaling were significantly upregulated. Knockdown of four genes, including the sugar transporter NlST45, the serine and arginine-rich protein NlSRp54, the cytochrome P450 gene NlCYP6AY1, and the cuticle protein NlCPR70 through RNA-interference revealed thess genes are important for BPH survival. Overall, the results of this study will be helpful for the future researches on BPH virulence shifts.

Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (Stål).

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies.