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.

Molecular Characterization of Ca2+/Calmodulin-Dependent Protein Kinase II Isoforms in Three Rice Planthoppers-Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II (CaMKII) gene from Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20-81 amino acid "inserts" were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph-1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera, the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR, USP1, and HR3, in N. lugens. These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.

Differential Responses of OsMPKs in IR56 Rice to Two BPH Populations of Different Virulence Levels.

The conserved mitogen-activated protein kinase (MAPK) cascades play vital roles in plant defense responses against pathogens and insects. In the current study, the expression profiles of 17 OsMPKs were determined in the TN1 and IR56 rice varieties under the infestation of brown planthopper (BPH), one of the most destructive hemimetabolous rice pests. The virulent IR56 BPH population (IR56-BPH) and the avirulent TN1 BPH population (TN-BPH) were used to reveal the roles of OsMPKs in the compatible (IR56-BPH infested on the TN1 and IR56 rice varieties, and TN1-BPH infested on the TN1 rice variety) and the incompatible (TN1-BPH infested on the IR56 rice variety) interaction. The statistical analysis revealed that rice variety, BPH population type, and infestation period have significant effects on the transcription of OsMPKs. Out of these genes, five OsMPKs (OsMPK1, OsMPK3, OsMPK7, OsMPK14, and OsMPK16) were found to exhibit upregulated expression only during incompatible interaction. Six OsMPKs (OsMPK4, OsMPK5, OsMPK8, OsMPK9, OsMPK12, and OsMPK13) were associated with both incompatible and compatible interactions. The transcription analysis of salicylic acid, jasmonic acid, and ethylene phytohormone signaling genes revealed their roles during the rice⁻BPH interactions. The upregulated expression of OsC4H, OsCHS, and OsCHI in the incompatible interaction implied the potential defense regulatory roles of phenylpropanoids. In both varieties, the elevated transcript accumulations of OsGST and OsSOD, and the increased enzyme activities of POD, SOD, and GST at 1 day post-infestation (dpi), but not at 3 dpi, indicated that reactive oxygen species (ROS) signaling might be an early event in rice⁻BPH interactions. Furthermore, upregulated transcription of OsLecRK3 and OsLecRK4 was found only during an incompatible interaction, suggesting their involvement in the BPH resistance response in the IR56 rice variety. Lastly, based on the findings of this study, we have proposed a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions, respectively.

Knockdown of a putative argininosuccinate lyase gene reduces arginine content and impairs nymphal development in Nilaparvata lugens.

Nilaparvata lugens is a typical phloem feeder. Rice phloem is high in simple sugars and very low in essential amino acids. Nilaparvata lugens harbors an ascomycete Entomomyces delphacidicola that hypothetically biosynthesizes several amino acids to meet the nutrition requirement of the planthopper. Among these amino acids, here, we focused on arginine biosynthesis. A complete cDNA of an E. delphacidicola gene, arginine-succinate lyase, EdArg4, the last step in arginine biosynthesis, was obtained. RNAi-mediated suppression of EdArg4 reduced arginine content in the hemolymph, and decreased the expression of several arginine biosynthesis genes. Silencing of EdArg4 delayed nymphal development and led to nymphal lethality. About 20% of the EdArg4 RNAi surviving adults were deformed. The most obvious defect was wider and larger abdomen. The EdArg4 RNAi-treated planthoppers had thickened wings and enlarged antennae, legs, and anal tubes and a few adults did not normally emerge. Arginine deficiency in the EdArg4 RNAi planthoppers repressed nitric oxide signaling, determined at the transcriptional level. We infer that E. delphacidicola biosynthesizes essential arginine to compensate for nutrition deficiency in N. lugens.

ATP phosphoribosyltransferase from symbiont Entomomyces delphacidicola invovled in histidine biosynthesis of Nilaparvata lugens (Stål).

Histidine is an essential amino acid assumed to be synthesized by an obligatory yeast-like symbiont (Entomomyces delphacidicola str. NLU) in Nilaparvata lugens, an important rice pest. The adenosine-triphosphate phosphoribosyltransferase (ATP-PRTase) facilities the committed first step of the histidine biosynthesis pathway. In the current study, a putative ATP-PRTase was cloned and verified to be of E. delphacidicola origin (EdePRTase). The expression of the gene was spatial and temporal universal with a profile that matched the distribution of the fungal symbiont. RNA interference aided the knockdown of the EdePRTase-suppressed EdePRTase expression by 32-48 %. Hemolymph histidine level was also reduced followed by significant reduction of adult body weight. However, other performance characters including nymph development, survival, and adult sex ratio were not adversely affected by the knockdown. Furthermore, forced histidine exposure (through injection or feeding) significantly inhibited the EdePRTase mRNA levels at higher concentrations, but significantly increased EdePRTase expression levels at lower concentrations (feeding only). The significance of these findings support that the EdePRTase is from symbiont E. delphacidicola, and its involvement in histidine biosynthesis of N. lugens was discussed. The results provide a better understanding of EdePRTase and the encoded functional ATP-PRTase enzyme regulation in N. lugens and insects in general.

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (Stål) (Hemiptera:Delphacidae).

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80-87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties.

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.

Defense Regulatory Network Associated with circRNA in Rice in Response to Brown Planthopper Infestation.

The brown planthopper (BPH), Nilaparvata lugens (Stål), a rice-specific pest, has risen to the top of the list of significant pathogens and insects in recent years. Host plant-mediated resistance is an efficient strategy for BPH control. Nonetheless, BPH resistance in rice cultivars has succumbed to the emergence of distinct virulent BPH populations. Circular RNAs (circRNAs) play a pivotal role in regulating plant-environment interactions; however, the mechanisms underlying their insect-resistant functions remain largely unexplored. In this study, we conducted an extensive genome-wide analysis using high-throughput sequencing to explore the response of rice circRNAs to BPH infestations. We identified a total of 186 circRNAs in IR56 rice across two distinct virulence groups: IR-IR56-BPH (referring to IR rice infested by IR56-BPH) and IR-TN1-BPH, along with a control group (IR-CK) without BPH infestation. Among them, 39 circRNAs were upregulated, and 43 circRNAs were downregulated in the comparison between IR-IR56-BPH and IR-CK. Furthermore, in comparison with IR-CK, 42 circRNAs exhibited upregulation in IR-TN1-BPH, while 42 circRNAs showed downregulation. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the targets of differentially expressed circRNAs were considerably enriched in a multitude of biological processes closely linked to the response to BPH infestations. Furthermore, we assessed a total of 20 randomly selected circRNAs along with their corresponding expression levels. Moreover, we validated the regulatory impact of circRNAs on miRNAs and mRNAs. These findings have led us to construct a conceptual model that circRNA is associated with the defense regulatory network in rice, which is likely facilitated by the mediation of their parental genes and competing endogenous RNA (ceRNA) networks. This model contributes to the understanding of several extensively studied processes in rice-BPH interactions.

Enhanced yield performance of Bt rice under target-insect attacks: implications for field insect management.

The rapid development of transgenic biotechnology has greatly promoted the breeding of genetically engineered (GE) rice in China, and many GE rice lines are in the pipeline for commercialization. To understand field performances of GE rice, key agronomic traits of two insect-resistant Bt rice lines that have been granted biosafety certificates for commercial production in China were evaluated together with their nontransgenic counterparts under environmental conditions with significant differences in insect pressure. Results from the experiments showed enhanced field performances of the Bt GE rice lines compared with the non-GE counterparts for yield-related traits such as number of panicles and filled seeds per plant, under environmental conditions with no insecticide application. No detectable underlying cost of the Bt transgene was observed in the two insect-resistant GE rice lines, particularly in the GE hybrid rice line. Results further indicated significantly greater yield performances of the two insect-resistant GE rice lines under environmental conditions with non-target insect control compared with no insect control. It is concluded from this study that insect-resistant Bt GE rice, particularly the hybrid line, has great potential to maintain its high yield when ambient insect pressure is high. In addition, proper application of insecticides to control non-target insects will guarantee optimal performance of insect-resistant Bt GE rice.

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis.

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC(50) of 0.24 and 0.30 microg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.

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.

Identification and functional analysis of five genes that encode distinct isoforms of protein phosphatase 1 in Nilaparvata lugens.

Ten distinct cDNAs encoding five different protein phosphatases 1 (PPP1) were cloned from Nilaparvata lugens. NlPPP1α and NlPPP1ß are highly conserved whereas NlPPP1-Y, NlPPP1-Y1 and NlPPP1-Y2 are lowly conserved among insects. NlPPP1α and NlPPP1ß exhibited a ubiquitous expression, while NlPPP1-Y, NlPPP1-Y1, and NlPPP1-Y2 were obviously detected from the 4th instar nymph to imago developmental stages in males, especially detected in internal reproductive organ and fat bodies of the male. Injection nymphs with dsRNA of NlPPP1α or NlPPP1ß was able to reduce the target gene expression in a range of 71.5-91.0%, inducing a maximum mortality rate of 95.2% or 97.2% at 10th day after injection and eclosion ratio down by 65.5-100.0%. Injection with dsNlPPP1Ys targeted to NlPPP1-Y, NlPPP1-Y1and NlPPP1-Y2 was able to induce a maximum mortality rate of 95.5% at 10th day after injection, eclosion ratio down by 86.4%. Knock-down one of the male-biased NlPPP1 genes has no effect on survival and eclosion ratio. Injection of 4th instar nymph with dsNlPPP1Ys led to reduced oviposition amount and hatchability, down by 44.7% and 19.6% respectively. Knock-down of NlPPP1-Y1 or NlPPP1-Y2 gene did not significantly affect oviposition amount but significantly affected hatchability. The results indicate that the male-biased NlPPP1 genes have overlapping functions in N. lugens development, and NlPPP1-Y1 and NlPPP1-Y2 may play important roles in spermatogenesis and fertilization. The dsNlPPP1ß and dsNlPPP1Ys in this study could be the preferred sequence in RNAi and low-conserved male-biased NlPPP1 genes could be potential target for N. lugens control.

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.

Double-stranded RNA targeting calmodulin reveals a potential target for pest management of Nilaparvata lugens.

BACKGROUND: Calmodulin (CaM) is an essential protein in cellular activity and plays important roles in many processes in insect development. RNA interference (RNAi) has been hypothesized to be a promising method for pest control. CaM is a good candidate for RNAi target. However, the sequence and function of CaM in Nilaparvata lugens are unknown. Furthermore, the double-stranded RNA (dsRNA) target to CaM gene in pest control is still unavailable. RESULTS: In the present study, two alternatively spliced variants of CaM transcripts, designated NlCaM1 and NlCaM2, were cloned from N. lugens. The two cDNA sequences exhibited 100% identity to each other in the open reading frame (ORF), and only differed in the 3' untranslated region (UTR). NlCaM including NlCaM1 and NlCaM2 mRNA was detectable in all developmental stages and tissues of N. lugens, with significantly increased expression in the salivary glands. Knockdown of NlCaM expression by RNAi with different dsRNAs led to an inability to molt properly, increased mortality, which ranged from 49.7 to 92.5%, impacted development of the ovaries and led to female infertility. There were no significant reductions in the transcript levels of vitellogenin and its receptor or in the total vitellogenin protein level relative to the control group. However, a significant reduction in vitellogenin protein was detected in ovaries injected with dsNlCaM. In addition, a specific dsRNA of NlCaM for control of N. lugens was designed and tested. CONCLUSION: NlCaM plays important roles mainly in nymph development and uptake of vitellogenin by ovaries in vitellogenesis in N. lugens. dsRNA derived from the less conserved 3'-UTR of NlCaM shows great potential for RNAi-based N. lugens management. © 2018 Society of Chemical Industry.

Ras-like family small GTPases genes in Nilaparvata lugens: Identification, phylogenetic analysis, gene expression and function in nymphal development.

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRß, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRß could be served as a candidate target for dsRNA-based pesticides for N.lugens control.

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.

A Genome-Wide Identification and Analysis of the Basic Helix-Loop-Helix Transcription Factors in Brown Planthopper, Nilaparvata lugens.

The basic helix-loop-helix (bHLH) transcription factors in insects play essential roles in multiple developmental processes including neurogenesis, sterol metabolism, circadian rhythms, organogenesis and formation of olfactory sensory neurons. The identification and function analysis of bHLH family members of the most destructive insect pest of rice, Nilaparvata lugens, may provide novel tools for pest management. Here, a genome-wide survey for bHLH sequences identified 60 bHLH sequences (NlbHLHs) encoded in the draft genome of N. lugens. Phylogenetic analysis of the bHLH domains successfully classified these genes into 40 bHLH families in group A (25), B (14), C (10), D (1), E (8) and F (2). The number of NlbHLHs with introns is higher than many other insect species, and the average intron length is shorter than those of Acyrthosiphon pisum. High number of ortholog families of NlbHLHs was found suggesting functional conversation for these proteins. Compared to other insect species studied, N. lugens has the highest number of bHLH members. Furthermore, gene duplication events of SREBP, Kn(col), Tap, Delilah, Sim, Ato and Crp were found in N. lugens. In addition, a putative full set of NlbHLH genes is defined and compared with another insect species. Thus, our classification of these NlbHLH members provides a platform for further investigations of bHLH protein functions in the regulation of N. lugens, and of insects in general.