The transcription factor CREB3-2 regulated neutral lipase gene expression in ovary of Nilaparvata lugens.

The cyclic AMP-responsive element-binding protein 3 (CREB3) members have unique regulatory roles in cellular lipid metabolism as transcription factors. Two CREB3 proteins in Nilaparvata lugens were identified and analyzed. In ovary, when silencing NlCREB3-2, triacylglycerol (TAG) content dramatically increased but glycerol and free fatty acid (FFA) significantly decreased, which implicated that NlCREB3-2 was involved in the lipase-related TAG metabolism. In N. lugens, five neutral lipases with complete features for TAG hydrolytic activity and high expression in ovary were focused. Among them, the expression levels of three neutral lipase genes were significantly down-regulated by NlCREB3-2 RNAi. The direct regulation of NlCREB3-2 towards the three neutral lipase genes was evidenced by the dual-luciferase reporter assay. After jointly silencing three neutral lipase genes, TAG and glycerol contents displayed similar changes as NlCREB3-2 RNAi. The study proved that NlCREB3-2 participated in TAG metabolism in ovary via the direct activation towards the ovary-specific neutral lipase genes.

Reducing Expression of Salivary Protein Genes by Flonicamid Partially Contributed to Its Feeding Inhibition of the Brown Planthopper on Rice.

Flonicamid inhibits the feeding of piercing-sucking pests as a selective systemic insecticide. The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most serious pests on rice. During feeding, it uses its stylet to collect sap by penetrating the phloem, and at the same time, it delivers saliva into the rice plant. Insect salivary proteins play important roles in feeding and interacting with plants. Whether flonicamid affects the expression of salivary protein genes and then inhibits the feeding of BPH is not clear. Here, from 20 functionally characterized salivary proteins, we screened five salivary proteins (NlShp, NlAnnix5, Nl16, Nl32, and NlSP7) whose gene expressions were significantly inhibited by flonicamid. We performed experimental analysis on two of them (Nl16 and Nl32). RNA interference of Nl32 significantly reduced the survival rate of BPH. Electrical penetration graph (EPG) experiments showed that both flonicamid treatment and knockdown of Nl16 and Nl32 genes significantly reduced the feeding activity of N. lugens in the phloem and also reduced the honeydew excretion and fecundity. These results suggested that the inhibition of flonicamid on the feeding behavior in N. lugens might be partially attributed to its effect on the expression of salivary protein genes. This study provides a new insight into the mechanism of action of flonicamid on insect pests.

The salivary chaperone protein NlDNAJB9 of Nilaparvata lugens activates plant immune responses.

The brown planthopper (BPH) Nilaparvata lugens (Stål) is a main pest on rice. It secretes saliva to regulate plant defense responses, when penetrating rice plant and sucking phloem sap through its stylet. However, the molecular mechanisms of BPH salivary proteins regulating plant defense responses remain poorly understood. A N. lugens DNAJ protein (NlDNAJB9) gene was highly expressed in salivary glands, and the knock down of NlDNAJB9 significantly enhanced honeydew excretion and fecundity of the BPH. NlDNAJB9 could induce plant cell death, and the overexpression of NlDNAJB9 gene in Nicotiana benthamiana induced calcium signaling, mitogen-activated protein kinase (MAPK) cascades, reactive oxygen species (ROS) accumulation, jasmonic acid (JA) hormone signaling and callose deposition. The results from different NlDNAJB9 deletion mutants indicated that the nuclear localization of NlDNAJB9 was not necessary to induce cell death. The DNAJ domain was the key region to induce cell death, and the overexpression of DNAJ domain in N. benthamiana significantly inhibited insect feeding and pathogenic infection. NlDNAJB9 might interact indirectly with NlHSC70-3 to regulate plant defense responses. NlDNAJB9 and its orthologs were highly conserved in three planthopper species, and could induce ROS burst and cell death in plants. Our study provides new insights into the molecular mechanisms of insect-plant interactions.

Knockdown of the salivary protein gene NlG14 caused displacement of the lateral oviduct secreted components and inhibited ovulation in Nilaparvata lugens.

Saliva plays important roles in insect feeding, but its roles in insect reproduction were rarely reported. Here we reported that the knockdown of a salivary gland-specific gene NlG14 disrupted the reproduction through inhibiting the ovulation of the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most devastating rice pests in Asia. NlG14 knockdown caused the displacement of the lateral oviduct secreted components (LOSC), leading to the ovulation disorder and the accumulation of mature eggs in the ovary. The RNAi-treated females laid much less eggs than their control counterparts, though they had the similar oviposition behavior on rice stems as controls. NlG14 protein was not secreted into the hemolymph, indicating an indirect effect of NlG14 knockdown on BPH reproduction. NlG14 knockdown caused the malformation of A-follicle of the principal gland and affected the underlying endocrine mechanism of salivary glands. NlG14 reduction might promote the secretion of insulin-like peptides NlILP1 and NlILP3 from the brain, which up-regulated the expression of Nllaminin gene and then caused the abnormal contraction of lateral oviduct muscle. Another explanation was NlG14 reduction disrupted the ecdysone biosynthesis and action through the insulin-PI3K-Akt signaling in ovary. Altogether, this study indicated that the salivary gland specific protein NlG14 indirectly mediated BPH ovulation process, which established a connexon in function between insect salivary gland and ovary.

Co-CRISPR: A valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda.

The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single-guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic-eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co-CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes.

Characterization of neutral lipases revealed the tissue-specific triacylglycerol hydrolytic activity in Nilaparvata lugens.

The lipid metabolism plays an essential role in the development and reproduction of insects, and lipases are important enzymes in lipid metabolism. In Nilaparvata lugens, an important insect pest on rice, triacylglycerol hydrolytic activities were different among tissues, with high activity in integument, ovary, and fat body, but low activity in intestine. To figure out the tissue-specific triacylglycerol hydrolytic activity, we identified 43 lipases in N. lugens. Of these 43 lipases, 23 belonged to neutral lipases, so this group was selected to perform further experiments on triacylglycerol hydrolysis. The complete motifs of catalytic triads, ß9 loop, and lid motif, are required for the triacylglycerol hydrolytic activity in neutral lipases, which were found in some neutral lipases with high gene expression levels in integument and ovary, but not in intestine. The recombinant proteins of 3 neutral lipases with or without 3 complete motifs were obtained, and the activity determination confirmed the importance of 3 motifs. Silencing XM_022331066.1, which is highly expressed in ovary and with 3 complete motifs, significantly decreased the egg production and hatchability of N. lugens, partially through decline of the lipid metabolism. In summary, at least one-third of important motifs were incomplete in all neutral lipases with high gene expression in intestine, which could partially explain why the lipase activity in intestine was much lower than that in other tissues. The low activity to hydrolyze triacylglycerol in N. lugens intestine might be associated with its food resource and nutrient components, and the ovary-specific neutral lipases were important for N. lugens reproduction.

Insecticide resistance associated overexpression of two sigma GST genes assists Nilaparvata lugens to remedy oxidative stress from feeding on resistant rice variety.

Insect glutathione S-transferases (GSTs) participate in detoxifying insecticides and plant metabolites in two different ways, metabolizing toxic components and remedying oxidative stress. Here in Nilaparvata lugens, a major insect pest on rice, the roles of cytosolic GSTs in resistance to insecticides and to plant defences were evaluated. The over-expression in four resistant strains indicated that NlGSTs1 and NlGSTs2 were essential to resistances to four test insecticides and H2O2 through an antioxidation mechanism. RNAi verified the antioxidation function of NlGSTs1 and NlGSTs2 in the resistances as a common mechanism, regardless of the structural differences among insecticides and H2O2. NlGSTs1 and NlGSTs2 also provided protection for N. lugens against rice defense by the same mechanism, reducing H2O2 levels when N. lugens were fed on the resistant rice variety Mudogo. The antioxidation activity of recombinant NlGSTs1 and NlGSTs2 is higher than their direct detoxification, which supported the ability of these two GSTs to remedy oxidative stress. For oxidative stress remediation as a common mechanism of NlGSTs1 and NlGSTs2 in both insecticide resistance and host adaptability, the development of insecticide resistance might enhance the ability of insects to remedy oxidative stress from feeding on resistant rice variety and thus to lower the resistance level of rice variety to N. lugens. The results call for careful assessment on N. lugens control when both insecticides and resistant rice variety are applied.

Characterization of cuticular proteins in CPR family in the wolf spider, Pardosa pseudoannulata, and the response of one subfamily genes to environmental stresses.

Cuticular protein (CP) plays an essential role in the construction and function of exoskeleton in arthropods. CPR family, CP with Rebers and Riddiford (R&R) Consensus, is the largest CP family in insects, but it lacks systematic research in non-insect arthropods. In this study, we explored CPRs in the wolf spider, Pardosa pseudoannulata, a predator to many insect pests. We totally identified 152 CPRs in P. pseudoannulata genome, which were divided into two subgroups based on R&R Consensus sequences, with 12 CPRs in RR-1 and 140 in RR-2. All RR-2 members presented a novel Consensus with 34 amino acids, G-x(8)-G-x(6)-Y-x-A-x(3)-G-x(7)-N-E-x-G, which was a common characteristic for RR-2 CPRs in chelicerates. Transcriptome data was used to document the expression patterns of CPR genes in different tissues and ecdysis processes. The specific expressions were found for part CPR genes, such as five RR-2 genes that were specifically expressed in male genital bulbs and eleven RR-1 genes that were highly expressed in the integument. Due to the limited number and integument-specific expression of RR-1 genes, we further analyzed their responses to different environmental stresses at the transcriptional level. Except for PapsCPR11, ten RR-1 genes responded to at least one environmental stress, among with the expression of PapsCPR12 was significantly changed by three stresses (dryness, low temperature and imidacloprid treatments). Silencing PapsCPR12 increased the tolerance of P. pseudoannulata to imidacloprid. Overall, the results presented novel Consensus characteristics of CPRs in P. pseudoannulata, which was helpful for the identification and evolution analysis of CPRs in non-insect arthropods.

Nilaparvata lugens salivary protein NlG14 triggers defense response in plants.

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a serious insect pest on rice. It uses its stylet to collect sap by penetrating the phloem and at the same time it delivers saliva into the host plant, which can trigger a reaction. The molecular mechanisms by which BPH salivary proteins result in plant responses are poorly understood. In this study, we screened transcriptomic data from different BPH tissues and found a protein specific to the salivary gland, NlG14, that could induce cell death in plants. We determined that NlG14 is uniquely found in the insect family Delphacidae. Detailed examination of N. lugens showed that NlG14 was mainly localized in the A-follicle of the principal gland of the salivary gland, and that it was secreted into rice plants during feeding. Knockdown of NlG14 resulted in significant nymph mortality when BPH was fed on either rice plants or on an artificial diet. Further analysis showed that NlG14 triggered accumulation of reactive oxygen species, cell death, callose deposition, and activation of jasmonic acid signaling pathways in plants. Transient expression of NlG14 in Nicotiana benthamiana decreased insect feeding and suppressed plant pathogen infection. Thus, NlG14, an essential salivary protein of N. lugens, acted as a potential herbivore-associated molecular pattern to enhance plant resistance to both insects and plant pathogens by inducing multiple plant defense responses. Our findings provide new insights into the molecular mechanisms of insect-plant interactions and offer a potential target for pest management.

The roles of GSTs in fipronil resistance in Nilaparvata lugens: Over-expression and expression induction.

As one of the most important detoxification enzymes in insects, Glutathione S-transferases (GSTs) play key roles in insecticide resistance via direct metabolism and protection against oxidative stress induced by insecticide exposure. Insect GSTs are often considered as the phase II detoxification enzymes, they have potential function to metabolize fipronil as well as its fipronil's metabolites. In the fipronil-resistant Nilaparvata lugens strain G28, GSTs' inhibitor DEM (diethyl maleate) showed the optimal synergistic effects (5.73-fold), indicating the essential roles of GSTs in the resistance to fipronil in this insect species. Four GST genes, NlGSTs1, NlGSTs2, NlGSTe1 and NlGSTd1, were found over-expressed in G28 when compared to its relative susceptible counterpart strain S28. The roles of these four GSTs in fipronil resistance were confirmed via RNAi. The four GST genes were highly over-expressed in the midgut and/or fat body with detoxification action, which might provide more chances for insects to metabolize fipronil and its metabolites. Additionally, the higher induction levels in the GST gene expression by insecticides in the midgut and/or fat body compared to the whole insect also supported the significant roles of the four GSTs in the detoxification. Above all, the results provided evidences to understand the functions of GSTs in fipronil resistance in N. lugens, and gave a reference for other insects in fipronil resistance.

Neurotoxins in the venom gland of Calommata signata, a burrowing spider.

Calommata signata, a burrowing spider, represents a special type of predation mode in spiders, and its utilization of toxins is different from that of web-weaving spiders and wandering spiders. The existing researches on spider toxins are mainly focused on the web-weaving and wandering spiders, but little attention on that of the burrowing spiders. Through transcriptome sequencing of C. signata venom gland and the remaining part as the counterpart tissue, 25 putative neurotoxin precursors were identified. These most neurotoxins were novel because their low similarities with the known sequences except for that of over 50% similarities in four neuropeptide toxins. The 25 neuropeptide toxins were divided into five families according to the constitution of cysteines for the possible disulfide bonds and the similarities of the deduced amino acid sequences. Besides neuropeptide toxins, other potential toxins in the venom gland were also analyzed. Unlike web-weaving spiders and wandering spiders, only a few neurotoxin genes were significantly expressed in the venom gland of C. signata. In the non-peptide toxin genes, only CsTryp_SPc-1, CsPA2-1, CsVa5-2 and four PDI genes were abundantly expressed in the venom gland. The present study provided an improved understanding on the spider toxin diversity and useful information for the exploitation of spider toxins.

Neonicotinoids stimulate H2-limited methane emission in Periplaneta americana through the regulation of gut bacterium community.

Methane emitted by insects is considered to be an important source of atmospheric methane. Here we report the stimulation of methane emission in the cockroach Periplaneta americana and termite Coptotermes chaohuensis, insects with abundant methanogens, by neonicotinoids, insecticides widely used to control insect pests. Cycloxaprid (CYC) and imidacloprid (IMI) caused foregut expansion in P. americana, and increased the methane emission. Antibiotics mostly eliminated the effects. In P. americana guts, hydrogen levels increased and pH values decreased, which could be significantly explained by the gut bacterium community change. The proportion of several bacterium genera increased in guts following CYC treatment, and two genera from four could generate hydrogen. Hydrogen is a central intermediate in methanogenesis. All increased methanogens in both foregut and hindgut used hydrogen as electron donor to produce methane. Besides, the up-regulation of mcrA, encoding the enzyme for the final step of methanogenesis suggested the enhanced methane production ability in present methanogens. In the termite, hydrogen levels in gut and methane emission also significantly increased after neonicotinoid treatment, which was similar to the results in P. americana. In summary, neonicotinoids changed bacterium community in P. americana gut to generate more hydrogen, which then stimulated gut methanogens to produce and emit more methane. The finding raised a new concern over neonicotinoid applications, and might be a potential environmental risk associated with atmospheric methane.

Contribution of Glutathione S-Transferases to Imidacloprid Resistance in Nilaparvata lugens.

The importance of glutathione S-transferases (GSTs) in imidacloprid resistance in Nilaparvata lugens, a major rice pest, and other insects was often excluded, mostly due to the slight effects of diethyl maleate (DEM) on synergizing imidacloprid in resistant populations. Here, we found that the synergistic effects of DEM were time-dependent. At 24 or 48 h, the time often selected to record mortalities in imidacloprid bioassay, DEM really did not cause an obvious increase in imidacloprid toxicity. However, significant effects were observed after 72 h. The results revealed that GSTs, as phase II detoxification enzymes to metabolize secondary products generated from phase I detoxification enzymes, were also important in imidacloprid resistance in N. lugens, but might have occurred a little later than that of P450s and CarEs as phase I enzymes. The constitutive overexpression in the imidacloprid-resistant strain G25 and expression induction by imidacloprid in the susceptible strain S25 indicated that four GST genes, NlGSTs1, NlGSTs2, NlGSTe1, and NlGSTm1, were important in imidacloprid resistance, which was confirmed by RNAi test. The higher expression levels and more expression induction by imidacloprid in the midgut and fat body compared to the whole insect supported the important roles of these four GSTs, which was also supported by the more overexpression times in the midgut and fat body versus the whole insect between G25 and S25 strains. Taking the data together, the study ascertained the roles of GSTs in imidacloprid resistance in N. lugens.