Gene cloning, protein expression, and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum.

RNA interference (RNAi) is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double-stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+ -binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species.

The Jinggangmycin-induced Mthl2 gene regulates the development and stress resistance in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

Methuselah (Mth) belongs to the GPCR family B, which regulates various biological processes and stress responses. The previous transcriptome data showed jinggangmycin (JGM)-induced Mthl2 expression. However, its detailed functional role remained unclear in brown planthopper, Nilaparvata lugens Stål. In adult N. lugens, the Mthl2 gene showed dominant expressions, notably in ovaries and fat body tissues. The 3rd instar nymphs treated with JGM increased starvation, oxidative stress, and high temperature (34 °C) tolerance of the adults. On the contrary, under dsMthl2 treatment, completely opposite phenotypes were observed. The lipid synthesis genes (DGAT1and PNPLA3) of both females and males treated with JGM in the nymphal stage were observed with high expressions, while the lipolysis of the Lipase 3 gene was observed with low expressions. The JGM increased triglyceride (TG) content, fat body droplet size, and the number of fat body droplets. The same treatment also increased the Glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities. An increase in the heat shock protein (HSP70 and HSP90) expression levels was also observed under JGM treatment but not dsMthl2. The current study demonstrated the influential role of the Mthl genes, particularly the Mthl2 gene, in modulating the growth and development and stress-responsiveness in N. lugens. Thus, providing a platform for future applied research programs controlling N. lugens population in rice fields.

The superoxide dismutase (SOD) genes family mediates the response of Nilaparvata lugens to jinggangmycin and sugar.

Introduction: Brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major rice pest causing significant damage to rice throughout the world. Intensive pesticide usage often causes resistance in these seasonal pests, mainly through the modulation of antioxidant machinery. The superoxide dismutase (SOD) gene family is known for regulating BPH response to pesticides. Methods: In the present study, we identified eight NlSOD genes from the NCBI using the BLASTP program. The bioinformatics analysis includes a phylogenetic tree, conserved domain, motifs, gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathways, and protein-protein interaction, highlighting the distinctive functional elements of NlSOD genes. Results and discussion: Additionally, the NlSOD genes showed expression in all developmental stages of BPH. Under three sugars (glucose, sucrose, and trehalose) treatment, the respective upregulation of NlSOD8, NlSOD6, and NlSOD2 was noted. The NlSOD1 induced significantly under jinggamycin (JGM) deduced its potential as a key regulator of BPH response to the pesticide. Our study has provided detailed knowledge of the NlSOD gene family in-silico analysis and the defensive response to insecticide and high sugar of BPH. We hope the results of this research will help to shed light on the resistance of BPH towards insecticide toxicity and high sugar and help to control it more efficiently.

Adenosine Monophosphate-Activated Protein Kinase (AMPK) Phosphorylation Is Required for 20-Hydroxyecdysone Regulates Ecdysis in Apolygus lucorum.

The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.

The Phosphoserine Phosphatase Alters the Free Amino Acid Compositions and Fecundity in Cyrtorhinus lividipennis Reuter.

The mirid bug Cyrtorhinus lividipennis (Reuter) is an important predator that consumes eggs and young nymphs of the brown planthopper Nilaparvata lugens as a primary food source and thus becomes an important member of the rice ecosystem. We identified and characterized the ClPSP gene in C. lividipennis encoding the phosphoserine phosphatase enzyme. The ClPSP has an open reading frame (ORF) of 957 bp encoding a protein with a length of 294bp and it possesses a haloacid dehalogenase-like (HAD) hydrolase, phosphoserine phosphatase, eukaryotic-like (HAD_PSP_eu) conserved domain. Furthermore, the in silico analysis of the ClPSP gene unveiled its distinct characteristics and it serves as a key player in the modulation of amino acids. The ClPSP showed expression in all developmental stages, with higher expression observed in the ovary and fat body. Silencing the ClPSP by RNA interference (RNAi) significantly decreased PSP enzyme activity and expression compared to dsGFP at two days after emergence (2DAE). The dsPSP treatment altered free hemolymph amino acid compositions, resulting in a significant reduction of serine (Ser) and Arginine (Arg) proportions and a significant increase of Threonine (Thr), Cystine (Cys), and Tyrosine (Tyr) in the C. lividipennis female at 2 DAE. Additionally, a hindered total protein concentration in the ovary and fat body, and reduced vitellogenin (Vg) expression, body weight, and number of laid eggs, were also observed. The same treatment also prolonged the preoviposition period and hindered ovarian development. Our data, for the first time, demonstrated the influential role of the PSP gene in modulating the fecundity of C. lividipennis and provide a platform for future insect pest control programs using the PSP gene in modulating fecundity.

The Homeodomain-Leucine Zipper Genes Family Regulates the Jinggangmycin Mediated Immune Response of Oryza sativa to Nilaparvata lugens, and Laodelphax striatellus.

The homeodomain-leucine zipper (HDZIP) is an important transcription factor family, instrumental not only in growth but in finetuning plant responses to environmental adversaries. Despite the plethora of literature available, the role of HDZIP genes under chewing and sucking insects remains elusive. Herein, we identified 40 OsHDZIP genes from the rice genome database. The evolutionary relationship, gene structure, conserved motifs, and chemical properties highlight the key aspects of OsHDZIP genes in rice. The OsHDZIP family is divided into a further four subfamilies (i.e., HDZIP I, HDZIP II, HDZIP III, and HDZIP IV). Moreover, the protein-protein interaction and Gene Ontology (GO) analysis showed that OsHDZIP genes regulate plant growth and response to various environmental stimuli. Various microRNA (miRNA) families targeted HDZIP III subfamily genes. The microarray data analysis showed that OsHDZIP was expressed in almost all tested tissues. Additionally, the differential expression patterns of the OsHDZIP genes were found under salinity stress and hormonal treatments, whereas under brown planthopper (BPH), striped stem borer (SSB), and rice leaf folder (RLF), only OsHDZIP3, OsHDZIP4, OsHDZIP40, OsHDZIP10, and OsHDZIP20 displayed expression. The qRT-PCR analysis further validated the expression of OsHDZIP20, OsHDZIP40, and OsHDZIP10 under BPH, small brown planthopper (SBPH) infestations, and jinggangmycin (JGM) spraying applications. Our results provide detailed knowledge of the OsHDZIP gene family resistance in rice plants and will facilitate the development of stress-resilient cultivars, particularly against chewing and sucking insect pests.

Octopamine signaling is involved in the female postmating state in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

Mating triggers physiological and behavioral changes in female insects. In many species, females experience postmating behavioral and physiological changes that define a post-mated state. These changes are comprised of several conditions, including long-term refractoriness to re-mating and increased production and laying of eggs. Here, we report that mating led to several changes in brown planthopper (BPH) females, including increased octopamine (OA), cAMP concentrations, and activities of several enzymes. Mating also led to changes in the expression of several genes acting in female physiology, including those in the cAMP/PKA signal transduction pathway. OA injections into virgin females led to similar changes. RNAi silencing of the gene encoding tyramine ß-hydroxylase, involved in the final step in OA synthesis, led to decreased expression of these genes, and reduced the cAMP/PKA signaling. At the whole-organism level, the RNAi treatments led to reduced fecundity, body weights, and longevity. RNAi silencing of genes acting in OA signaling led to truncated ovarian development, egg maturation, and ovarian vitellogenin (Vg) uptake. The impact of these decreases is also registered at the population level, seen as decreased population growth. We infer that OA signaling modulates the postmating state in female BPH and possibly other hemipterans.

Seed dressing with triflumezopyrim controls brown planthopper populations by inhibiting feeding behavior, fecundity and enhancing rice plant resistance.

BACKGROUND: Triflumezopyrim (TFM), a novel mesoionic insecticide, has high efficiency at a low dosage, and is mainly used to control hopper species. A previous study demonstrated that seed dressing with TFM effectively controlled rice planthopper populations in mechanically transplanted rice fields; however, mode of action for control was unclear. RESULTS: The study shows that seed dressing with TFM resulted in elevated levels of oxalic acid, flavonoids, phenolic substances, callose and other compounds associated with Nilaparvata lugens resistance in rice plants, and low TFM residue content in rice plant stem and grain. Host choice behavioral experiments showed that N. lugens females prefer feeding on untreated rice plants. Electrical penetration graph (EPG) data showed that seed dressing with TFM at medium and high doses significantly prolonged the non-probing period and inhibited phloem ingestion in N. lugens females. These changes led to a significant decrease in female secretion of honeydew, expression of genes encoding vitellogenin and juvenile hormone acid methyltransferase, body weight and longevity, and significantly influenced several physiological parameters resulting in impaired oocyte growth, fecundity and population. Field survey data showed that seed dressing with TFM was efficacious and relatively durable in protecting rice plants from infestation by planthoppers. CONCLUSION: This study revealed that seed dressing with TFM enhances rice plant resistance to N. lugens by limiting phloem ingestion and increasing the N. lugens non-probing period; this leads to reduced fecundity of females and lowers N. lugens numbers in the field. © 2021 Society of Chemical Industry.

The Amino Acid-Mediated TOR Pathway Regulates Reproductive Potential and Population Growth in Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae).

The predatory mirid bug, Cyrtorhinus lividipennis Reuter, feeds on brown planthopper (BPH) eggs that are deposited on rice and gramineous plants surrounding rice fields. The development and reproduction of C. lividipennis are inhibited by feeding on BPH eggs from gramineous species, and the underlining regulatory mechanism for this phenomenon is unclear. In the present study, HPLC-MS/MS analysis revealed that the concentrations of six amino acids (AAs:Ala, Arg, Ser, Lys, Thr, and Pro) were significantly higher in rice than in five gramineous species. When C. lividipennis fed on gramineous plants with BPH eggs, expression of several genes in the target of rapamycin (TOR) pathway (Rheb, TOR, and S6K) were significantly lower than that in the insects fed on rice plants with BPH eggs. Treatment of C. lividipennis females with rapamycin, dsRheb, dsTOR, or dsS6K caused a decrease in Rheb, TOR, and S6K expression, and these effects were partially rescued by the juvenile hormone (JH) analog, methoprene. Dietary dsTOR treatment significantly influenced a number of physiological parameters and resulted in impaired predatory capacity, fecundity, and population growth. This study indicates that these six AAs play an important role in the mediated-TOR pathway, which in turn regulates vitellogenin (Vg) synthesis, reproduction, and population growth in C. lividipennis.

Frizzled 2 Functions in the Regulation of TOR-Mediated Embryonic Development and Fecundity in Cyrtorhinus lividipennis Reuter.

The mirid bug, Cyrtorhinus lividipennis Reuter, is an important predator of rice planthoppers in Asia. In a previous study, C. lividipennis fed on gramineous weeds with brown planthopper (BPH) eggs had reduced development compared to those fed on rice with BPH eggs. In the current study, the concentrations of selected amino acids (AAs) were higher in rice than five gramineous species, which might explain the enhanced growth of C. lividipennis on rice. When C. lividipennis was fed on AA-deprived artificial diets, the Wnt/ß-catenin pathway was inhibited. Furthermore, C. lividipennis females silenced for expression of Frizzled 2 (Fz2) showed a significant reduction in the Wnt/ß-catenin and target of rapamycin (TOR) pathways. Silencing Fz2 led to decreased expression of the vitellogenin gene (Vg), lower Vg accumulation in oocytes, reduced soluble protein in ovaries and fat bodies, reduced titers of juvenile hormone, prolonged preoviposition periods, and lower predation capacity, body weight, and egg numbers as controlled to controls. Fz2 silencing resulted in undeveloped ovaries and the inhibition of oocyte growth in the ovarioles, resulting in decreased numbers of offspring and reduced hatching rates. The silencing of Fz2 also resulted in aberrant embryos with undeveloped eyespots and organs, suggesting that Fz2 is an essential gene for embryonic development, oogenesis, and egg maturation. In summary, this study established a potential link between Wnt and TOR pathways, which interact synergistically to regulate C. lividipennis reproduction in response to AA signals. These results provide valuable new information that can be applied to large-scale rearing of C. lividipennis predators, which is important for reducing planthopper damage in rice fields.

Ubiquitin-Conjugating Enzyme E2 E Inhibits the Accumulation of Rice Stripe Virus in Laodelphax striatellus (Fallén).

The ubiquitin-proteasome system (UPS) is an essential protagonist in host-pathogen interactions. Among the three classes of enzymes in the UPS, ubiquitin-conjugating enzyme E2 plays a dual role in viral pathogenesis; however, the role of insect E2s in interactions with plant viruses is unclear. Twenty E2-encoding genes in Laodelphax striatellus, the small brown planthopper, were identified and classified into 17 groups by transcriptomic and phylogenetic analysis. Full-length cDNAs of four LstrE2s (LstrE2 A/E/G2/H) were obtained by rapid-amplification of cDNA ends (RACE-PCR) analysis. Expression of the four LstrE2s showed tissue- and development-specific patterns. RT-qPCR analyses revealed that Rice stripe viruse (RSV) infection increased the level of LstrE2 A/E/G2/H. Further study indicated that repression of LstrE2 E via RNAi caused significant increases in the expression of RSV coat protein mRNA and protein levels. These findings suggest that LstrE2 E inhibits RSV accumulation in the planthopper body. Understanding the function of LstrE2 E in RSV accumulation may ultimately result in the development of novel antiviral strategies.

The antibiotic jinggangmycin increases brown planthopper (BPH) fecundity by enhancing rice plant sugar concentrations and BPH insulin-like signaling.

The brown planthopper (BPH), Nilaparvata lugens, is a resurgent pest with an unexpected response to jinggangmycin (JGM), a broadly applied antibiotic used to control rice sheath blight disease. JGM stimulates BPH fecundity, but the underlining molecular mechanisms remain unclear. Here we report that JGM sprays led to increased glucose concentrations, photosynthesis and gene expression, specifically Rubsico, sucrose phosphate synthase, invertase 2 (INV2) and INV3 in rice plants. JGM sprays led to high-glucose rice plants. Feeding BPH on these plants led to increased insulin-like signaling and vitellogenin synthesis. Treating BPH with metformin, a gluconeogenesis inhibitor, reversed the influence of feeding on high-glucose rice, which was rescued by glucose injections. Silencing insulin-like peptide 2 using per os dsRNA led to reduction in juvenile hormone (JH) III titers and other fecundity parameters, which were reversed by topical applications of the JH analog, methoprene. We infer that JGM acts via two broad mechanisms, one through increasing rice plant sugar concentrations and a second by upregulating BPH insulin-like signaling.

Pesticide-Induced Planthopper Population Resurgence in Rice Cropping Systems.

Planthoppers are serious rice pests in Asia. Their population resurgence was first reported in the early 1960s, caused mainly by insecticides that indiscriminately killed beneficial arthropods and target pests. The subsequent resurgence involved two mechanisms, the loss of beneficial insects and insecticide-enhanced planthopper reproduction. In this review, we identify two forms of resurgence, acute and chronic. Acute resurgence is caused by traditional insecticides with rapid resurgence in the F1 generation. Chronic resurgence follows application of modern pesticides, including fungicides and herbicides, with low natural enemy toxicity, coupled with stimulated planthopper reproduction. The chemical-driven syndrome of changes leads to later resurgence in the F2 or later generations. Chronic resurgence poses new threats to global rice production. We review findings on the physiological and molecular mechanisms of chronic planthopper resurgence and suggest research directions that may help manage these new threats.

Male Selenoprotein F-Like (SPF-L) Influences Female Reproduction and Population Growth in Nilaparvata lugens (Hemiptera: Delphacidae).

Selenoproteins serve in anti-oxidant and cellular redox functions in almost all organisms. A recent study characterized a selenoprotein F-like (SPF-L) in the brown plant hopper's (BPH), Nilaparvata lugens, male accessory glands (MAGs), raised the question of whether the SPF-L is associated with female fecundity. In this study, SPF-L mRNA was found to be enriched in the internal reproductive organ (IRO) of virgin males, also expressed relatively stably in virgin males and females, and dietary dsSPF-L-treatments led to reduced MAG protein and Arginine content. Knockdown of NlSPF-L in unmated males did not influence juvenile hormone (JH) III and ecdysteroid titers, however, dsSPF-L-treated mated males had increased JH III titer, and reduced ecdysteroid titer compared to controls. After mating with dsSPF-L-treated males, female partners had reduced fat body and ovary soluble proteins and JH III tier and vitellogenin (Vg) mRNA levels, but no alterations in ecdysteroid titer, body weight or longevity. The experimental females had prolonged pre-oviposition periods and they laid fewer eggs, which suffered reduced hatching rates and population growth index (PGI). Such mating also led to impaired IRO development in males and females, which was confirmed by immunofluorescence staining. We infer that SPF-L affects reproductive success of males and their partners.

Silencing of triazophos-induced Hexokinase-1-like reduces fecundity in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

Hexokinase is a rate-limiting enzyme that plays pivotal roles in glucose homeostasis and energy metabolism via glucose (Glc) phosphorylation and Glc signaling mediation. Previous investigations have revealed the modulatory role of Hexokinase (Hex) genes involved in proper glucose regulation during insect diapause and embryo development, whereas whether it functions in insect fecundity remains largely unknown. We aimed to explore the relationship between Triazophos (TZP)-induced Hex-1 and fecundity of female Nilaparvata lugens. In this study, Hex-1 expression were characterized at different developmental stages and in various tissues of N. lugens, with the highest expression registered in brain tissues and 5th instar nymph. The present findings indicated that TZP + dsHex-1 silencing significantly reduced protein synthesis, including the fat body and ovarian protein content of female adults. Meanwhile, the glycometabolism with respect to the soluble sugar, trehalose and glucose content in female adults were strikingly influenced as a result of Hex-1 knockdown. The relative transcript level of Hex-1, vitellogenin (NlVg) and vitellogenin receptor (NlVgR) considerably decreased in TZP + dsHex-1 treated females compared to TZP and TZP + dsGFP-treated groups. More importantly, TZP + dsHex-1 silencing led to reduced number of eggs laid and vitellogenin (Vg) accumulation as well as retarded ovary development compared with TZP-treated and TZP + dsGFP-treated groups. Taken together, it is proposed that Hex-1 implicates in N. lugens fecundity by exerting profound effects on glycometabolism, protein sythesis and NlVg expression.

Long chain fatty acid coenzyme A ligase (FACL) regulates triazophos-induced stimulation of reproduction in the small brown planthopper (SBPH), Laodelphax striatellus (Fallen).

The small brown planthopper (SBPH), Laodelphax striatellus (Fallen) is a major pest insect of rice, wheat, and maize in China and other countries. SBPH not only damage rice plants through sucking plant sap, but also transmits rice virus diseases, for example, striped virus disease (RSV), black streaked dwarf, and maize rough disease virus. Therefore, understanding of pesticide-induced stimulation of reproduction in SBPH is of great significance for the pest management. Our previous study discovered that triazophos (TZP) increased reproduction of SBPH. But the molecular mechanisms are unclear. Here, by using proteomic analysis, we screened and cloned the gene of long chain fatty acid coenzyme A ligase (FACL), and silenced FACL to examine influences of TZP on reproduction and glycerin content in SBPH females. In TZP-treated females vs control females, there were 41 differential proteins in 18 pathways related to reproduction, of which 8 were up-regulated and 33 were down-regulated. TZP + dsFACL eliminated TZP-induced stimulation of reproduction of SBPH females (↓about 73.92%) and decreased glycerin content and body weight (↓about 19.93% and 13.62%). TZP + dsFACL treatment led to reduced expression of FACL (↓about 61.88%). FACL is a key gene of TZP-induced increase of reproduction of SBPH.

Two Fungicides Alter Reproduction of the Small Brown Planthopper Laodelphax striatellus by Influencing Gene and Protein Expression.

Aside from their intended actions, fungicides can drive pest insect outbreaks due to virtually continuous use and pest evolution. Small brown planthopper (SBPH), Laodelphax striatellus, outbreaks occurred recently in many provinces in China, with devastating rice losses. Because exposure to the fungicide jinggangmycin (JGM) increased reproduction of the brown plant hopper, Nilaparvata lugens, via its influence on fatty acid synthase, we posed the hypothesis that JGM and carbendazim (CBM) influence SBPH reproduction via their influence on enzymes involved in other aspects of lipid metabolism. Exposure to the fungicide CBM stimulated SBPH reproduction (egg-laying up by 78%) and to another fungicide, JGM, led to decreased egg-laying (down by 47.3%). These inverse effects are mediated by down-regulated expression of l-3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) in JGM-treated females and up-regulated expression of hydroxysteroid dehydrogenase-like protein 2-like (HSD) in CBM-treated females. RNAi knockdown of, separately, LCHAD and HSD led to reduced egg-laying (down by 52% for dsLCHAD and by 73% for dsHSD). dsLCHAD, dsHSD, and JGM treatments also led to severely reduced ovarian development in experimental SBPH, with shorted and thinned valvula and lack of egg cells in ovaries. Valvula of CBM-treated females enlarged, with banana-shaped eggs in ovaries. These data strongly support our hypothesis.

An adenylyl cyclase like-9 gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH), Nilaparvata lugens Stål. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.

Silencing pyruvate kinase (NlPYK) leads to reduced fecundity in brown planthoppers, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

Pyruvate kinase (PYK) operates in the glycolytic pathway, responsible for regulating the balance between glycolysis and gluconeogenesis. The previous work indicates PYK acts in development of Drosophila embryos and in embryonic muscle growth, from which it may be inferred that PYK acts in insect fecundity. More to the point, as a central enzyme in the glycolytic pathway, PYK acts in many energy-spending functions in most organisms. On the background findings that triazophos (TZP) stimulates fecundity via increase activities of several genes in brown planthoppers, Nilaparvata lugens, we investigated the combined influence of TZP and silencing a N. lugens PYK (NlPYK) on reproduction-linked biological performance parameters. Here, we report that TZP+dsNlPYK treatments led to reduced (by 26%) ovarian, but not fat body, protein content relative to controls. Ovarian (35%) and fat body (54%) soluble sugar contents were reduced. TZP+dsNlPYK treatments also led to reduced (by about 24%) fecundity, expressed as numbers of eggs laid. These data show directly that NlPYK acts in insect fecundity, probably via increases in glucose metabolism.

Carboxylesterase Precursor (EST-1) Mediated the Fungicide Jinggangmycin-Suppressed Reproduction of Sogatella furcifera (Hemiptera: Delphacidae).

The jinggangmycin (JGM) is a widely used fungicide for controlling the rice sheath blight, Rhizoctonia solani, in China. Previous experiments under lab conditions showed that JGM foliar spray suppressed Sogatella furcifera (Horvath) reproduction. However, the molecular mechanisms of JGM-driven changes in S. furcifera reproduction are unclear. Therefore, we selected carboxylesterase precursor (EST-1) as a target gene for silencing by RNAi based on gene expression profiles. The present results demonstrated that JGM and control + dsSfEST-1 treatments significantly reduced the number of eggs laid (down by 58% and 54%, respectively), oviposition period (down by 57% and 38%, respectively), and longevity (down by 32% and 38%, respectively) in adult females compared with untreated controls, while no pronounced differences in the preoviposition period were observed. Meanwhile, the dietary control + dsSfEST-1 treatment also severely impeded protein synthesis, specifically soluble ovarian protein content (down by 20% and 24%, respectively) and soluble sugar content (down by 42% and 35%, respectively), which led to stunted growth and reduced body weight in adult females. We thereby speculate that downregulated SfEST-1 expression may be one molecular mechanism underlying JGM-driven reproduction in S. furcifera.