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Ten distinct cDNAs encoding five different protein phosphatases 1 (PPP1) were cloned from Nilaparvata lugens. NlPPP1α and NlPPP1β are highly conserved whereas NlPPP1-Y, NlPPP1-Y1 and NlPPP1-Y2 are lowly conserved among insects. NlPPP1α and NlPPP1β 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 NlPPP1β 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 dsNlPPP1β 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.

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.

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.

Rice production and sustainability are challenged by its most dreadful pest, the brown planthopper (Nilaparvata lugens Stål, BPH). Therefore, the studies on rice-BPH interactions and their underlying mechanisms are of high interest. The rice ontogenetic defense, such as the role of microRNAs (miRNAs) has mostly been investigated against the pathogens, with only a few reports existing against the insect infestations. Thus, revealing the involvement of rice miRNAs in response to BPH infestations will be beneficial in understanding these complex interactions. In this study, the small RNA profiling of the IR56 rice in response to separate BPH infestations of varied virulence levels identified the BPH-responsive miRNAs and revealed the differential transcript abundance of several miRNAs during a compatible and incompatible rice-BPH interaction. The miRNA sequence analysis identified 218 known and 28 novel miRNAs distributed in 54 miRNA families. Additionally, 138 and 140 numbers of differentially expressed (DE) miRNAs were identified during the compatible and incompatible interaction, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the target gene candidates of DE miRNAs (including osa-miR2871a-3p, osa-miR172a, osa-miR166a-5p, osa-miR2120, and osa-miR1859) that might be involved in the IR56 rice defense responses against BPH infestation. Conversely, osa-miR530-5p, osa-miR812s, osa-miR2118g, osa-miR156l-5p, osa-miR435 and two of the novel miRNAs, including novel_16 and novel_52 might negatively modulate the IR56 rice defense. The expressional validation of the selected miRNAs and their targets further supported the IR56 rice defense regulatory network. Based on our results, we have proposed a conceptual model depicting the miRNA defense regulatory network in the IR56 rice against BPH infestation. The findings from the study add further insights into the molecular mechanisms of rice-BPH interactions and will be helpful for the future researches.

Structural cuticular proteins (CPs) are the primary components of insect cuticle, linings of salivary gland, foregut, hindgut and tracheae, and midgut peritrophic membrane. Variation of CPs in insect cuticle can cause penetration resistance to insecticides. Moreover, depletion of specific CP by RNA interference may be a suitable way for the development of potential pest control traits. Leptinotarsa decemlineata (Say) CPs are poorly characterized at present, and therefore, we mined the genome and transcriptome data to better annotate and classify L. decemlineata CPs in this study, by comparison with the annotated CPs of Tribolium castaneum Browse (Coleoptera: Tenebrionidae). We identified 175 CP genes. Except one miscellaneous CP with an 18-amino acid motif, these CPs were classified into 7 families based on motifs and phylogenetic analyses (CPs with a Rebers and Riddiford motif, CPR; CPs analogous to peritrophins, CPAP3 and CPAP1; CPs with a tweedle motif, TWDL; CPs with a 44-amino acid motif, CPF; CPs that are CPF-like, CPFL; and CPs with two to three copies of C-X5-C motif, CPCFC). Leptinotarsa decemlineata CPRs could be categorized into three subfamilies: RR-1 (50), RR-2 (85), and RR-3 (2). The RR-1 proteins had an additional motif with a conserved YTADENGF sequence. The RR-2 members possessed a conserved RDGDVVKG region and three copes of G-x(3)-VV. Few genes were found in TWDL (9), CPAP1 (9), CPAP3 (8), CPF (5), CPFL (4), and CPCFC (2) families. The findings provide valuable information to explore molecular modes of penetration resistance to insecticides and to develop dsRNA-based control method in L. decemlineata.

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.

Nilaparvata lugens (Stal) is a serious threat to rice production in Asian countries. Synthetic chemicals are widely used for its control. However, evolution of resistance in this pest to commonly used insecticides has been recorded during the last decades. In this study, we determined and compared current insecticide resistance levels of thirteen N. lugens populations from China and Bangladesh to eight insecticides by rice-stem dipping method. These results showed that N. lugens had developed high levels of resistance to imidacloprid (resistance ratio, RR = 2491.0–8078.5-fold), thiamethoxam (RR = 303.6–1253.6-fold), buprofezin (RR = 175.7–365.1-fold) and dinotefuran (RR = 49.9–596.0-fold), low to moderate levels of resistance to nitenpyram (RR = 12.1–67.3-fold) and sulfoxaflor (RR = 3.8–25.5-fold), but were susceptible to triflumezopyrim (RR = 0.2–1.1-fold) in China. Meanwhile, increased activities of detoxification enzymes (EST, GST and P450) were found in multiple-resistant FY19 field population, which also showed the significant changes in the expression levels of P450s compared to susceptible strain, especially CYP6ER1 (11.04-fold). In contrast, first report of insecticide resistance in Bangladesh showed high levels of resistance to imidacloprid (RR = 3680.1–4430.8-fold) and thiamethoxam (RR = 144.3–737.9-fold), low to moderate levels of resistance to dinotefuran (RR = 38.7–57.9-fold), buprofezin (RR = 28.5–149.1-fold) and nitenpyram (RR = 3.4–14.1-fold). In addition, significant differences in insecticide resistance level of N. lugens to nitenpyram and dinotefuran were observed between two countries in 2020. The present study will be beneficial for implementing effective resistance management strategies to prevent or delay resistance development in N. lugens.

In the brown planthopper Nilaparvata lugens , introduction of double-stranded RNA (dsRNA) successfully knocks down genes transcribed in several types of tissues. However, whether RNA interference (RNAi) can effectively reduce the transcripts of neuronally-expressed genes remains undetermined. In the present paper, we selected three neuronally-expressed genes respectively encoding prothoracicotropic hormone ( NlPTTH ), pigment-dispersing factor ( NlPDF ) and short neuropeptide F ( NlsNPF ). The transcripts of the three genes were detectable throughout all tested developmental stages, and were mainly restricted to the nervous system of N. lugens . We introduced dsRNA originated from each of the three neuronally-expressed genes at a dose of 300 ng into the nymphal body cavity by microinjection. The target mRNA was successfully knocked down. Moreover, silencing of NlPTTH downregulated the transcripts of two ecdysteroidogenesis genes ( NlPHM and NlDIB ), and surpressed the expression of an ecdysone-response genes ( NlFTZ-F1 ). Furthermore, RNAi of NlPTTH caused typical 20-hydroxyecdysone deficient phenotypes: nymphal development was delayed and nymphal lethality occurred. In addition, injection of a series of ds NlsDPF solutions at the doses of 300, 60, 12, 2.4 and 0.5 ng into the fourth-instar nymphs decreased its target transcript in a dose-dependent manner: the mRNA levels reduced by 94%, 86%, 57%, 33% and 29%, respectively. Therefore, delivery of dsRNA by microinjection can effectively repress the transcripts of the neuronally-expressed genes in N. lugens.

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.

The speed of toxic action of an insecticide is an indicator for control efficacy and has considerable practical importance. For agricultural pest control, fast-acting is an important feature for an insecticide to consistently reduce the amount of feeding damage. Butene-fipronil is a novel compound obtained via the structural modification of fipronil. However, information about the toxicity and speed of toxic action is still limited. In the present paper, we compared the toxic feature of butene-fipronil with seven other insecticides, of which imidacloprid and abamectin are slow-acting insecticides, and acephate, endosulfan, methomyl, α-cypermethrin and spinosad are fast-acting insecticides. We found that the contact and stomach toxicities of butene-fipronil were among the highest ever estimated to Leptinotarsa decemlineata and Drosophila melanogaster. The speed of toxic action of butene-fipronil was determined using median lethal time (LT50) at a dose (concentration) equivalent to LD80 values. For L. decemlineata, the values for butene-fipronil, imidacloprid, abamectin, acephate, endosulfan, methomyl, cypermethrin and spinosad were calculated to be 39.9, 36.5, 37.5, 20.2, 22.4, 23.8, 16.4 and 23.1 h, respectively. Those for D. melanogaster were 29.8, 31.5, 29.4, 14.0, 20.3, 18.1, 13.5, and 20.1 h, respectively. ANOVA analysis showed that butene-fipronil, imidacloprid, abamectin had similar LT50 values, whereas acephate, endosulfan, methomyl, spinosad and cypermethrin had comparable LT50 values. Thus, butene-fipronil belongs to slow-acting insecticides. Our results provide more empirical information for butene-fipronil potential application.

Metamorphosis is a crucial process in insect development. Ecdysone-induced protein 93 (E93) is a determinant that promotes adult metamorphosis in both hemimetabolous and holometabolous insects. Krüppel-homolog 1 (Kr-h1), an early juvenile hormone (JH)-inducible gene, participates in JH signaling pathway controlling insect metamorphosis. In the current study, an E93 cDNA (NlE93) and two Kr-h1 cDNA variants (NlKr-h1-a and NlKr-h1-b) were cloned from Nilaparvata lugens (Stål), one of the most destructive hemimetabolous insect pests on rice. Multiple sequence alignment showed that both NlE93 and NlKr-h1 share high identity with their orthologs from other insects. The expression patterns revealed that decreasing NlKr-h1 mRNA levels were correlated with increasing NlE93 mRNA levels and vice versa. Moreover, RNA interference (RNAi) assays showed that the knockdown of one of the two genes resulted in significantly upregulated expression of the other. Correspondingly, phenotypical observation of the RNAi insects revealed that depletion of NlE93 prevented nymph–adult transition (causing a supernumerary nymphal instar), while depletion of NlKr-h1 triggered precocious formation of incomplete adult features. The results suggest that Nlkr-h1 and NlE93 are mutual repressors, fitting into the MEKRE93 pathway. The balance between these two genes plays a critical role in the metamorphosis of N. lugens determining the proper timing for activating metamorphosis during the nymphal stage.

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.

The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.

Ecdysteroid hormone 20-hydroxyecdysone plays fundamental roles in insect postembryonic development and reproduction. Several cytochrome P450 mono-oxygenases (CYPs), encoded by the Halloween genes, have been documented to be involved in ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. Here the putative Halloween gene Phantom (Phm, cyp306a1) from a hemipteran insect species, the small brown planthopper Laodelphax striatellus, was cloned. LsPHM shows five insect conserved P450 motifs, that is, Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of LsPhm were evaluated by quantitative polymerase chain reaction. Through the fourth-instar and the early fifth-instar stages, LsPhm showed two expression peaks in day 2 and days 4-5 fourth-instar nymphs, and three troughs in day 1 and 3 fourth instars and day 1 fifth instars. On day 5 of the fourth-instar nymphs, LsPhm clearly had a high transcript level in the thorax where the prothoracic glands were located. Dietary introduction of double-stranded RNA (dsRNA) of LsPhm at the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene and caused a higher nymphal mortality rate and delayed development. Ingestion of 20-hydroxyecdysone on LsPhm-dsRNA-exposed nymphs did not increase LsPhm expression level, but almost completely rescued the LsEcR mRNA level, and relieved the negative effects on survival and development. Thus, our data suggest that the putative LsPhm encodes a functional 25-hydroxylase that catalyzes the biosynthesis of ecdysteroids in L. striatellus.

BACKGROUND Leptinotarsa decemlineata is an able disperser by flight. Novel control strategies must be explored to control the damage and inhibit the dispersal efficiently. Proline is a major energy substrate during flight. Δ-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyses the second step of proline degradation for the production of ATP. RESULTS A full-length Ldp5cdh cDNA was cloned. Ldp5cdh was ubiquitously expressed in the eggs, the first through fourth larval instars, wandering larvae, pupae and adults. In the adults, Ldp5cdh mRNA was widely distributed in thorax muscles, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion, fat body and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdp5cdh1 and dsLdp5cdh2) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of dsLdp5cdh1 and dsLdp5cdh2 successfully silenced Ldp5cdh, significantly increased the contents of proline, arginine and alanine, but strongly decreased the contents of asparate, asparagine, glutamate and glutamine in the haemolymph. Moreover, knocking down Ldp5cdh significantly reduced ATP content, decreased flight speed, shortened flight distance and increased adult mortality. CONCLUSIONS It seems that identified Ldp5cdh encodes a functional P5CDh enzyme, and Ldp5cdh may serve as a potential target for dsRNA-based pesticide for controlling the damage and dispersal of L. decemlineata adults. © 2014 Society of Chemical Industry

Delta 1-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the conversion of glutamate (Glu) to Glu semialdehyde (GSA). GSA spontaneously cyclizes to form P5C. P5C is then reduced to proline (Pro) or is converted to ornithine, the intermediate for arginine (Arg) biosynthesis. In the present study, a full-length Ldp5cs complementary DNA was cloned from the Colorado potato beetle Leptinotarsa decemlineata, a notorious insect defoliator of potato in most potato-growing regions of the world. Ldp5cs encodes a 792-amino-acid protein which shares high identity to homologues from other insect species. Quantitative reverse transcription polymerase chain reaction revealed that Ldp5cs was ubiquitously expressed in the eggs, first to fourth-instar larvae, wandering larvae, pupae and sexually mature adults. In the adults, Ldp5cs mRNA levels were higher in the fat body, foregut, midgut and hindgut, moderate in the ventral ganglion, lower in the thorax muscles, epidermis and Malpighian tubules. Two double-stranded RNAs (dsRNAs) (dsLdp5cs1 and dsLdp5cs2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of dsLdp5cs1 or dsLdp5cs2 successfully silenced Ldp5cs, significantly reduced the contents of Pro and Arg in the hemolymph, decreased flight speed and shortened flight distance of the resulting adults. Furthermore, knocking down Ldp5cs significantly increased adult mortality. Thus, our results suggest that identified Ldp5cs encodes a functional P5CS enzyme that is involved in the biosynthesis of Pro and Arg in L. decemlineata.

Postembryonic development of insects is highly dependent on ecdysteroid hormones ecdysone (E) and 20-hydroxyecdysone (20E). A cytochrome P450 monooxygenase CYP306A1, the product of the Halloween gene phantom (phm), is involved in the ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. In the present paper, Sfphm was cloned from a hemipteran insect species, the white-backed planthopper Sogatella furcifera. SfPHM has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfphm were evaluated by q-PCR. Sfphm showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. The relative 20E levels exhibited similar temporal patterns to Sfphm expression levels. On day 3 of the fourth-instar nymphs, Sfphm clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA of Sfphm into the second instars successfully knocked down the target gene, and greatly reduced 20E level and ecdysone receptor (EcR) expression level. Moreover, knockdown of Sfphm caused lethality and slowed down ecdysis during nymphal stages. Furthermore, ingestion of 20-hydroxyecdysone did not alter Sfphm expression level, but almost completely rescued SfEcR expression level, and relieved the negative effects on nymphal survival and ecdysis in Sfphm-dsRNA-exposed planthoppers. Thus, our results suggest that Sfphm plays a critical role in ecdysteroidogenesis in S. furcifera.

Sogatella furcifera is a serious phloem-feeding pest of rice. Rice phloem sap cannot meet its nutrition requirement for some essential amino acids. The yeast-like symbionts (YLSs) have been hypothesized to aid S. furcifera in amino acid metabolism to compensate for the unbalanced amino acid composition in the rice phloem. In the present paper, two full-length cDNAs of putative alanine aminotransferases (ALTs) were obtained from S. furcifera. Sequence similarity and phylogenetic analysis suggested that one ALT came from the YLSs (SfylsALT) whereas the other ALT originated from S. furcifera (SfALT1). Quantitative reverse transcription polymerase chain reaction revealed that SfylsALT clearly had a high transcript level in the abdomen. In contrast, SfALT1 was broadly expressed in the head, wings, legs, thorax and abdomen. Since YLSs were distributed in the abdominal fat body, ovary and eggs, and were not located in the head, leg, thorax and wing, the tissue expression patterns also suggested that SfylsALT came from the YLSs' genome and SfALT1 was from the host. Moreover, the enzyme activities of Sf9 cell-expressed SfALT1 and SfylsALT proteins were tested in vitro. The isolated suspensions containing either of the two proteins showed higher ALT activities than that from blank cells or egfp-expressed cells. It seems that SfALT1 and SfylsALT encode functional alanine aminotransferases.

Ecdysone 20-monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix-C, Helix-I, Helix-K, PxxFxPE/DRF (PERF) and heme-binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double-stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd-dsRNA by the fourth instars also down-regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd-dsRNA-ingested second instars, and of halofenozide into the LdShd-dsRNA-ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.

BACKGROUND Laodelphax striatellus is an economically important rice pest in China. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. The cytochrome P450 monooxygenase Shadow (Sad) plays a critical role in ecdysteroidogenesis. Here, tests were conducted to establish whether Lssad was a potential target gene for RNA-interference-based management of L. striatellus. RESULTS Lssad was cloned and characterised. LsSad had Helix-C, Helix-I, Helix-K, PERF and haem-binding motifs. Lssad is expressed at a higher level in the thorax, where prothoracic glands are located, compared with the level in the head or abdomen. It showed two expression peaks in day 2 and day 4–5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Oral delivery of double-stranded RNA (dsRNA) of Lssad at the nymph stage successfully knocked down the expression of the target gene, reduced the expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development in a dose-dependent manner. Ingestion of 20-hydroxyecdysone in Lssad-dsRNA-exposed nymphs did not increase Lssad expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. CONCLUSIONS The ecdysteroidogenic pathway is conserved in L. striatellus. Lssad can serve as a possible target for dsRNA-based pesticides for planthopper control. © 2014 Society of Chemical Industry

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.

Leptinotarsadecemlineata is the most important pest in potato and causes serious yield loss each year. Chlorantraniliprole acts on insect ryanodine receptors (RyRs) and is among the most active compounds against L. decemlineata. Here we cloned and characterized a 15,792-bp full-length LdRyR cDNA that encoded a 5128-amino acid protein. LdRyR shares 85-92% amino acid similarities with other insect RyR homologues, and 59-61% similarities with those from Caenorhabditis elegans and Homo sapiens. All hallmarks of the RyR proteins are conserved in LdRyR. LdRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR domain and a RIH-associated domain in the N-terminus, and it possesses two consensus calcium ion-binding EF-hand motifs and six predicted transmembrane helices in the C-terminus. Temporal, spatial and tissue-specific expression patterns of LdRyR were evaluated. LdRyR expression level was increased constantly from egg to wandering stages, dropped in pupal stage and was increased again in the adult stage. It was widely expressed in the head, thorax and abdomen of day 3 fourth-instar larvae. Moreover, it was ubiquitously expressed in all inspected tissues including epidermis, foregut, midgut, ileum, rectum, fat body, ventral ganglia and Malpighian tubules in day 3 fourth-instar larvae. Dietary introduction of double-stranded RNA of LdRyR significantly reduced the mRNA levels of the target gene in the larvae and adults, respectively, and significantly decreased chlorantraniliprole-induced mortalities. Thus, our results suggested that LdRyR encoded a functional ryanodine receptor in L. decemlineata.

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&ndash, 81 amino acid &ldquo, inserts&rdquo, 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&minus, 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.

The striped rice stem borer, Chilo suppressalis (Walker), is a major pest for rice production in China and the rest of Southeast Asia. Chemical control is the main means to alleviate losses due to this pest, which causes serious environmental pollution. An effective and environmentally friendly approach is needed for the management of the striped rice stem borer. Cysteine proteases in insects could be useful targets for pest management either through engineering plant protease inhibitors, targeting insect digestive cysteine proteases, or through RNA interference-based silencing of cysteine proteases, disrupting developmental regulation of insects. In this study, eight cysteine protease-like genes were identified and partially characterized. The genes CCO2 and CCL4 were exclusively expressed in the larval gut, and their expression was affected by the state of nutrition in the insect. The expression of CCL2, CCL3, and CCO1 was significantly affected by the type of host plant, suggesting a role in host plant – insect interactions. Our initial characterization of the striped rice stem borer cysteine protease-like genes provides a foundation for further research on this important group of genes in this major insect pest of rice.

The diamide insecticides activate ryanodine receptors (RyRs) to release and deplete intracellular calcium stores from the sarcoplasmic reticulum of muscles and the endoplasmic reticulum of many types of cells. They rapidly interrupt feeding of the target pest and eventually kill the pest due to starvation. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated a 15,985 bp full-length cDNA (named SfRyR) from Sogatella furcifera, a serious rice planthopper pest throughout Asia. SfRyR encodes a 5140-amino acid protein, which shares 78–97% sequence identities with other insect homologues, and less than 50% identities with Homo sapiens RyR1–3. All hallmarks of the RyR proteins are conserved in SfRyR. In the N-terminus, SfRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeated domain and a RIH-associated domain. In the C-terminus, SfRyR possesses two consensus calcium ion-binding EF-hand motifs, and six transmembrane helices. Temporal and spatial expression analysis showed that SfRyR was widely found in all development stages including egg, first through fifth instar nymphs, macropterous adult females and males. On day 2 fifth-instar nymphs, SfRyR was ubiquitously expressed in the head, thorax and abdomen. Dietary ingestion of dsSfRyR1 and dsSfRyR2 significantly reduced the mRNA level of SfRyR in the treated nymphs by 77.9% and 81.8% respectively, and greatly decreased chlorantraniliprole-induced mortality. Thus, our results suggested that SfRyR gene encoded a functional RyR that mediates chlorantraniliprole toxicity to S. furcifera.

Nilaparvata lugens is a serious phloem-feeding pest of rice throughout Asia. Rice phloem sap can meet its nutrition requirement for sugars but not for some essential amino acids such as isoleucine, leucine, methionine, phenylalanine, tryptophan, lysine, arginine and histidine. N. lugens harbours yeast-like symbionts in mycetocytes formed by abdominal fat body cells. Removal of the symbionts results in negative physiological effects, suggesting that the symbionts play a pivotal role in the nitrogen metabolism. In the present paper, 521 mRNA expressed sequence tags (ESTs) encoding 126 enzymes that were involved in amino acid biosynthesis were identified based on a transcriptome data, reverse transcription (RT)-PCR and rapid amplification of cDNA ends. Similarity analysis, codon usage bias, along with tissue-biased expression and phylogenetic analysis of a subset of ESTs, suggest that 437 ESTs out of the 521 originate from symbionts, and the remaining 84 mRNA fragments come from N. lugens. Accordingly, the biosynthesis pathways for 20 amino acids were manually constructed. It is postulated that both N. lugens and its symbiont can independently assimilate ammonia and biosynthesize seven non-essential amino acids: glutamate; glutamine; aspartate; asparagine; alanine; serine; and glycine. N. lugens and symbiont enzymes may work collaboratively to catalyse the biosynthesis of proline, methionine, valine, leucine, isoleucine, phenylalanine and tyrosine. We infer from this that symbionts function in the biosynthesis of lysine, arginine, tryptophan, threonine, histidine and cysteine. Our data support the previously proposed hypothesis, i.e. the yeast-like symbionts compensate for, at least partially, the amino acid needs of N. lugens.

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate of L. decemlineata during flight and proline dehydrogenase (ProDH) catalyzes the first step in proline catabolism. Here we identified a putative LdProDH gene; it had three cDNA isoforms which shared the same 5'UTR and coding region, but differed in the lengths of 3'UTRs (515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively). LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDH was expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH-dsRNA by adults significantly decreased its messenger RNA (mRNA) level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals (AATAAA) were tandemly located in the 3'UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3'UTR to control proline catabolism for the season-dependent activity of L. decemlineata adults.

In Leptinotarsa decemlineata, juvenile hormones (JHs) play primary roles in the regulation of metamorphosis, reproduction and diapause. In JH biosynthetic pathway in insect corpora allata, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a potent feedback inhibitor S-adenosyl-L-homocysteine (AdoHcy). Rapid removal of AdoHcy is hypothesized to be essential for JH synthesis. AdoHcy hydrolase (SAHase) is the only eukaryotic enzyme catalyzing the removal. In the present paper, we firstly cloned a putative LdSAHase gene from L. decemlineata. The cDNA consists of 1806 bp and encodes a 525 amino acid protein. LdSAHase was expressed in all developmental stages. The gene had the highest and the lowest level of transcription respectively in the 3rd- and 4th-instars' heads that contain corpora allata, which was positively correlated with JH titer in the haemolymph and the mRNA level of a JH early-inducible gene, the Krüppel homolog 1 gene (Kr-h1). Secondly, dietary ingestion of bacterially-expressed LdSAHase-dsRNA significantly decreased LdSAHase and LdKr-h1 mRNA levels, reduced JH titer, and caused the death of the larvae, and the failure of pupation and adult emergence. After continuous exposure for 12 days, 42% of the larvae died, 65% of the prepupae failed to pupate and 100% of the pupae failed to emerge. Moreover, RNAi-mediated LdSAHase knockdown also reduced larval developing time, and decreased larval weight. Lastly, application of JH analogue pyriproxyfen to LdSAHase-dsRNA-exposed larvae did not greatly increase LdSAHase expression level and JH content, but up-regulated LdKr-h1 mRNA level. Expectedly, pyriproxyfen application could partially rescue the negative effects on the survival and the development. Thus, our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Being delivered as sprays or expressed in plant, Bacillus thuringiensis (Bt) crystalline proteins (Cry toxins) display insecticidal activities against numerous Lepidopteran, Dipteran, and Coleopteran larvae. Comparative study of toxicities of Bt Cry toxins between larvae and adults may afford important new insights into the interactions of the toxins with receptor proteins in host insect, and represent intriguing targets for the control of insect pests. However, the effectiveness of Bt Cry toxins in insect adults has paid less attention. In the present article, the effectiveness of Cry1Ac and Cry1Ca on lifespans and reproductive performance of Helicoverpa armigera (Hubner) and Spodoptera exigua (Hubner) adults were evaluated by in vivo experiments. Considering transgenic plants express modified, truncated versions of cry genes yielding active toxin fragment, we used activated Bt toxins at the concentration of 500, 100, and 20 microg/ml in a 10% sucrose aquous solution. At the highest concentration, Cry1Ac and Cry1Ca shortened 48.1 and 48.9% of H. armigera female lifespan, and 43.5 and 38.5% of S. exigua female lifespan, and they reduced 37.8 and 40.3%, and 50.5 and 47.4% of H. armigera and S. exigua male lifespans respectively. Bt toxins negatively affected copulation. Exposure to 500 microg/ml of Cry1Ac and Cry1Ca greatly reduced 50.0 and 46.8%, and 58.7 and 57.3% spermatophore acceptance by H. armigera and S. exigua females, respectively. Similarly, Cry1Ac and Cry1Ca exposure decreased 40.0 and 50.3%, and 61.3 and 60.0% of spermatophore transfer by H. armigera and S. exigua males, respectively. Moreover, exposure females rather than males to 500 microg/ml of Cry1Ac and Cry1Ca significantly dropped 57.5 and 57.5% of the number of eggs laid by H. armigera, and 35.4 and 45.8% of the number of egg masses deposited by S. exigua. In contrast, both Cry1Ac and Cry1Ca did not negatively influence the egg hatchability. At the middle and the lowest concentrations, however, the biological effects of Cry1Ac and Cry1Ca on lifespans and reproductive performance partially or completely disappeared.

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.

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.

Spodoptera exigua is a highly polyphagous pest and causes extensive damage to many field and truck crops. Since the larvae are not or only sublethally affected by Cry1Ac, the fused Cry1Ac/Cry1Ab, or the stacked Cry1Ac + CpTI transgenic cottons that are widely planted in China, S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in 1997 in China. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against, and for expanding control spectrum for insect pests. In the present paper, we first found that S. exigua larval midgut fluids could remarkably degrade activated Cry1Ca, and slightly hydrolyze Cry1Ac and Cry1Ab. Subsequently, we investigated interactions between the 3 Cry toxins and 7 proteinase inhibitors, i.e., phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N -α-tosyl- l -phenylalanine chloromethyl ketone, N -α-tosyl- l -lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA), by monitoring larval growth and mortality rate. A 6-day dietary exposure of the newly molted 2nd instars to either the inhibitors (3 test concentrations) or the toxins (Cry1Ac, 31.30 ng/cm 2 ; Cry1Ab, 3.2 ng/cm 2 ; Cry1Ca, 0.6 ng/cm 2 ) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight. In general, the synergisms were more obvious at higher inhibitor concentrations. Regarding larval mortality, the inhibitors except EDTA at the highest test concentration showed significant synergism to both Cry1Ab (0.2 μg/cm 2 ) and Cry1Ca (0.04 μg/cm 2 ). As the inhibitor concentration reduced, however, the synergistic effects decreased. These results indicated that trypsin-, chymotrypsin-, and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the 3 activated Cry toxins, and protection of Bt Cry toxins from proteinase degradation in the midgut by inhibitors may greatly enhance toxicity against S. exigua larvae.

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.

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&ndash, 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&ndash, 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.

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.

Ran (RanGTPase) in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1) and vitellogenin, are involved. A putative Ran gene (NlRan) was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1) Extended body form, and failed to molt; (2) The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control.

Alanine aminotransferase (ALT) plays important physiological and biochemical roles in insect. In this study, a full-length Ldalt cDNA was cloned from Leptinotarsa decemlineata. It was ubiquitously expressed in the eggs, larvae, pupae and adults. In the adults, Ldalt mRNA was widely distributed in thorax muscles, fat body, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdalt1 and dsLdalt2) targeting Ldalt were constructed and bacterially expressed. After adults fed on dsLdalt1- and dsLdalt2-immersed foliage for 3 day, Ldalt mRNA abundance was significantly decreased by 79.5 and 71.1 %, and ALT activities were significantly reduced by 64.5 and 67.6 %, respectively. Moreover, silencing Ldalt affected free amino acid contents. Lysine was decreased by 100.0 and 100.0 %, and arginine was reduced by 87.5 and 89.4 %, respectively, in the hemolymph from dsLdalt1- and dsLdalt2-ingested beetles, compared with control ones. In contrast, proline was increased by 88.7 and 96.4 %. Furthermore, ingestion of dsLdalt1 and dsLdalt2 significantly decreased flight speed, shortened flight duration time and flight distance. In addition, knocking down Ldalt significantly increased adult mortality. These data imply that LdALT plays important roles in amino acid metabolism and in flight in L. decemlineata.

Leptinotarsa decemlineata is an able disperser by flight. Novel control strategies must be explored to control the damage and inhibit the dispersal efficiently. Proline is a major energy substrate during flight. Δ-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyses the second step of proline degradation for the production of ATP.A full-length Ldp5cdh cDNA was cloned. Ldp5cdh was ubiquitously expressed in the eggs, the first through fourth larval instars, wandering larvae, pupae and adults. In the adults, Ldp5cdh mRNA was widely distributed in thorax muscles, midgut, foregut, hindgut, Malpighian tubules, ventral ganglion, fat body and epidermis, with the expression levels from the highest to the lowest. Two double-stranded RNAs (dsRNAs) (dsLdp5cdh1 and dsLdp5cdh2) targeting Ldp5cdh were constructed and bacterially expressed. Ingestion of dsLdp5cdh1 and dsLdp5cdh2 successfully silenced Ldp5cdh, significantly increased the contents of proline, arginine and alanine, but strongly decreased the contents of asparate, asparagine, glutamate and glutamine in the haemolymph. Moreover, knocking down Ldp5cdh significantly reduced ATP content, decreased flight speed, shortened flight distance and increased adult mortality.It seems that identified Ldp5cdh encodes a functional P5CDh enzyme, and Ldp5cdh may serve as a potential target for dsRNA-based pesticide for controlling the damage and dispersal of L. decemlineata adults. © 2014 Society of Chemical Industry.

Post-embryonic development of insects is highly dependent on ecdysteroid hormone 20-hydroxyecdysone. Halloween gene spookier (spok, cyp307a2) has been documented to be involved in ecdysteroidogenesis in Drosophila melanogaster and Bombyx mori. We describe here the cloning and characterization of Halloween gene spookier (Lsspok, Lscyp307a2) in the small brown planthopper Laodelphax striatellus, a hemipteran insect species. LsSPOK has three insect-conserved P450 motifs, that is, Helix-K, PERF motif and heme-binding domain. Temporal and spatial expression patterns of Lsspok were evaluated by quantitative polymerase chain reaction. Through the fouth-instar and the early fifth-instar stages, Lsspok showed two expression peaks in the second- and fifth-day fourth-instar nymphs, and two troughs in the first-day fourth and fifth instars. On day 5 of the fourth-instar nymphs, Lsspok clearly had a high transcript level in the thorax where prothoracic glands were located. Dietary introduction of double-stranded RNA of Lsspok in the nymph stage successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development. Ingestion of 20-hydroxyecdysone in Lsspok-dsRNA-exposed nymphs did not increase Lsspok expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects and LsSPOK is responsible for specific steps in ecdysteroidogenesis in L. striatellus.

Laodelphax striatellus is an economically important rice pest in China. Ecdysteroid hormone 20-hydroxyecdysone regulates insect development and reproduction. The cytochrome P450 monooxygenase Shadow (Sad) plays a critical role in ecdysteroidogenesis. Here, tests were conducted to establish whether Lssad was a potential target gene for RNA-interference-based management of L. striatellus.Lssad was cloned and characterised. LsSad had Helix-C, Helix-I, Helix-K, PERF and haem-binding motifs. Lssad is expressed at a higher level in the thorax, where prothoracic glands are located, compared with the level in the head or abdomen. It showed two expression peaks in day 2 and day 4-5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Oral delivery of double-stranded RNA (dsRNA) of Lssad at the nymph stage successfully knocked down the expression of the target gene, reduced the expression level of ecdysone receptor (LsEcR) gene, caused nymphal lethality and delayed development in a dose-dependent manner. Ingestion of 20-hydroxyecdysone in Lssad-dsRNA-exposed nymphs did not increase Lssad expression level, but almost completely rescued the LsEcR mRNA level and relieved the negative effects on survival and development.The ecdysteroidogenic pathway is conserved in L. striatellus. Lssad can serve as a possible target for dsRNA-based pesticides for planthopper control.

Background Ecdysteroid hormones ecdysone and 20-hydroxyecdysone play fundamental roles in insect postembryonic development and reproduction. Five cytochrome P450 monooxygenases (CYPs), encoded by Halloween genes, have been documented to be involved in the ecdysteroidogenesis in insect species of diverse orders such as Diptera, Lepidoptera and Orthoptera. Up to now, however, the involvement of the Halloween genes in ecdysteroid synthesis has not been confirmed in hemipteran insect species. Results In the present paper, a Halloween gene spook (Sfspo, Sfcyp307a1) was cloned in the hemipteran Sogatella furcifera. SfSPO has three insect conserved P450 motifs, i.e., Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfspo were evaluated by qPCR. Sfspo showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. On day 3 of the fourth-instar nymphs, Sfspo clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA (dsRNA) of Sfspo into the second instars successfully knocked down the target gene, and greatly reduced expression level of ecdysone receptor (EcR) gene. Moreover, knockdown of Sfspo caused lethality and delayed development during nymphal stages. Furthermore, application of 20-hydroxyecdysone on Sfspo- dsRNA-exposed nymphs did not increase Sfspo expression, but could almost completely rescue SfEcR expression, and relieved the negative effects on nymphal survival and development. Conclusion In S. furcifera, Sfspo was cloned and the conservation of SfSPO is valid. Thus, SfSPO is probably also involved in ecdysteroidogenesis for hemiptera.

Serpins, also called serine proteinase inhibitors, are widely distributed in eukaryotes. In insects, serpins play important roles in regulating immune responses, gut physiology, and other processes. Here, we report the cloning and characterization of 12 serpin-like cDNAs from the striped rice stem borer (Chilo suppressalis), a major rice pest. The putative proteins share significant sequence similarity with known insect serpins, especially those from lepidopterons. Analysis of functional domains revealed that nine of the cloned serpins are putative trypsin- or chymotrypsin-like inhibitors; two are mixed-type serpins that may act as inhibitors for trypsins, elastases, or thrombin; and the remaining one is truncate. The potential functions of these serpins in interacting with host plants were also investigated by analyzing tissue-specific expression and the impact of different host plant genotypes on gene expression. Our results provide a foundation for future studies on the role of serpins in gut physiology in the striped rice stem borer, and also useful information for comparative analyses of serpins from different insect species.