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

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.

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.

The dissipation and residue levels of thiacloprid, spirotetramat and its four metabolites residues in cowpeas were investigated under field conditions. The QuEChERS technique with high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) was used to detect thiacloprid, spirotetramat and its four metabolites residues content in cowpeas. The recoveries were 81.3–95.1% at a spike level of 0.005–0.5 mg/kg, the relative standard deviations (RSDs) were 2.1–9.5%. The dissipation kinetics data showed that thiacloprid and spirotetramat in cowpeas were degraded with the half-lives of 1.14–1.54 days and 1.25–2.79 days. The terminal residues of thiacloprid and spirotetramat were 0.0255–0.4570 mg kg⁻¹ and 0.0314–0.3070 mg kg⁻¹ after application 2 times with a pre-harvest interval (PHI) of 3 days under the designed dosages. The chronic and acute dietary exposure assessment risk quotient (RQ) values of thiacloprid in cowpeas for different consumers were 2.44–4.41% and 8.72–15.78%, respectively, and those of spirotetramat were 1.03–1.87% and 0.18–0.32%, respectively, all of the RQ values were lower than 100%. The dietary risk of thiacloprid through cowpeas to consumers was higher than spirotetramat. The results from this study are important reference for Chinese governments to develop criteria for the safe and rational use of thiacloprid and spirotetramat, setting maximum residue levels (MRLs), monitoring the quality safety of agricultural products and protecting consumer health.

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