As an r-strategy insect species, the brown planthopper (BPH) Nilaparvata lugens (Stål) is a serious pest of rice crops in the temperate and tropical regions of Asia and Australia, which may be due to its robust fecundity. Here we combined 2-DE comparative proteomic and RNA-seq transcriptomic analyses to identify fecundity-related proteins and genes. Using high- and low-fecundity populations as sample groups, a total of 54 and 75 proteins were significantly altered in the third and sixth day brachypterous female stages, respectively, and 39 and 54 of these proteins were identified by MALDI-TOF/TOF MS. In addition, 71 966 unigenes were quantified by Illumina sequencing. On the basis of the transcriptomic analysis, 7408 and 1639 unigenes demonstrated higher expression levels in the high-fecundity population in the second day brachypterous female adults and the second day fifth instar nymphs, respectively, and 411 unigenes were up-regulated in both groups. Of these dozens of proteins and thousands of unigenes, five were differentially expressed at both the protein and mRNA levels at all four time points, suggesting that these genes may regulate fecundity. Glutamine synthetase (GS) was chosen for further functional studies. RNAi knockdown of the GS gene reduced the fecundity of N. lugens by 64.6%, disrupted ovary development, and inhibited vitellogenin (Vg) expression. Our results show that a combination of proteomic and transcriptomic analyses provided five candidate proteins and genes for further study. The knowledge gained from this study may lead to a more fundamental understanding of the fecundity of this important agricultural insect pest.Keywords: 2-DE; fecundity; glutamine synthetase; Nilaparvata lugens; RNA-seq; RNAi;

•Nl23867 is a novel NTPase coding gene in Nilaparvata lugens.•The expression of Nl23867 is significantly related with the fecundity of N. lugens.•The affected genes are mostly lipid metabolism-related after Nl23867 knockdown.•Nl23867 directly impacts the synthetic pathway of palmitic acid.BackgroundInsect fecundity can be regulated by multiple genes in several important signaling pathways which form an extremely complicated regulatory network. However, there are still many genes that have significant impact on insect fecundity but their action mode are still unknown.MethodsQuantitative real-time PCR (qRT-PCR), immunofluorescence and western blot were used to study the expression profile of Nl23867 in the brown planthopper, Nilaparvata lugens. RNA interference (RNAi), RNA-seq and isobaric tags for relative and absolute quantification (iTRAQ) were performed to investigate the action mode of Nl23867 in the regulation of fecundity. High performance liquid chromatography (HPLC) analysis was performed to detect the fatty acid contents.ResultsWe show that knockdown of Nl23867, a gene encoding a hypothetical P-loop NTPase, significantly decreased fecundity of N. lugens. Underdeveloped ovaries, fewer eggs laid and reduction in vitellogenin (Vg) protein expression were observed after RNAi knockdown of Nl23867, and most of the affected genes and pathways are fatty acid metabolism-related. We further determined that Nl23867 directly impacts the palmitic acid biosynthesis by regulating the expression of palmitoyl-protein thioesterase (PPT), subsequently affecting the content of total lipids in N. lugens.ConclusionsNl23867 regulates the fecundity of N. lugens by modulating the biosynthetic pathway of palmitic acid and affecting lipid metabolism during vitellogenesis and oocyte development.General significanceThe presented study pioneers the exploration into how a function-unknown gene takes part in the regulation of fecundity in an insect, and will contribute to the construction of gene regulatory network for insect fecundity.Download high-res image (152KB)Download full-size image

•Two genes in chitin biosynthesis pathway are new targets of miR-8-5p and miR-2a-3p.•miR-8-5p and miR-2a-3p play key roles in chitin biosynthesis and insect molting.•miR-8-5p and miR-2a-3p are transcriptionally repressed by Broad-Complex (BR-C).•We report a miRNA-mediated link between chitin biosynthesis pathway and 20E signaling.Molting is an important developmental process in insects, usually along with synthesis and degradation of chitin. 20-hydroxyecdysone (20E), an insect hormone, has been reported to contribute to many processes including molting. However, little is known about the link between the chitin biosynthesis pathway and 20E signaling. Here, we report that conserved miR-8-5p (miR-8-5p) and miR-2a-3p and their new target genes are critical for ecdysone-induced chitin biosynthesis in a hemipteran insect Nilaparvata lugens. We found that membrane-bound trehalase (Tre-2) and phosphoacetylglucosamine mutase (PAGM) in the chitin biosynthesis pathway were targets of miR-8-5p and miR-2a-3p, respectively, through bioinformatic analysis and experimental verification. The levels of miR-8-5p and miR-2a-3p were reduced, whereas the levels of Tre-2 and PAGM were up-regulated in response to 20E. In addition, miR-8-5p and miR-2a-3p were transcriptionally repressed by an early-response gene, the Broad-Complex (BR-C), in the 20E signaling pathway. Moreover, the overexpression of miR-8-5p and miR-2a-3p led to a significant reduction in the survival rate along with a molting obstacles defect phenotype caused by miR-2a-3p mimics feeding, and the chitin content of N. lugens was simultaneously reduced. Thus, miR-8-5p and miR-2a-3p act as molecular link that tune the chitin biosynthesis pathway in response to 20E signaling.Download high-res image (241KB)Download full-size image

Chitin synthase (CHS) is an important enzymatic component required for chitin formation in the cuticles and cuticular linings of other tissues. In the present study, a new CHS gene was characterized from the beet armyworm Spodoptera exigua (Hübner) (Se). Homologous alignment and phylogenetic analysis of S. exigua CHS (SeCHS) with other related proteins suggest that SeCHS belongs to the class A CHS family (SeCHSA). Northern blot analysis revealed that SeCHSA is transcribed preferentially in the cuticle and tracheae. Further investigation indicated that SeCHSA mRNA is highly expressed in the early and late stages of each larval instar, and consistently expressed in high level during the pupal stage. Using antibody specific for CHS, SeCHS was further localized in the underlying epidermal cells of the integument and tracheal cells, but not in the fat body or Malpighian tubules. These data suggest that SeCHS plays an important role in cuticle formation and development of S. exigua.

The chitin synthase (CHS) gene B (4781 bp) of Spodoptera exigua (SeCHSB) was cloned by reverse-transcription PCR (RT-PCR) and 3′/5′ RACE from the midgut. SeCHSB contains an open reading frame of 4572 nucleotides, encoding a protein of 1523 amino acids with a predicted molecular mass of approximately 174.6 kDa. Alignment of SeCHSB with class B CHSs of other insects showed a high degree of conservation in the putative catalytic domain region. The structure of the SeCHSB gene was analyzed and was found to be the same as that of Manduca sexta CHSB (MsCHSB), including 23 exons and 22 introns but without alternative exons. Southern blot analysis revealed that SeCHSB was a single copy gene and the presence of only two chitin synthase genes in S. exigua. Further investigation indicated that SeCHSB was specifically expressed in the midgut, and its transcript existed constitutively in the midgut from the 3rd instar larval stage to prepupae and reached highest expression on the 1st day of the fifth instar larval stage. These data suggest that SeCHSB is very important in midgut formation and development. Chitin synthase gene comparisons between different classes of insects using software tools revealed some interesting aspects of the similarity and divergence of the gene in the Class Insecta.

The mechanism of how endoparasitoids avoid the host's cellular immune reaction is not well known. Evidence is presented here for the existence of a Helix pomatia lectin binding protein (HpLBP) on Macrocentrus cingulum extraembryonic membrane and its involvement in the protection of embryos against encapsulation by its host Ostrinia furnaclis. HpLBP is present in eggs, embryos and larvae and is located on the outmost layer of the extraembryonic membrane. While Sephadex A-25 beads and immature Macrocentrus eggs coated with follicular cells were encapsulated, Macrocentrus embryos were not after they were transplanted separately into naive O. furnaclis larvae. Moreover, embryos became encapsulated after being coated with anti-HpLBP serum. Furthermore, encapsulation of agarose-H. pomatia lectin beads decreased significantly after the beads were coated with HpLBP. However, encapsulation of the HpLBP-coated agarose beads increased and the extent of encapsulation was enhanced significantly when the HpLBP-coated beads were pre-incubated with anti-HpLBP antibody.