Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.     

The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

Adipokinetic hormone signalling system in the Chagas disease vector,Rhodnius prolixus

Neuropeptides and their G protein‐coupled receptors are widespread throughout Metazoa and in several cases, clear orthologues can be identified in both protostomes and deuterostomes. One such neuropeptide is the insect adipokinetic hormone (AKH), which is related to the mammalian gonadotropin‐releasing hormone. AKH has been studied extensively and is known to mobilize lipid, carbohydrates and proline for energy‐consuming activities such as flight. In order to determine the possible roles for this signalling system in Rhodnius prolixus, we isolated the cDNA sequences encoding R. prolixus AKH (Rhopr‐AKH) and its receptor (Rhopr‐AKHR). We also examined their spatial expression pattern using quantitative PCR. Our expression analysis indicates that Rhopr‐AKH is only expressed in the corpus cardiacum of fifth‐instars and adults. Rhopr‐AKHR, by contrast, is expressed in several peripheral tissues including the fat body. The expression of the receptor in the fat body suggests that AKH is involved in lipid mobilization, which was confirmed by knockdown of Rhopr‐AKHR via RNA interference. Adult males that had been injected with double‐stranded RNA (dsRNA) for Rhopr‐AKHR exhibited increased lipid content in the fat body and decreased lipid levels in the haemolymph. Moreover, injection of Rhopr‐AKH in Rhopr‐AKHR dsRNA‐treated males failed to elevate haemolymph lipid levels, confirming that this is indeed the receptor for Rhopr‐AKH.     

Amplification and quantification of cold‐associated microRNAs in the Colorado potato beetle (Leptinotarsa decemlineata) agricultural pest

The Colorado potato beetle [Leptinotarsa decemlineata (Say)] is an important insect pest that can inflict considerable damage to potato plants. This insect can survive extended periods of cold exposure, and yet the molecular switches underlying this phenomenon have not been fully elucidated. A better characterization of this process would highlight novel vulnerabilities associated with L. decemlineata that could serve as targets for the management of this devastating pest. Using high‐throughput sequencing, the current work reveals a cold‐associated signature group of microRNAs (miRNAs) in control (15 °C) and ?5 °C‐exposed L. decemlineata. The results show 42 differentially expressed miRNAs following cold exposure including miR‐9a‐3p, miR‐210‐3p, miR‐276‐5p and miR‐277‐3p. Functional analysis of predicted targets associated with these cold‐responsive miRNAs notably linked these changes with vital metabolic and cellular processes. Overall, this study highlights the miRNAs probably responsible for facilitating cold adaptation in L. decemlineata and implicates miRNAs as a key molecular target to consider in the development of novel pest management strategies against these insects.     

RNA interference in Pardosa pseudoannulata,an important predatory enemy against several insect pests,through ingestion of dsRNA-expressing Escherichia coli

RNA interference is an important technology for gene functional research in many organisms. The pond wolf spider (Pardosa pseudoannulata) is an important natural enemy of rice field pests. To facilitate large-scale gene functional research in this spider species and others, we developed an RNA interference (RNAi) method via ingestion of bacteria expressing dsRNA. The dsRNA targeting a cytochrome P450 monooxygenase (cyp41g2) was expressed in Escherichia coli HT115 (DE3). And then the bacterial suspension was fed to 14–20 days old spiderlings. The mRNA abundance of the target gene was significantly reduced after 3-day's ingestion of bacteria expressing dsRNA, and between day 5 and 7, RNAi efficiency remained stable. Thus, we selected 5 days as the optimum interference time. Furthermore, the bacteria resuspension containing 20 ng/μl dsRNA was selected as the optimum concentration. To evaluate the applicability of this method, three other genes with different tissue expression pattern were also selected as targets. And the mRNA abundance of all the four target genes was significantly reduced with RNAi efficiency between 66.0% and up to 86.9%. The results demonstrated that the oral delivery of bacteria expressing dsRNA would be an effective RNAi method for the gene functional study in P. pseudoannulata.     

Cloning of two novel P450 cDNAs from German cockroaches, Blattella germanica (L.): CYP6K1 and CYP6J1

Two novel P450 cDNAs, CYP6K1 and CYP6J1, were isolated from German cockroaches, Blattella germanica (L). Both CYP6K1 and CYP6J1 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Both CYP6K1 and CYP6J1 showed the highest per cent identity (based on the deduced amino acid sequence) to CYP6L1 from B. germanica and CYP6H1, a putative ecdysone 20‐hydroxylase from Locusta migratoria. Using a CYP6K1 probe, two mRNA signals (~2.5 and ~2.1 kb) were detected in all life stages. Both signals were just detectable in the eggs and became stronger in later instars. The strongest signals were detected in the fifth and sixth instars as well as in adults. These two bands were also detected in the abdomens and in the remainder of bodies of both male and female adults. Southern blots suggest the two mRNA bands detected in the Northern blot might be a result of alternative splicing. No signal could be detected at any life stage using the CYP6J1 probe, suggesting that CYP6J1 was expressed at a low level.     

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

Storage proteins are haemolymph‐specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune‐responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real‐time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co‐immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP‐6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two‐hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.