Project description:Henosepilachna vigintioctopunctata is a vegetable pest that has spread worldwide. It belongs to the Coccinellidae family, whose members exhibit remarkable diversity, both in terms of their diets and the colored spots that appear on the elytra in the adult stage. Transcriptomic data from H. vigintioctopunctata at different life stages would be useful for further investigating the genetic basis of this dietary diversity and the formation of the colored spots in ladybird beetles, as well as revealing the population dynamics of H. vigintioctopunctata, which could be useful in pest control. Here, we generated a comprehensive RNA-seq data set (a total of ~24?Gb of clean data) for H. vigintioctopunctata by sequencing samples collected at different life stages. We characterized the transcriptomes of each of the four life stages (egg, larva, pupa, adult) and generated a high-coverage pool by combining all the RNA-seq reads. Furthermore, we identified a catalog of simple sequence repeat (SSR) markers. This represents the first study to collect transcriptome data from all life stages of a ladybird beetle.
Project description:Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae) is a major pest affecting Solanaceae plants in Asian countries. In this study, we sequenced the ovary and testis transcriptomes of H. vigintioctopunctata to identify gonad-related genes. Comparison of the unigene sequences in ovary and testis libraries identified 1,421 and 5,315 ovary- and testis-specific genes, respectively. Among the ovary-specific genes, we selected the RC2-like and PSHS-like genes to investigate the effects of gene silencing on the mortality, percentage infertility, pre-oviposition period, fecundity, daily number of eggs laid, and hatching rate in female adults. Although the percentage mortality and infertility of females did not differ significantly among dsRNA treatments, fecundity was significantly reduced in the dsRC2-like and dsPSHS-like treatment groups. Moreover, the pre-oviposition period was markedly prolonged in response to dsPSHS-like treatment. This is the first reported RNA sequencing of H. vigintioctopunctata. The transcriptome sequences and gene expression profiles of the ovary and testis libraries will provide useful information for the identification of gonad-related genes in H. vigintioctopunctata and facilitate further research on the reproductive biology of this species. Moreover, the gonad-specific genes identified may represent candidate target genes for inhibiting the population growth of H. vigintioctopunctata.
Project description:Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. <i>Henosepilachna vigintioctopunctata</i> is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of <i>H. vigintioctopunctata</i> were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for <i>H. vigintioctopunctata</i> have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, <i>Actin</i>, <i>GAPDH</i>, <i>RPL13</i>, <i>RPL6</i>, <i>RPL32</i>, <i>RPS18</i>, and <i>ATPB</i>, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using <i>RefFinder</i>, which integrates four different analytical tools (<i>Normfinder</i>, <i>geNorm</i>, the ?<i>Ct</i> method, and <i>BestKeeper</i>). The results displayed that <i>RPL13</i> and <i>RPS18</i> were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, <i>lov</i> and <i>TBX1</i>, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in <i>H. vigintioctopunctata</i>.
Project description:The gut bacteria of insects positively influence the physiology of their host, however, the dynamics of this complicated ecosystem are not fully clear. To improve our understanding, we characterized the gut prokaryotic of Henosepilachna vigintioctopunctata that fed on two host plants, Solanum melongena (referred to as QZ hereafter) and Solanum nigrum (referred to as LK hereafter), by sequencing the V3-V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq system. The results revealed that the gut bacterial composition varied between specimens that fed on different host plants. The unweighted pair group method with arithmetic mean analyses and principal coordinate analysis showed that the bacterial communities of the LK and QZ groups were distinct. Four phyla (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were present in all H. vigintioctopunctata gut samples. It is noteworthy that bacteria of the phylum Cyanobacteria were only found in the LK group, with a low relative abundance. Proteobacteria and Enterobacteriaceae were the predominant phylum and family, respectively, in both the LK and QZ groups. Linear discriminant analysis effect size (LEfSe) analyses showed that the QZ group enriched the Bacilli class and Lactococcus genus; while the LK group enriched the Alphaproteobacteria class and Ochrobactrum genus. PICRUSt analysis showed that genes predicted to be involved in xenobiotic biodegradation and metabolism, metabolism of other amino acids, signaling molecules, and interaction were significantly higher in the QZ group. Genes predicted to be involved in the metabolism of cofactors and vitamins were significantly higher in the LK group. Furthermore, the complexity of the network structure and the modularity were higher in the LK group than in the QZ group. This is the first study to characterize the gut bacteria of H. vigintioctopunctat, our results demonstrate that the two host plants tested had a considerable impact on bacterial composition in the gut of H. vigintioctopunctata and that the bacterial communities were dominated by relatively few taxa.
Project description:Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata, as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1*-dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata, using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1*-dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1*-dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1*-dsRNA were fed to larvae of H. vigintioctopunctata, diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.
Project description:Pelteobagrus vachelli is a well-known commercial species in Asia. However, a sudden lack of oxygen will result in mortality and eventually to pond turnover. Studying the molecular mechanisms of hypoxia adaptation in fishes will not only help us to understand fish speciation and the evolution of the hypoxia-signaling pathway, but will also guide us in the breeding of hypoxia-tolerant fish strains. Despite this, the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in hypoxia responses in fish have remained unexamined. In the present study, we used next-generation sequencing technology to characterise mRNA-seq and miRNA-seq of control- and hypoxia-treated P. vachelli livers to elucidate the molecular mechanisms of hypoxia adaptation. We were able to find miRNA-mRNA pairs using bioinformatics analysis and miRNA prediction algorithms. Furthermore, we compared several key pathways which were identified as involved in the hypoxia response of P. vachelli. Our study is the first report on integrated analysis of mRNA-seq and miRNA-seq in fishes and offers a deeper insight into the molecular mechanisms of hypoxia adaptation. qRT-PCR analysis further confirmed the results of mRNA-Seq and miRNA-Seq analysis. We provide a good case study for analyzing mRNA/miRNA expression and profiling a non-model fish species using next-generation sequencing technology.