The melon fly, Zeugodacus cucurbitae (Coquillett), is an important destructive pest worldwide. Functional studies of the genes associated with development and reproduction during different life stages are limited in Z. cucurbitae. There have yet to be comprehensive transcriptomic resources for genetic and functional genomic studies to identify the molecular mechanisms related to its development and reproduction. In this study, we comprehensively sequenced the transcriptomes of four different developmental stages: egg, larva, pupa, and adults. Using the Illumina RNA-Seq technology, we constructed 52 libraries from 13 stages with four biological replicates in each and generated 435.61 Gb clean reads. We comprehensively characterized the transcriptomes with highcoverage mapping to the reference genome. A total of 13,760 genes were mapped to the reference genome, and another 4481 genes were characterized as new genes. Finally, 14,931 genes (81.85%) were functionally annotated against six annotation databases. This study provides the first comprehensive transcriptome data of all developmental stages of Z. cucurbitae, and will serve as a valuable resource for future genetic and functional studies. These results indicated that sequencing quality was high. Clean reads were separately aligned to the reference genome using HISAT2 33 .Transcriptome quality assessment. The saturation of sequencing was also analyzed using RSeQC 2.3.6 package, and the data showed a high saturation of each sample sequencing (Fig. 3a). Most of the reads were mapped to the coding sequence region (Fig. 3b). The alignment of the superreads to the reference genome resulted in a total of 41,530 transcripts, and 34.03% were longer than 2700 bp, and 75.96% were longer than 900 bp (Fig. 3c). Analyses of the structures of the transcripts suggested that 11,506 transcripts were potential novel transcript isoforms (Fig. 3d), and 22,068 transcripts were completely matched to the exons.
Background: Long non-coding RNAs (lncRNAs) are involved in many fundamental biological processes, such as transcription regulation, protein degradation, and cell differentiation. Information on lncRNA in the melon fly, Zeugodacus cucurbitae (Coquillett) is currently limited. Results: We constructed 24 RNA-seq libraries from eight tissues (midgut, Malpighian tubules, fat body, ovary, and testis) of Z. cucurbitae adults. A total of 3124 lncRNA transcripts were identified. Among those, 1464 were lincRNAs, 1037 were intronic lncRNAs, 301 were anti-sense lncRNAs, and 322 were sense lncRNAs. The majority of lncRNAs contained two exons and one isoform. Differentially expressed lncRNAs were analyzed between tissues, and Malpighian tubules versus testis had the largest number. Some lncRNAs exhibited strong tissue specificity. Specifically expressed lncRNAs were identified and filtered in tissues of female and male Z. cucurbitae based on their expression levels. Four midgut-specific lncRNAs were validated by quantitative real-time polymerase chain reaction (RT-qPCR), and the data were consistent with RNA-seq data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of targets of midgut-specific lncRNAs indicated an enrichment of the metabolic process. Conclusions: This was the first systematic identification of lncRNA in the melon fly. Expressions of lncRNAs in multiple adult tissues were evaluated by quantitative transcriptomic analysis. These qualitative and quantitative analyses of lncRNAs, especially the tissue-specific lncRNAs in Z. cucurbitae, provide useful data for further functional studies.
Long non‐coding RNAs (lncRNAs) generally display tissue‐specific distributions, and testis‐specific lncRNAs form the highest proportion of lncRNAs in many species. Here, we presented a detailed analysis of testis‐specific lncRNAs in the melon fly, Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. Most testis‐specific lncRNAs were found to be long intergenic non‐coding RNAs (lincRNA). The size distribution of these lncRNAs ranged between 600 and 1000 nucleotides. Testis‐specific lncRNAs that harboured one isoform number and two exons were the most abundant. Compared to other male tissues, the testis had more highly expressed lncRNAs. The quantitative real‐time polymerase chain reaction results of 10 randomly selected testis‐specific lncRNAs showed expression patterns consistent with RNA‐seq data. Further analysis of the most highly expressed testis‐specific lncRNA, lnc94638, was undertaken. Fluorescent in situ hybridization assays localized lnc94638 to the apical region of the testis that contains mature spermatozoa. RNA interference‐mediated knockdown of lnc94638 expression reduced spermatozoa numbers and impaired the fertility of Z. cucurbitae male. This study provides a catalogue of testis‐specific lncRNAs, shows that the testis‐specific lnc94638 is involved in spermatogenesis and has the potential to be used for treating male sterility.
The ATP-binding cassette (ABC) transporter is a protein superfamily that transports specific substrate molecules across lipid membranes in all living species. In insects, ABC transporter is one of the major transmembrane protein families involved in the development of xenobiotic resistance. Here, we report 49 ABC transporter genes divided into eight subfamilies (ABCA-ABCH), including seven ABCAs, seven ABCBs, 10 ABCCs, two ABCDs, one ABCE, three ABCFs, 16 ABCGs, and three ABCHs according to phylogenetic analysis in Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. The expressions level of 49 ABC transporters throughout various developmental stages and within different tissues were evaluated by quantitative transcriptomic analysis, and their expressions in response to three different insecticides were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). These ABC transporter genes were widely expressed at developmental stages but most highly expressed in tissues of the midgut, fat body and Malpighian tube. When challenged by exposure to three insecticides, abamectin, β-cypermethrin, and dinotefuran, the expressions of ZcABCB7 and ZcABCC2 were significantly up-regulated. ZcABCB1, ZcABCB6, ZcABCB7, ZcABCC2, ZcABCC3, ZcABCC4, ZcABCC5, and ZcABCC7 were significantly up-regulated in the fat body at 24 h after β-cypermethrin exposure. These data suggest that ZcABCB7 and ZcABCC2 might play key roles in xenobiotic metabolism in Z. cucurbitae. Collectively, these data provide a foundation for further analysis of ABCs in Z. cucurbitae.
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