Project description:Purpose: Next-generation sequencing (NGS) has revolutionized genetic information from organisms that lack of gene sequencing data. Sequences of transcriptomic or genomic data of C. includens are no longer available to conduct relevant and applied studies to agriculture, such as, characterize biological process, screening for resistance and identification of new target genes. In this sense, we constructed a reference transcriptome of C. includens from head and body tissues of third instar larvae and we performed and analysis of gene expression functionality of transcripts between head and body. Methods: Two replicates of pool 5 larvae (third instar) were dissected into two parts: head (enriched with neural tissue) and body (enriched with adipose and gut tissues). Paired-end libraries of head and body tissues, with an approximate average insert length of 300 bp, were sequenced on an Illumina HiSeq platform with 150 bp paired-end sequencing. Trimmed RNA-Seq reads from transcriptomes of head and body larvae were used for de novo assembly using de Bruijn graph-based assembler (Trinity). All contig sequences built on the reference transcriptome assembled were annotated using Blast2GO pipeline. For the expression analysis RNA-seq reads were mapped to the built reference transcriptome using TopHat and expression values of transcripts in FPKM (fragments per kilobase per million mapped reads) between head and body tissues were estimated on Cufflinks. Results: The assembled transcriptome from reads of head and body tissues of C. includens larvae generated a fasta file format (136.2MB) containing 102,249 transcript sequences and 60,347 “genes”. The length distribution of transcripts was between a minimum of 200bp and a maximum of 37,230bp with an average of 1,005bp. As the RNA extraction of body included enriched tissues of tegument, gut, and hemolymph of third instar larvae, the transcripts were found to be involved on transmembrane transport, proteolysis, cation transport and many types of metabolic process. These biological processes in soybean looper were related to the cellular functionality of oxidoreductase, transmembrane transporter and serine-type endopeptidase activities and many types of binding functions. In the head tissue of C. includens we found most transcripts with gene ontology related to signal transduction. This biological process could be associated with the functionality of genes related to odorant binding, signaling receptor binding, DNA binding and other types of molecular binding. As we found, we suggest that these genes expressed only in the head tissue might be involved mainly in the detection of a signal and subsequent cascade expression of biological processes. Conclusions: Our study represents the first detailed transcriptome of Chrysodeixis includens of different larvae tissues, with biologic replicates, generated by RNA-seq technology. Our results show that NGS offers a comprehensive and accurate sequencing data to species with reference genome not available. Also, RNA-seq data offer a global gene expression analysis between tissues and permit understand some biological process involved in insect physiology.
Project description:Purpose: To investigate resistance mechanisms among individuals that tolerate high doses of insecticides, we performed high-throughput RNA sequencing on two Brazilian populations of C. includens, that differ in susceptibility to pyrethroid λ-cyhalothrin. Methods: Two replicates of pool 4 larvae (third instar) of each population, susceptible (LAB) and resistant (MS), were dissected into two parts: head (enriched with neural tissue) and body (enriched with adipose and gut tissues). Paired-end libraries of head and body tissues, with an approximate average insert length of 300 bp, were sequenced on an Illumina HiSeq platform with 150 bp paired-end sequencing. Trimmed RNA-Seq reads from transcriptomes of head and body larvae were used to alings the reads in the reference transcriptome (GSE121104), using TopHat. Expression analysis between LAB and MS population and between tissues were performed on Cufflinks, where the FPKM (fragments per kilobase per million mapped reads) between transcriptomes were estimated. Results: . Our results revealed several potential molecular mechanisms that underlie their low susceptibility to pyrethroids. We identified 10 synonymous and five non-synonymous mutations within the coding region of the voltage-gated sodium channel in the resistant population, specifically in domains II, III, and IV. Additionally, we observed overexpression of metabolic enzymes including Cyp, Gst, Est, and Ugt. Some of these enzymes were up-regulated in the head as well as the abdomen of the resistant population, suggesting a detoxification process that begins in the mouth parts and continues through the gut. Conclusions: The complexity of altered gene expression and nonsynonymous mutations can effectively be assessed with RNA sequencing in order to implement integrated pest management strategies and insecticide resistance management.
Project description:Transcriptional analyses of different Chrysodeixis includens larvae (Lepidoptera: Noctuidae) unveil molecular resistance mechanism for pyrethroid resistance in Brazil
