| Digestion and detoxification are two important metabolic pathways in insects. The digestion provides enough energy for growth, development and reproduction, and detoxification can prevent damage from xenobiotics. During a long period, researches have been just focused on a few genes or enzymes. Without sufficient molecular information, it was hard to get a broad view of this part. At present, high throughput sequencing technology has become the best choice to screen out the gene information of the organism of interest without reference genome. This technology provides a short cut that can effectively and rapidly characterize the unique transcripts from individuals or specific tissues, and make great promotion to molecular studies.This study focused on an important agriculture pest, the oriental fruit fly, Bactrocera dorsalis. Firstly, we compiled four digital gene expression (DGE) libraries to investigate the expression profiles of genes at different developmental stages (egg, third-instar larva, pupa, and adult). The differently expressed genes were annotated and the expression distribution of digestion and detoxification genes were determined. Larva was found an important stage for the expression of these genes. As the second largest organ in insects, the insect midgut is the major tissue for digestion of food and detoxification of xenobiotics, and the first barrier and target against exogenous toxin. The functions and related mechanisms of midgut are always deemed as hot points by researchers. Secondly, we performed a midgut-specific transcriptome analysis of this insect. The annotation of gene transcripts provided sufficient molecular information for the fruit fly database and was useful for studying the biochemical and physiological mechanisms at molecular level. Meanwhile, for the pest control, we screened out the genes involved in digestion, RNAi mechanism and detoxification, which were important functions of the midgut, and conducted sequence characterization and phylogenic analysis of these genes. It is meaningful for finding the potential insecticide target, understanding the mechanism of detoxification, or developing new ways for pest control via RNAi. Using the transcriptome data, we futhur examined the expression reaction of detoxification and digestion genes under the stress. Based on the reference evaluation, our qPCR data showed the expression model of these genes in the midgut after stimulated by β-Cypermethrin. Compared with the data from the whole body of larvae and fat body, tissue specific genes related to detoxification were identified, and revealed some energy distribution strategies of this insect when stimulated by insecticides.In conclusion, this study forms a solid basis for the function study of insect digestion and detoxification at molecular level, and provids a insight view for the future studies in this aspect. The analysis of gene sequences related to digestion, RNAi mechanism and detoxification will give new ideas and directions for pest control, especially for the identification of a number of specific genes that were considered to be significant to resistance.1Comparison of gene expression among different developmental stages of Bactrocera dorsalis1.1Summary of DGE librariesRNA of different development stages was extracted from the lab population, which were initially collected from Hai Nan province, China. Based on the previous transcriptome data of developmental stages, four DGE libraries were constructed by Illumina HiSeq2000to identify the Unigene expression profiles of the different developmental stages (accession numbers:SRA043792for egg, SRA043786for third-instar larva, SRA043785for pupa, and SRA043783for adult). After excluding low-quality reads, each library generated above five million clean reads, and the distribution of genes with coverage above50%was75%in egg,60%in larva,77%in pupa, and63%in adult. These results indicated high quality of DGE library sequencing.1.2Differently expressed genes among developmental stagesThe four developmental stages were evaluated in three pairwise comparisons:egg vs. third-instar larva (E vs. L), third-instar larva vs. pupa (L vs. P), and pupa vs. adults(P vs. A). Genes found to have significant differences in expression were identified in each comparison. The results suggested that the expression of15516genes was significantly different between egg and third-instar larva. Of these genes7352were up-regulated and8164were down-regulated in the E vs. L comparison. In the comparison of third-instar larva and pupa, the expression profiles of7581genes were changed. There were3786genes up-regulated in pupa and3795genes down-regulated. When comparing pupa and adult,3077genes were up-regulated in adults and7195genes were down-regulated.By the gene function annotation, we found in egg, the highly expressed genes were involved in embryo development and cell mitosis regulation. In larva, the expressions of genes involved in energy metabolism and muscle composition were the most active. In pupae, genes encoding chitinase were highly expressed, which indicated that in this period, cuticle was remodeling, and meanwhile, the the expressions of genes related to cell mitosis regulation were also very active. The genes highly expressed in adults were similar to larva, most of them were related to energy metabolism and muscle composition, and some genes related to visual and auditory sense were specifically identified in this stage. The differently expressed genes in different developmental stages reflected the most important actions in each stage. In egg and pupa, genes related to cell mitosis regulation were highly expressed, wich indicates these two period were crucial for metamorphosis. In larva and adult, the highly expressed genes were involved in energy, detoxification and movability.1.3Expression distribution of digestion and detoxification genesThe expression distribution of genes involved in digestion and detoxification were determined in different developmental stages. The most abundant digestion enzyme was trypsin.15seqences were found highly expressed in adult and larva, but most of them were not found in egg. The expression distribution of some other digestion enzymes were similar to that of trypsin, such as aminopeptidase N and carboxypeptidase.The most abundant detoxification enzyme was P450s.76sequences were found expressed in different developmental stages. P450s were also enriched in adult and larva, and62sequences were expressed in these two stages, respectively. Besides,23sequences encoding GSTs and17sequences encoding CCEs were identified. They were also most active in larva, but not enriched in egg.2The midgut-specific transcriptome analysis of B. dorsalis2.1Data assembly and annotation of midgut transcriptsThe insect was initially collected from Hai Nan province, China, and RNA was extracted from the midgut of larva. A cDNA library (SRA submission number: SRA056311) was constructed by Illumina HiSeq2000, and this generated52838060total clean reads and4755425400nucleotides. These reads with certain length of overlap were assembled to53318contigs with an average length of379nt via the Trinity program. Using paired-end joining and gap-filling, the contigs were further assembled to25236Unigenes. To annotate Unigenes, sequences were searched in the nonredundant (Nr) NCBI protein database using BLASTX with a cutoff E-value of10-5. A total of16531distinct sequences returned a blast result, which meant that these Unigenes were successfully mapped to known function genes. Besides the Nr database, there were13026Unigenes successfully annotated in Swiss-Prot,11488in KEGG,11575in GO,9825in Nt, and5595in COG. Totally,17308Unigenes were annotated across these databases.2.2Unigene distributionOf the16531annotated Unigenes in Nr,48.6%had an E-value of10-45, showing a significant homology matching in the NCBI database, while51.4%had an E-value ranging from10-45to10-5. The similarity distribution showed that24%of the sequences had a significant homology higher than80%. Among the annotated Unigenes,5273sequences were longer than1000nt.4386sequences between500nt to1000nt were successfully annotated, and6873sequences shorter than500nt returned a blast result. According to the best hit at Nr database, the majority of Unigenes (75.74%) had strong homology with Drosophila. Of these,11.03%Unigenes were best matched to sequences from D. virilis, followed by D. willistoni (10.32%), D. mojavensis (9.86%), and other species of Drosophila.24.26%of Unigenes matched to other Diptera species, such as Glossina morsitans morsitans (5.51%), Aedes aegypti (1.03%), Culex quinquefasciatus (0.79%) and Anopheles gambiae (0.62%). Only136Unigenes had a best hit to Bactrocera, such as B. oleae (68Unigenes), B. papaya (7), B. tryoni (6), B. cucurbitae (5), and B. philippinensis (4). There were only46sequences matched the genes of B. dorsalis, which indicated that in the data base, the sequence imformation of B. dorsalis was still far from sufficiency.2.3Classification of gene ontology (GO) and Clusters of orthologous groups (COG)The result of GO classification showed that11575Unigenes (45.87%of total) were mapped to61functional groups. These functional groups were classified into three categories:nameliy "biological process","cellular component" and "molecular function". In the "biological process","cellular process" was the most abundant group, followed by "metabolic process","multicellular organismal process", and "biological regulation". More than4000Unigenes were identified to be involved in these processes. In contrast, only2Unigenes were classified in "carbon utilization", which was the smallest group in this category,"cell" and "cell part" were the two largest groups in "cellular component" with>6000Unigenes in each group. In "molecular function", "binding" was the largest group containing6854Unigenes, while there was only1Unigene in "receptor regulator activity".5595sequences had a COG annotation. Among the COG categories, most Unigenes (2130) were mapped to the "General function prediction". The largest three groups presenting specific functions were "Transcription"(1173),"Translation, ribosomal structure and biogenesis"(926),"Replication, recombination and repair"(886).3Gene function classification3.1Analysis of genes involved in digestionIn the midgut-specific transcriptome data of this study, we identified106Unigenes encoding various proteases, lipases, and carbohydrases related to digestion by BLAST in Nr. Among the proteases, Unigenes were divided into6groups:namely trypsins (22Unigenes), aminopeptidases (22), chymotrypsins (2), cysteine proteases (3), aspartic proteases (3), and carboxypeptidases (7). These sequences shared homology with aminopeptidases from Drosophila, C. quinquefasciatus, A. aegypti and G. morsitans morsitans.4sequences were characterized as aminopeptidases N, which were the target for Bt. The identification of these sequences will provide useful information for control and resistance management of B. dorsalis at molecular level.3.2Transcripts encoding genes involved in RNAi mechanismsMidgut is an important entrance for dsRNA. The uptake and cleavage of dsRNA are key factors for RNAi. To ensure the RNAi working in B. dorsalis, we characterized the genes involved in RNAi mechanisms. Four kinds of genes with great significance to RNAi were classified, such as Dicer-2i, that can cut dsRNA fragments into short nucleotides; R2D2and AGO2, that were important for RNA-initiated silencing complex (RISC). However, no SID-1orthologs were found in Diptera insects, as well as B. dorsalis, and we could not find a sequence encoding SR-CI. In addition, Eater and another scavenger receptor was classified. Further study can be focused on the RNAi mechanism and pest management via RNAi.3.3Midgut specific sequences encoding detoxification genesBy BLAST in Nr,9sequences encoding GSTs were identified from the annotation results of the midgut transcriptome data of B. dorsalis, and8of them were found to represent full length of ORF. It was confirmed by ORF finding and protein BLAST at NCBI. The average length of ORF was639nt, ranging from531to702nt. Based on the BLAST results and phylogenetic analysis, these sequences were divided into different GSTs families. We found4delta genes and1gene in each of the theta, zeta family and a microsomal GST, and there was1unclassified gene (U).In the transcriptome data,10sequences were found encoding CCEs, and6of these were fully sequenced, containing the complete ORF. ORF finder analysis and protein BLAST results at NCBI showed that the average length of CCEs ORF was1695nt, ranging from1635to1722nt. Further phylogenetic analysis with esterase genes from other insect species demonstrated that these6esterase genes were all a-esterase, and the highly conserved motif "FGESAG" and "EDCLYLN" of cholinesterases and the catalytic triad were found in all amino acid sequences. All of these genes were classified to the dietary class. Hence, it is known that the genes of this class are also involved in detoxification and xenobiotic resisatance.22sequences encoding P450s were isolated, and16of these were fully sequenced with the complete ORF. The average length of fully sequenced ORFs was1556nt, ranging from1467to1626. In the deduced amino acid sequences of these P450genes, the typical conserved P450motifs were identified, including the absolutely conserved ExxR motif found in the Helix-K (hydrogen bonding), and the heme-binding domain (PFxxGxRxCxG/A). Phylogenetic analysis identified6families:6genes in the CYP4family,5genes in the CYP6family,2genes in the CYP12family, and1gene in the CYP317, CYP309and CYP9family each.4The expression model of detoxification and digestion genes when stimulated by insecticide4.2Effect of P-Cypermethrin exposure on larval survival and the stability of housekeeping genesTo determine the appropriate concentration of β-Cypermethrin against larvae in the exposure experiment, different doses of β-Cypermethrin were mixed into the diet that was fed to the larvae. The survive rate and the relative mortality were normalized by a control group.0.33μg/g (P-Cypermethrin/artificial diet) was determined as a suitable concentration to stimulate the larvae. Such a concentration would not cause apparent death as1μg/g and33μg/g, and most individuals could normally develop under the toxicity stress. To ensure the accuracy of qPCR results, we firstly evaluated the expression stability of housekeeping genes under the experimental conditions. GeNormplus and Normfinder were used to estimate the stability of9housekeeping genes in the whole body of larvae, midgut and fat body under toxicity stress. EFla was found the best one for both larvae and midgut. In fat body, the most stable housekeeping gene was RPL13. Thus, these two genes were used as reference gene in qPCR study, respectively.4.2The expression model of detoxification and digestion genes when stimulated by insecticideBased on the reference evaluation, we analyzed the expression model of detoxification genes under toxicity stress. The qPCR results of8GSTs genes showed the genes from Delta family were involved in the detoxification, but other genes did not show very active reaction. The expressions of Delta genes were up-regulated5.6～32.5fold in the midgut and fat body. But in larvae, the expression changes of these genes were not so apparent, which indicated that their expression in other tissues were down-regulated. The results revealed some energy distribution strategy of this insect under survival stress. The energy was focused to important tissues when stimulated by insecticide to expression detocification genes.In larvae, the expression of6CCEs genes was up-regulated in some degree, and a-E3was up-regulated4.1fold. But in the midgut and fat body, the expressions of most CCEs genes were not significantly changed. We assume that the up-regulation in larvae showed the relation between the CCEs and detoxcification of β-Cypermethrin. This kind of gene did not work in the midgut or fat body, but in other tissues, such as Malpighian tubes.The qPCR results of16P450genes showed that most P450s genes were involved in the detoxification of β-cypermethrin, especially the CYP4and CYP6family. In CYP4family, the expression of CYP4D47(midgut), CYP4E9(larvae and fat body), CYP4P5(Larvae and fat body) was highly up-regulated. The expression of CYP4AD1increased in some degree in the midgut and fat body, but no significant changes were found in larvae. CYP4S18and CYP4AC4did not show any positive reaction when stimulated. In CYP6family, the expression of CYP6A48and CYP6A41has a significant increase in the fat body,18.0and9.6fold up-regulated, respectively. The expression of others like CYP6G6and CYP6A50also increased but not highly. The expression of CYP6EK1was up-regulated by4.8fold, but showed no apparent changes in midgut and fat body, which indicated that it might work in Malpighian tubes. It suggested that this gene play a role in metabolic of β-cypermethrin, and worked in fatbody. CYP12C2was12.6fold up-regulated in fat body, and2fold upregulated in larvae and midgut. Other genes, such as CYP12N1, CYP9F6, and CYP309B1also returned a positive reaction to stimulation in the fat body. But no significant expression of CYP317B1was detected under the stress.We also detected the expression of6digestion genes. They all expressed highly after stimulated, more than3fold up-regulated. The increasing of trypsin was up to10.7fold. The results showed that under toxicity stress, the digestion of midgut was improved.In conclusion, this study conducted DGE analysis of different developmental stages and transcriptome analysis of midgut from the larvae of B. dorsalis via high throughput sequencing technology. Through data analysis and annotation, we characterized the differently expressed genes, and showed the expression distribution of detoxification and digestion genes in developmental stages. From the transcriptome data, we screened out the genes related to digestion, RNAi mechanism, and detoxification, and characterized their molecular information. The expression models of detoxification and digestion genes under toxicity stress were further detected. The results revealed some energy distribution strategy of this insect when stimulated by insecticide. Our data provides sufficient gene information of B. dorsalis, and will promote the function study of midgut at molecular level. |
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