- impairment in the domestic silkworms:. - Comparison of the same sex between the domestic and wild silkworms revealed 1410 and 1173 differentially expressed genes (DEGs) in males and females, respectively. - Furthermore, one deleterious mutation was found in OR30 of the domestic population, which was located in transmembrane helix 6 (TM6).. - Conclusions: Our results suggested that down-regulation of the olfactory-related genes and relaxed selection might be the major reasons for olfactory impairment of the domestic silkworm reared completely indoor. - Bombyx mori is one of the model organisms for the study of insect olfaction, especially in pheromone per- ception [7, 8] and functional diversities of odorant re- ceptors [9, 10]. - In this study, we analyzed the antennal transcriptomes of the domestic and wild silkmoths, and mainly focused on gene families that have been implicated in olfaction.. - The objective of this study was to detect expression difference of olfactory-related genes in the antennae of the domestic and wild silkworms and to deepen understanding of how the domestic silkworms to impair olfactory sensitivity.. - Comparison of the indoor species and its corresponding wild species would help us understand the potential mechanisms of olfactory adaptation in wild condition.. - The larvae of the wild silkworms were reared on mulberry trees in open-air field chambers.. - Two days after pupation, the pupae of the domestic and wild silkworms were transferred to a same indoor cham- ber until dissection of adult antennae. - Two replicates were taken for the antennae of female (W_F) and male (W_M) of the wild silkworm, female (D_F) and male (D_M) of the domestic silkworm, respectively. - Total RNA of the antennae was extracted with Trizol re- agent (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. - The quality of the RNA samples was checked using an Agilent Bioana- lyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). - The reads of the whole genome and tran- scriptome sequencing were often mapped to the domes- tic silkworm reference genome [20, 21]. - The statistical significance of the functional GO enrich- ment was evaluated using a false discovery rate (FDR <. - HMM files were used to screen the translated proteins of the antenna-expressed genes using HMMER 3.0 (E-value <1e-5). - Using known olfactory-related pro- tein sequences as queries, BLASTP was also used to search against the predicted protein database of the antenna-expressed genes (E-value <1e-5). - Population genetics and molecular evolution of the olfactory genes. - The consensus sequences of the candidate genes were extracted in the genome sequence of each sam- ple, including the coding sequences (CDS) and gene se- quences comprised of CDS, introns, and 2-Kb 3′ and 5′. - In the domestic and wild populations, genetic diversities (π) of the differentially expressed olfactory genes were esti- mated by DnaSP 5.1 [36]. - Genes with FPKM ≥ 1 in at least one of the four antennal samples were considered to be expressed. - Overview of the antennal transcriptomes in the silkworms To explore the mechanisms of olfactory adaptation, we collected the intact antennae from the domestic silkworm strain Dazao and wild silkworm. - All of the mapped reads were merged and assembled using Cufflinks [24].. - Totally, 22,767 unigenes were assembled from the an- tennal transcriptomes of the domestic and wild silk- worms (Additional file 4: Table S4). - KEGG assign- ments were used to classify the functions of the antenna-expressed genes. - were used to identify novel olfactory-related genes in the assembled unigenes of the antennal transcriptomes (Table 1). - Our results indicated that more than half of the whole-genome OR, IR, and OBP genes were expressed (FPKM ≥1) in the antennae of the domestic and wild silkworms (Table 1). - In addition, almost all of the CSPs showed expression signals in the antennae. - Within the ten olfactory-related families of interest here, more than 60% of the antennal-expressed OBPs, CSPs and AOXs showed high expressions (FPKM >. - In order to under- stand the functions of the DEGs, GO enrichment analysis was performed in BLAST2GO [27]. - 3 Hierarchical clustering and Venn diagram of the differentially expressed genes in the antennal transcriptome of the domestic and wild silkworms.. - a Venn diagram showing the number of the DEGs between the domestic and wild silkworms. - b Venn diagram of the DEGs in D_M vs. - To understand the olfactory impairment, compari- son of the same sex between the domestic and wild silkworms may be more meaningful. - Compared with the domestic silkworms, 13 out of 19 olfactory genes and 13 out of 15 putative ODEs were up-regulated in females of the wild silkworm (Fig. - In the compari- sons of the same sex between the domestic and wild silkworms, overall 30 olfactory genes and 19 ODEs were differentially expressed, in which 19 olfactory genes and 14 ODEs were up-regulated in the wild silkworm. - This indicated that the decreased expressions of the olfactory-related genes may lower. - the sensitivity of OSNs in the domestic silkworms [13, 14].. - Molecular population genetics of the olfactory genes down-regulated in the domestic silkworm. - To further understand olfactory impairment, molecular evo- lution of the down-regulated ORs and OBPs was investi- gated. - 4 Validation of the DEGs by qPCR method. - all of the genes indicated that there were greater numbers of rare alleles. - In the domestic strains, most of the differ- entially expressed olfactory genes contained positive values from those tests, which may be due to a population bottleneck or balancing selection.. - Evolutionary rate and intolerant mutations of the olfactory genes. - 5 Olfactory-related genes showed differential expressions in the same sex between the domestic and wild silkworms. - a Hierarchical clustering of the DEGs and differentially expressed olfactory and putative ODE genes in females. - b Hierarchical clustering of the DEGs and differentially expressed olfactory-related genes in males. - Table 2 Genetic diversity and neutrality analysis of the ORs and OBPs genes down-regulated in the domestic silkworms Gene. - The ratio of the numbers of non-synonymous SNPs to the numbers of synonymous SNPs (N/S) can be used to assess the relaxation of purifying selection [43]. - It was indicated that one deleterious amino acid mutation was found in OR30 of the domestic popu- lation (Fig. - In the adult antennal transcriptomes of the domestic and wild silkworms, 22,767 unigenes were assembled, in which over 57.45% of the unigenes were expressed (FPKM ≥1) (Additional file 4: Table S4). - We found that more than half of the olfactory-related genes were expressed in the antennae of the domestic and wild silkworms (Table 1, Additional file 4: Table S4). - 6 Box plot of the d N /d S values of the ORs and OBPs down-regulated in the domestic silkworms. - a The d N /d S values of the ORs down-regulated in the domestic silkworms. - D_OR: OR genes in the domestic population. - W_OR: OR genes in the wild population. - values of the four OBPs down-regulated in the domestic silkworms.. - D_OBP: OBP genes in the domestic population. - W_OR: OBP genes in the wild population. - Table 3 Numbers of synonymous and non-synonymous nucleotide changes of the ORs and OBPs down-regulated in the domestic silkworms. - Previous studies have found that a number of putative ODEs were expressed in the antennae of the D. - In this study, 127 putative ODEs were expressed in the antennae of the domestic and wild silk- worms (Additional file 10: Table S4). - 7 The deleterious amino acid substitution of OR30 in the domestic silkworms. - a Population-unique deleterious mutation of OR30 in the domestic silkworms. - b The topology of OR30 and the location of the deleterious amino acid substitution. - In this study, the pupae of the domestic and wild silkworms were put in an identi- cal indoor chamber until dissection of adult antennae.. - The differential expressions of the candidate genes might be caused by the divergence of the genomic background during long-term adaptation. - Through the comparisons of the same sex between the domestic and wild silk- worms, 30 differentially expressed olfactory genes were identified (Fig. - Based on the microarray data of the tissues and developmental stages in the silkworms [54, 55], CSP3, CSP8, CSP11, CSP12 showed high expressions in various tissues and developmental stages (Additional file 12:. - In the silkworm, some of the ORs have been char- acterized for ligand responsiveness [9, 10]. - However, the high background ex- pression levels of the olfactory genes should be beneficial for wild silkworm to maintain a high perception of plant volatiles.. - Except for expression level of the olfactory genes, se- quence polymorphisms would also contribute to olfac- tory sensitivity [16, 18]. - In coding regions, the nucleotide diversities of the 17 olfactory genes showed lower nu- cleotide diversity in the domestic silkworms than wild silkworms (Table 2). - Due to inbreeding and bottleneck effect, genetic diver- sities (π) of the 17 differentially expressed olfactory genes were lower in the domestic silkworm (Table 2). - However, the global d N /d S ratios of the differentially expressed olfactory genes were comparable between the domestic and wild silkworms (Fig. - Furthermore, we focused on the population-unique non-synonymous sites of the 13 OR genes. - In future, the odorant ligands of OR30 and effects of the deleterious mutations on olfaction sensitivity need to be validated. - Comparative analysis of the antennal transcriptomes was conducted in the domestic and wild silkworms. - It was indicated that down-regulation and functional relaxation of the olfactory-related genes might impair the olfactory. - R1 and R2 at the end of the sample name represent repeat 1 and 2, respectively. - Summary of the clean reads mapped to the silkworm reference genome. - Distribution of gene expressions in adult antennae of the domestic and wild silkworms. - The female (W_F) and male (W_M) of the wild silkworm, female (D_F) and male (D_M) of the domestic silkworm were showed. - The sequences of the olfactory-related novel genes. - Gene Ontology enrichment analysis of the DEGs. - The sex-biased genes in the domestic and wild silkworms. - Expression profiles of the differentially expressed CSPs in the tissues and developmental stages of the silkworm. - Experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Chongqing University (permit number CBE-A201607020).. - Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori. - Phylogeny and evolutionary history of the silkworm. - Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms. - Comparative analysis of the silk gland transcriptomes between the domestic and wild silkworms. - Molecular evolution of the odorant and gustatory receptor genes in lepidopteran insects: implications for their adaptation and speciation. - Insights into the evolution of the CSP gene family through the integration of evolutionary analysis and comparative protein modeling. - Comparative analysis of the UDP- glycosyltransferase multigene family in insects. - Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila. - A diversity of putative carboxylesterases are expressed in the antennae of the noctuid moth Spodoptera littoralis. - Silencing of the olfactory co-receptor gene in Dendroctonus armandi leads to EAG response declining to major host volatiles. - Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. - Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex
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