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Abstract
Chemosensory genes play a central role in sensing chemical signals and guiding insect behavior. The Chinese honeybee, Apis cerana cerana, is one of the most important insect species in China in terms of resource production, and providing high-quality products for human consumption, and also serves as an important pollinator. Communication and foraging behavior of worker bees is likely linked to a complex chemosensory system. Here, we used transcriptome sequencing on adult A. c. cerana workers of different ages to identify the major chemosensory gene families and the differentially expressed genes(DEGs), and to investigate their expression profiles. A total of 109 candidate chemosensory genes in five gene families were identified from the antennal transcriptome assemblies, including 17 OBPs, 6 CSPs, 74 ORs, 10 IRs, and 2SNMPs, in which nineteen DEGs were screened and their expression values at different developmental stages were determined in silico. No chemosensory transcript was specific to a certain developmental period. Thirteen DEGs were upregulated and 6were downregulated. We created extensive expression profiles in six major body tissues using qRT-PCR and found that most DEGs were exclusively or primarily expressed in antennae. Others were abundantly expressed in the other tissues, such as head, thorax, abdomen, legs, and wings. Interestingly, when a DEG was highly expressed in the thorax, it also had a high level of expression in legs, but showed a lowlevel in antennae. This study explored five chemoreceptor superfamily genes using RNA-Seq coupled with extensive expression profiling of DEGs. Our results provide new insights into the molecular mechanism of odorant detection in the Asian honeybee and also serve as an extensive novel resource for comparing and investigating olfactory functionality in hymenopterans.
Citation: Zhao H, Du Y, Gao P, Wang S, Pan J, Jiang Y (2016) Antennal Transcriptome and Differential Expression Analysis of Five Chemosensory Gene Families from the Asian Honeybee Apis cerana cerana. PLoS ONE 11(10): e0165374. https://doi.org/10.1371/journal.pone.0165374
Editor: J Joe Hull, USDA Agricultural Research Service, UNITED STATES
Received: June 11, 2016; Accepted: October 11, 2016; Published: October 24, 2016
Copyright: © 2016 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grants from the National Natural Science Foundation of China (31502021 and 31272513), and Scientific Research Starting Foundation for Introduced Doctor of Shanxi Agriculture University (2013YJ37). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Olfactory sensing by the antennae is crucial for insect survival, reproduction, and intraspecific communication. Peripheral odor information is detected by olfactory receptor neurons, most of which are housed in the chemosensillae of the antennae [1]. Diverse olfactory protein families are involved in different steps of peripheral olfactory signal transduction events, such as odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs) [2, 3]; however, OR proteins are considered the most important [4].
The OBPs of insects are small and water-soluble and accumulate in the sensillar lymph. They are considered to be the first protein in the olfactory recognition procedure [5]. Odorant molecules are hydrophobic, and are thought to be bound by OBPs and transported through the aqueous sensillar lymph to chemoreceptor proteins located on the surface of olfactory sensory neuron OSN dendrites [6]. Like OBPs, CSPs are small soluble binding proteins and are more conserved than OBPs [7]. CSPs are not only involved in semiochemical detection [8], but may also participate in some non-chemosensory functions [9, 10].
ORs and IRs are two chemosensory receptors families located in the dendritic membrane of OSNs and both are regarded as key factors in the chemosensory signal transduction process [11]. ORs are multi-transmembrane domain receptors and have been shown to function as heteromeric ion channels, consisting of a conventional olfactory receptor and a highly conserved member which is referred to as Orco [12, 13]. IRs appear to be far more ancient than ORs; they are related to the ionotropic glutamate receptors (iGluRs) family but contain a divergent ligand-binding domain.IRs are expressed in coeloconic OSNs and may function in the detection of acids and ammonia [3, 14].
SNMPs are also membrane proteins with two transmembrane helical domains [15]. Insects generally have only one or two genes that encode SNMPs, and their exact functions are mostly unknown [2].
Recently, some of the characteristics of these olfactory-related gene families and their functions have been determined, but the detailed relationships among these proteins, including their synergistic or antagonistic effects in the olfactory recognition process, have received less attention from researchers.
The honeybee is a eusocial insect and key model in the study of olfactory perception and learning behavior [16, 17]. A colony is composed of a single queen, hundreds of male drones, and thousands of sterile female workers. They possess specific social roles: the queen is responsible for laying eggs, drones function in mating with the queen, and workers perform different tasks at different ages, such as taking care of the brood and the queen, building nests, gathering various resources, and guarding the colony [18]. In this society, the sense of chemoreception plays a fundamental role in mediating the wide range of behaviors [19], and honeybee might be expected to have a more complex olfactory system than solitary insects [20].
In the genus Apis, the western honeybee (Apis mellifera L.) and eastern honeybee (Apis cerana Fabr.) are the two most important species. Olfactory related genes was relatively earlier researched in A. mellifera, such as the OBP genes ASP1 and ASP2, the CSP gene ASP3 and the OR gene Or2 were the first to be identified and characterized in Hymenoptera [21–25]. The elucidation of the complete genome sequence of A. mellifera has facilitated molecular biological research and also resulted in the identification of chemosensory genes [20].To data, 170 ORs, 21 OBPs, 6 CSPs, 10IRs, and 2SNMPs have been annotated [14, 26–29]. Recently, the genome of A. cerana was sequenced using next-generation technology, and two chemoreceptor families, ORs and IRs, were characterized in the transcriptome data [30].
Apis cerana cerana is an economically important species indigenous to China. Compared to A. mellifera, A. c. cerana possesses unique characteristics such as using sporadic resources of nectar and pollen, good foraging ability, and strong resistance to parasitic mites. As a consequence of these excellent characteristics, there is considerable interest in the olfactory system of the Asian bee. Many olfactory genes have been identified, particularly OBP and CSP genes [31–34]. Previously, we cloned three OR genes and one OBP gene from A. c. cerana and described their expression characteristics [35–37].
Worker bees undertake different tasks in and outside the hive across their life span. Such variation in their behavioral repertoires at different times of the lifecycle is likely regulated by different expression patterns of many genes, including the olfactory genes. In this study, we investigated the antennal chemosensory gene families and compared their expression patterns at different developmental stages in A. c. cerana using an Illumina RNA-Seq approach. Overall, 109 chemosensory genes were identified including 17 OBPs, 6 CSPs, 74 ORs, 10 IRs, and 2 SNMPs. Differentially expressed genes (DEGs) were screened and their spatio-temporal expression profiles were analyzed in combination with quantitative real-time PCR. The aim of this study was to explore the relationship between chemosensory genes and the division of labor of worker bees. The results clearly showed an age-biased expression of some chemosensory genes and ultimately allowed us to identify potential targets in production management of the colony. The data also provide a valuable resource for further genetic and molecular studies on the olfactory recognition mechanism of honeybees.
Materials and Methods
Insects and tissue samples
Honeybees (A. c. cerana) were sampled from March to July from an apiary of Shanxi Agricultural University, China. Frames with bee pupae close to eclosion were taken from two hives and incubated in an environmental chamber at a constant temperature of 33°C and80% humidity. After eclosion, the workers were marked and returned to their hives until sampling. For transcriptome sequencing, the antennae of about 200 worker bees from two hives at 1, 10, 15, and 25 days of age were dissected, respectively. The major body parts (antenna, head, thorax, abdomen, legs, and wings) of 10-day-old worker bees were separated for qPCR analysis.
RNA-Seq library construction and sequencing
Samples were collected, and immediately frozen in liquid nitrogen and then stored at -80°C until extraction. Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Residual DNA in the total RNA was removed using DNase (Promega, Madison, WI, USA). First-strand cDNA was synthesized using reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Eight cDNA libraries from four developmental stages were processed for RNA-Seq analysis independently using the reagents provided in the Illumina sequencing kit according to the manufacturer’s instructions. Finally, the eight cDNA libraries were sequenced using the Illumina HiSeq 2500 platform at Biomarker Technologies Co., Ltd (Beijing, China).
Assembly
The raw reads were cleaned by removing the adapter sequences and low-quality sequences (reads with ambiguous bases ‘N’, and reads with more than 10% Q <20 bases).Clean reads were then mapped back to contigs using Trinity software, with the parameters set at a similarity of 90% [38]. Next, by performing pair-end joining and gap filling, contigs were assembled into transcripts and were subsequently clustered to obtain unigenes. In this study, all Illumina sequencing data was submitted to the SRA of NCBI (accession number:SRR3180625).
Gene annotation and chemosensory gene identification
Unigenes longer than 200 bp were first aligned by BLASTX against the non-redundant protein database, including Nr, Swiss-Prot, KEGG, GO, and COG at a threshold E-value of 10-5.Functional annotation was performed by retrieving proteins with the highest sequence similarity with the given unigene. Then, GO terms were assigned using the Blast2GO algorithm [39]. The transcripts were categorized as cellular component, molecular function, or biological process, allowing for a general quality assessment.
To identify the putative chemosensory genes of A. c. cerana, sequences whose annotations corresponded best to OBPs, CSPs, ORs, IRs, or SNMPs were retained as candidate genes. Next, these genes were manually revised by further analysis using the BLASTN algorithm in the non-redundant nucleotide database acquired at NCBI. ORs were further verified in a custom-made database of known A. mellifera query protein sequences [29].
The ORFs of the candidate chemosensory genes were determined using the ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Putative N-terminal signal peptides of candidate OBPs and CSPs were predicted by the program Signal IP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) [40]. Transmembrane domains of novel candidate ORs, IRs, and SNMPs were determined using TMHMM server v2.0 [41]. Phylogenetic trees were constructed based on the amino acid sequences of putative A. c. cerana chemosensory genes and homologous sequences reported in other hymenopteran species. Amino acid sequences were aligned using Clustal W, then unrooted neighbor-joining trees were constructed using MEGA6.0 [42] and the branch support was assessed with 1000 bootstrap replications. Dendrograms were viewed and graphically edited in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).
Identification of chemosensory DEGs and gene expression analysis
To compare gene expression levels between different libraries, the expression abundance of all unigenes were calculated by the RPKM (reads per kilobase of transcript per million mapped reads) algorithm [43]. DEGs in the 8 libraries were identified via a rigorous statistical algorithm based on Audic and Claverie’s method [44]. A threshold for FDR (false discovery rate) of <0.01and an absolute expression value of log2FC (fold change) ≥1 were used to determine significant differences in gene expression. As a result, we acquired all DEGs related to olfaction and their RPKM values.
To verify the expression values of the chemosensory DEGs identified from our transcriptome and to further investigate their tissue expression profiles, qPCR was performed. Specific primers (S1 Table) were designed using primer3plus (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi). qPCR was performed using aSYBR®Premix Ex Taq™ Kit(Takara, Dalian, China), which was run on a Mx3000PqPCRSystem (Stratagene, La Jolla, CA,USA). The cycling conditions were as follows: denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 15 s, and 60°C for 20 s. A melting curve analysis was then performed at 95°C for 20 s, 60°C for 30 s, and 95°C for 30 s in order to judge the specificity of the PCR products.
Three biological replicates were performed for each tested gene, and three technical replicates were performed for each biological replicate. Negative controls were non-template reactions (replacing cDNA with ddH2O). Relative quantification was analyzed relative to the control gene Arp1 using the comparative 2-ΔΔCt method [45].
Results and Discussion
Summary of transcriptome sequencing data
Illumina HiSeq RNA-Seq technology has become popular over the last decade because of its advantages of higher throughput and lower cost. This approach has been used in an increasing number of studies of different insect species, to identify total genes expressed in specific cells or tissues; determine relative expression abundance; discover SNP sites at the transcriptional level in different transcripts and alternative splice sites of specific genes; reveal genetic polymorphism and genetic markers (SSRs); and detect unknown genes and new transcripts.
In this study, data on the antennal transcriptome of A. c. cerana was generated using Illumina HiSeq 2500 technology. After filtering the raw reads, 8 high-quality unigene libraries were established, composed of 184.04M clean reads, and in each library the Q30>90% (see S2 Table). These clean reads were assembled into 4,235,071 contigs. After merging and clustering, 125,072 unigenes were acquired with a mean length of 760 nt and an N50 length of 1,151 nt. Approximately 43% of these unigenes (53,576) were more than 500nt in length (see S3 Table). Each unigene was given a unique gene ID.
Gene ontology annotation
Through annotation by BLASTX in 5 protein databases, 16,762 unigenes were matched to known proteins. GO annotation was used to classify the transcripts into functional groups according to GO category. One transcript could align to more than one biological process; therefore, a total of 34,051 unigenes were assigned into three main GO terms (Fig 1): 14,575 unigenes were aligned in the cellular component category, 10,541 in the molecular function category, and 19,757 in the biological process category. In these categories, some subcategories had high percentages, such as cell (20.21%) and cell part (20.25%) in the cellular component category, binding (41.61%) and catalytic activity (35.56%) in the molecular function category, and cellular process (23.24%) and metabolic process (23.07%) in the biological process category, suggesting that transcripts in the antennae of A. c. cerana cover a wide spectrum of biological processes.
The unigenes were assigned into three main categories: cellular component, molecular function, and biological process. The three main categories were categorized further to 47 subcategories using the Blast2GO program.
Putative chemosensory gene families and their expression profiles
The molecular basis of chemoreception in the Asian honeybee is not as well understood as that of its sibling species, the Western honeybee (A. mellifera). In this study, we primarily focused on five crucial repertoires of chemosensory genes (OBPs, CSPs, ORs, IRs, and SNMPs). Candidate genes were identified by keyword searches and manually analyzing the annotated unigenes. In addition, we also constructed a local nucleotide sequence database of the RNA-Seq unigenes, in which ORs and IRs were also queried based on similar sequences of A. mellifera [14, 29]. Using this method, we identified 109 chemosensory genes in the antennal transcriptome of A. c. cerana, including 17 OBPs, 6 CSPs, 74 ORs, 10 IRs, and 2SNMPs. These candidate chemosensory unigenes were named by a four-letter species abbreviation in accordance with established conventions. Gene names were preferentially given the same name when they were orthologous to a sequence in A. mellifera (e.g.,AcerOBP1, AcerIR8a). Of these transcripts, 91 unigenes had full length ORFs. Sequence accuracy was verified by conventional PCR and sequencing on 10 selected unigenes (data not shown). In phylogenetic trees, almost all candidate chemosensory genes clustered with at least one A. mellifera (S1–S5 Figs).In the olfactory receptor gene families, we identified the coreceptor gene OR2 in the OR family and IR25a and IR8a in the IR family cluster along with the respective orthologs of other hymenopteran species. In the OBP family dendrogram, members of the Minus-C class of A. c. cerana and A. mellifera were clustered into a clade, with their second and fifth cysteine residues missing. Other information tied to these unigenes, including the gene name, amino acid sequence length, transmembrane domains, signal peptide, and sequence similarity are listed in Tables 1–5.
Expression values of the 109 candidate chemosensory unigenes in the 8 different libraries were calculated using the RPKM metric, and their RPKM values are shown in Tables 1–5. The expression correlation between repetitive samples is the most important indicator for rationality and reliability of the RNA-Seq results. In general, the correlation value should be greater than 0.92 (R2≧0.92). In our study, the scatter plot showed that the R2 between each group of two biological replicates was more than 0.98 (see S4 Table). The RPKM value analysis showed that nearly all of the chemosensory genes were expressed in each library and no unique genes belonged to any library, showing no age bias in chemosensory gene expression. Among the 109 putative genes, 5 OBP genes (OBP1, OBP2, OBP5, OBP6, and OBP21) and 2 CSP genes(CSP1 andCSP3)were highly expressed in the worker antennal transcriptomes (RPKM values>1000);OBP1 had the highest expression levels. At the same time, we found that the other three gene families (ORs, IRs and SNMPs)exhibited relatively low expression levels across the whole developmental stages (RPKM values<100), except those of OR2, IR218, and SNMP1.These results were similar to the transcriptome data of Agrotis ipsilon [46]. This observation suggested that these antennae-enriched OBP and CSP proteins might play essential roles in olfactory sensory processes for worker bees and that the insects might use a large number of odor binding proteins to monitor these small molecules.
Homology analysis with the sibling species A. mellifera
A. mellifera was the first hymenopteran species to have its genome sequenced, and several chemosensory protein families were found and reported. In this study, the same number of CSPs, IRs and SNMPs were identified in A. c. cerana as in A. mellifera, whereas the number of ORs and OBPs were relatively fewer. By repeated comparison and analysis, it was found that the missing genes were not due to the lack of expression in the antennae of A. c. cerana, but to the limitation of denovo RNA-Seq. Since some superfamilies have undergone duplications or alternative splicing during the evolutionary process, this may have produced many genes highly homologous to each other, including the ORs. However, the unigenes with high homology were merged and clustered to single sequence during the de novo assembly process. Thus, the number of annotated genes will be less than the actual number. This problem occurs prominently in gene superfamilies.
In order to investigate the conservatism of these putative chemosensory genes, sequence similarity analysis was performed with orthologs from A. mellifera. In summary, OR family genes were highly divergent, with sequence identities varying from 59% to 100% (Table 3), which is common for insect olfactory receptor genes [12, 47, 48]. In addition, the OBP family also displayed high sequence variation with sequence identities varying from 65% to 98% (Table 1).OBP9 and OBP13 shared the highest identity of with their homologsAmelOBP9 and AmelOBP13, whereasOBP19 showed a low level of sequence similarity with AmelOBP19.
Compared with ORs and OBPs, the other two olfactory gene families (IRs and CSPs) were more highly conserved in insects, and their number was generally consistent [7, 14]. In our study, we identified 10 putative IRs and 6 CSPs that shared greater than 90% sequence identity compared to the orthologs of A. mellifera (see Tables 2 and 4).
SNMPs are another chemosensory protein family that is conserved throughout holometabolous insects [28]. In our study, two SNMP genes were also highly similar to the orthologs of A. mellifera (>94%) (see Table 5).
Chemosensory DEGs and their expression profiles
After estimating gene expression levels, we performed a DEG analysis between two of the eight samples. Using the Audic and Claverie method [44], a total of 1,052 DEGs were detected, in which 19chemosensory-related genes were screened, including 7 OBPs, 3 CSPs, 6 ORs, 2 IRs, and 1SNMP (see Tables 1–5). These 19 DEGs were discovered in all eight libraries. To show the expression patterns of these DEGs at each developmental stage, a heatmap was constructed on the basis of the RPKM values (Fig 2). Based on the heatmap, the correlation between biological replicates was well established, and all the correlation coefficients exceeded 0.9 (P<0.05), supporting the reliability of the RNA-Seq data. We were surprised to discover that the expression levels of the 19 chemosensory genes changed significantly at the T1 period(for both T1-1 and T1-2) compared to those of the other three periods, and all the DEGs were generated from T1.This may be attributable to the fact that the newly emerged bees have undergone great changes as a consequence of physical or environmental factors, or that they may be more sensitive to odorant molecules than the adults. On the one hand, among the 19 DEGs, 6 were downregulated (including all 3 CSPs, 2 OBPs and 1 IR), in which OBP13, OBP17, CSP5, and CSP6showed great changes. The remaining13DEGs were upregulated (including all 6 ORs, 5 OBPs, 1 IR, and 1 SNMP), in which OBP7 and OBP15changed greatly. Presumably, these downregulated olfactory genes were related to the stimulatory influence of environmental variation, while the upregulated genes were connected with working behavior.
Clustering of genes is shown on the left of the figure, and the transcript ID and gene name are shown on the right. The color from red to green indicates high to low expression levels.
We speculate that there may be synergistic effects among the up- or down-regulated genes, but the specific mechanism still requires further in-depth analysis. In contrast to T1, the level of expression in the other three developmental periods did not vary significantly. In other words, expression of all olfactory-related genes in adult bees remained relatively stable. These data imply that one olfactory gene may act on specific odor molecules; this ability was relatively stable in adult bees and did not change dramatically with the variation of individual behavior and the outer environment.
Nine DEGs were randomly chosen for the purpose of validating the results of the Solexa sequencing using qPCR. The qPCR results showed that the expression pattern of 8 ofthe9 selected genes was consistent with that of the RNA-Seq analysis (S6 Fig);these results demonstrated that the quality of Solexa sequencing was reliable. In addition, we used qPCR in order to obtain more expression information about the19 DEGs in different tissues. Five genes (OBP14,OR28, OR113, OR167,and IR76b) were highly expressed in antennae. Eight genes (OBP7, OBP12, OBP15, CSP5, CSP6, OR119, IR218, and SNMP2) were relatively enriched in antennae. Two genes (OR139 and OR141) were highly expressed in both antennae and head. Other two genes (OBP17and CSP2) showed higher expression in thorax. OBP21 was expressed more in legs, and OBP13 was expressed more in wings (Fig 3). An interesting feature of the expression profiles was that when a gene was highly expressed in the thorax, it was also highly in legs, but was weakly in antennae.
The X-axis shows different tissues. The Y-axis shows the relative mRNA expression level. An, antennae; H, head; T, thorax; Ab, abdomen; L, legs; W, wings. The standard error is represented by the error bars above the columns.
The expression profiles of OBPs and CSPs involved in sex, tissue and age in the honeybee have been investigated in previous studies. Due to the different materials selected in different studies, it is hard to perform a detailed comparison among the different studies. Here we compared the tissue-related expression patterns of OBPs and CSPs between this study and previous reports. The expression patterns of OBP7, OBP13, OBP15 andOBP17 were similar to the data of Forêt and Maleszka [26]. However, some results showed inconsistencies; for example, OBP12 was identified in the antennae in this study, but was only detected in the queen ovaries of A. mellifera [26]. This suggests that OBP12 may play a different physiological role in the two species. With respect to CSPs, CSP5 and CSP6 were reported to be relatively highly expressed in antennae [34], in agreement with our data. This result indicates that CSP5 and CSP6 may be involved in chemical sensory activities. Additionally, there were evident differencesinCSP2 between this study and that of Li and coauthors [34], while our data was similar with the expression pattern in A. mellifera [27].
Our analyses revealed two main categories of expression patterns within these genes. One pattern showed that most DEGs were abundantly expressed in the antennae; again verifying that the antennae are the major olfactory organ of insects [49]. Other patterns revealed that some genes were enriched in other non-chemosensory organs, such as the thorax, legs, and wings. This expression pattern indicates that some chemosensory sensillae are located on other body parts, such as on the thorax stegma, the leg tarsi, and the wing margin, or that these genes participate in physiological processes other than chemoreception [50–52].
In addition, we found that all OR DEGs were 5 to 50 times more enriched in the antennae, compared with the thorax, abdomen, legs, and wings, which was similar to the expression pattern of other hymenopteran ORs, such as A. mellifera [29], Cotesia vestalis [53], and Microplitis mediator [54]. Our expression profiling results suggest that the ORs of insects are highly restricted in the antennae and may play important and special role in olfactory recognition.
Conclusion
In this study, we not only identified 109 putative chemosensory genes of A. c. cerana, acquiring19 DEGs for different developmental stages by antennal transcriptome analysis, but also investigated expression profiles of these DEGs. As a result, we obtained worthy information. This research serves as a valuable resource in the identification of characteristics on the olfactory recognition mechanism of A. c. cerana, making it possible for further research on functional analyses of these genes.
Supporting Information
S1 Fig. Phylogenetic tree of candidate AcerOPBs with other hymenopteran OBPs.
Acep, Atta cephalotes; Acer, Apis cerana; Amel, Apis mellifera; Cflo, Camponotus floridanus; Evir, Euglossini viridissima; Mmed, Microplitis mediator. The clade in blue indicates the Minus-C class of A. c. cerana and A. mellifera.
https://doi.org/10.1371/journal.pone.0165374.s001
(TIF)
S2 Fig. Phylogenetic tree of candidate AcerCSPs with other hymenopteran CSPs.
Acep, Atta cephalotes; Acer, Apis cerana; Amel, Apis mellifera; Cflo, Camponotus floridanus; Evir, Euglossini viridissima.
https://doi.org/10.1371/journal.pone.0165374.s002
(TIF)
S3 Fig. Phylogenetic tree of candidate AcerORs with other hymenopteran ORs.
Acer, Apis cerana; Amel, Apis mellifera; Nvit, Nasonia vitripennis. The clade in blue indicates the Orco class.
https://doi.org/10.1371/journal.pone.0165374.s003
(PDF)
S4 Fig. Phylogenetic tree of candidate AcerIRs with other hymenopteran IRs.
Acep, Atta cephalotes; Acer, Apis cerana; Amel, Apis mellifera; Cflo, Camponotus floridanus;Nvit, Nasonia vitripennis. The clade in blue indicates the IR8a/IR25a class.
https://doi.org/10.1371/journal.pone.0165374.s004
(TIF)
S5 Fig. Phylogenetic tree of candidate AcerSNMPs with other hymenopteran SNMPs.
Acep, Atta cephalotes; Acer, Apis cerana; Ador, Apis dorsata; Aflo, Apis florea; Amel, Apis mellifera; Bter, Bombus terrestris; Hlab, Habropoda laboriosa; Hsal, Harpegnathos saltator; Pcan, Polistes canadensis.
https://doi.org/10.1371/journal.pone.0165374.s005
(TIF)
S6 Fig. Comparison diagrams between the RPKM values and the qPCR results of 9 DEGs.
https://doi.org/10.1371/journal.pone.0165374.s006
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S1 Table. Primers used for qRT-PCR analysis on DEGs.
https://doi.org/10.1371/journal.pone.0165374.s007
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S2 Table. Evaluation statistical table of high quality sequencing data.
https://doi.org/10.1371/journal.pone.0165374.s008
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S3 Table. Distribution of unigene size in the transcriptome assembly.
https://doi.org/10.1371/journal.pone.0165374.s009
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S4 Table. Correlation statistics between biological replicate samples.
https://doi.org/10.1371/journal.pone.0165374.s010
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Acknowledgments
We would like to acknowledge the experimental help by the other members of the Lab, Shuang Yang, Kai Xu, and Lina Guo. We also would like to thank Editage forthe language editing.
Author Contributions
- Conceptualization: HZ YJ.
- Data curation: HZ YD SW JP.
- Formal analysis: HZ PG.
- Funding acquisition: HZ YJ.
- Methodology: HZ YJ.
- Resources: YJ.
- Supervision: YJ.
- Validation: YD SW JP.
- Visualization: HZ PG.
- Writing – original draft: HZ.
- Writing – review & editing: YJ PG.
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