RNA sequencing and de novo transcriptome assembly

Comprehensive transcriptome analysis of R. speratus was performed using high-throughput mRNA sequencing in the previous study [40]. To compare the expression levels of R. speratus immune-related genes among castes, ages, and between sexes, we analyzed transcriptomic database of both male and female alates, young primary kings (PKs) and primary queens (PQs), mature PKs and secondary queens (SQs), both male and female soldiers, and both male and female workers [40]. Alates were collected from three colonies in secondary forests in Kyoto, Japan, in the swarming season from April to May 2013. The alates were separated by sex, and then a male and female were randomly selected from each colony; five nestmate pairs were made for each colony and kept at 25°C under continuous darkness. After 6 months, male and female alates became young PKs and PQs, respectively, and then they were extracted from the incipient colonies. Kings and queens of R. speratus were collected from mature termite colonies in secondary forests in Kyoto and Shiga, Japan, during the reproductive season from July to October 2013. We found royal chambers of these colonies and extracted mature PKs and SQs together with workers and soldiers. Male and female workers and soldiers were separated based on the caudal sternite configuration using a stereoscope. No specific permits were required for the described field studies and no specific permissions were required for the locations/activities for insect sampling because they are public lands and are not privately owned or protected in any way. These field studies did not involve endangered or protected species.

Total RNA was extracted from the whole body of each individual of each reproductive caste (alates, kings, and queens) using an RNeasy mini kit (Qiagen), using the standardized instructions from the manufacturer. As for workers and soldiers, we pooled 10 individuals of each sex to extract a sufficient amount of RNA, and we performed RNA-seq analysis on a total of 60 samples. Following previously described procedures [41] for first-strand cDNA synthesis, RNA sequencing was performed using the Illumina HiSeq 2000 at the Okinawa Institute of Science and Technology Graduate University. For further details of the methods, see Mitaka et al. (2016) [40]. Sequence data was deposited in the DNA Data Bank of Japan (DDBJ) under the BioProject PRJDB3531, which contains links and access to sampling data through the BioSample SAMD00026264—SAMD00026323 and the Sequence Read Archive DRR030795—DRR030854.

The raw sequencing reads were trimmed by removing adapter sequences. Preprocessing was performed in DDBJ Read Annotation Pipeline. Bases with a quality score of < 20 were trimmed from the 5’ and 3’ ends of each read. After the trimming, reads with a high percentage (> 30%) of low quality bases (< 15) and short reads (< 25 bp) were discarded, and the remaining reads were used for an assembly. The remaining reads from all of the samples were assembled de novo using Trinity version trinityrnaseq_r2012–04–27 [42,43], which generates transcriptomic assemblies from short read sequences using the de Bruijn graph algorithm. All parameters selected to run Trinity were default parameters (k-mer length = 25-mers) except max_reads_per_loop, which was set to 1,500,000. Illumina sequencing yielded a total of 729 M read pairs with a mean length of 93 bp for each short read, and the GC content was 41%. The transcriptome yielded a total of 856 Mbp of RNA sequences, 1,144,272 contigs with a minimum length of 201 bp, a maximum length of 35,267 bp, a mean length of 748 bp, and a N50 value of 1,296 bp [40].

We also searched open reading frames (ORFs) in the assembled sequences using transcriptsToOrfs version 0.0.2 and detected 156,276 ORFs with a total length of 76,684,150 amino acids (aa), a minimum length of 100 aa, a maximum length of 11,369 aa, a mean length of 490.70 aa, and a N50 length of 584 aa. Based on the subsequent homology search of the known amino acid sequence data from a termite (Zootermopsis nevadensis), an aphid (Acyrthosiphon pisum), a honeybee (Apis mellifera), a beetle (Tribolium castaneum), a silkmoth (Bombyx mori), and a fruit fly (Drosophila melanogaster), 10,238 protein-coding transcripts were inferred in R. speratus [40].

Annotation of immune-related proteins

Targets of immune-related genes were selected based on a previous study investigating immune-inducible genes in C. formosanus [29]. Pattern recognition proteins (PRPs) included C-type lectins (CTLs), lipopolysaccharide-binding proteins (LPSBPs), agglucetins, brevicans, laminins, GNBPs, apolipophorin III, PGRPs, and endo-β-1,4-glucanase. Signalling proteins included SPs, SPIs, and prophenoloxidase activating factor, 14-3-3 proteins, calpains, minor histocompatibility antigens (MHAs), LRPs, and FHLs. Also, effectors contained antimicrobial proteins such as carboxypeptidases (CPases), cathepsins, lysozymes, metacaspase-like cysteine peptidases (MCA-like CPs), asparaginyl endopeptidase-like cysteine peptidase (AE-like CPs), lysosomal Pro-X carboxypeptidases, prolixicin antimicrobial proteins, transferrins, and termicins, cysteine-rich proteins (CRPs), ferritins, melanotransferrins, venom allergens, and thaumatin-like proteins.

Peptide sequences of immune-related proteins of the following species were obtained from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), and used as BLAST queries for our peptide database with an E-value cutoff of 1E−20: Zootermopsis nevadensis, Cryptotermes secundus, C. formosanus, R. flavipes, R. chinensis, Nasutitermes comatus, Periplaneta americana, Eupolyphaga sinensis, Tribolium castaneum, Apis mellifera, Bombyx mori, and Drosophila melanogaster (S1 Table).

Abundance estimation and differential expression analyses

Expression levels of immune-related genes were estimated by RSEM version 1.2.8 software [44] separately for the filtered reads from each sample. Raw read counts generated by RSEM were normalized with the trimmed mean of M-value normalization method [45]. Subsequently, these read counts were used for differential expression analyses among castes and ages, and between sexes using the edgeR software package v3.4.2 [46]. All statistical analyses were performed using the R package v3.0.3 and heatmaps were generated with heatmap.2 in the gplots package.

Pattern recognition proteins

CTLD-containing proteins include CTLs, LPSBPs, agglucetins, brevicans, and laminins [47]. Through a BLAST search querying the amino acid sequences of various insect species (S1 Table), we found 26 CTLD-coding genes (Table 1). Among these genes, the expression levels of six genes (brevican1, CTL7, and CTL11–14) in soldiers were more than 1.2 times as high as those in other castes (false discovery rate (FDR) < 0.05; Fig 1 and S2 Table). The expressions of CTL17 and LPSBP4 in young reproductives (PKs and PQs) were more than twice as high as those in other castes, and those of CTL9 and LPSBP1 in young reproductives, mature reproductives (PKs and SQs), and soldiers were more than 1.3 times as high as those in alates and workers (FDR < 0.05; Fig 1 and S2 Table). Additionally, the expression levels of 16 genes differed significantly between reproductive statuses, i.e. reproductive castes (alates, young PKs and PQs, and mature PKs and SQs) and neuter castes (soldiers and workers), and 12 genes showed sexually dimorphic expression patterns regardless of reproductive status (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). Comparing the expression levels between sexes in each caste, the expression of four genes (brevican 1, CTL10, CTL17, and laminin 1) in young PQs were more than twice as high as those in young PKs, but two other genes (CTL7 and CTL9) showed the opposite pattern (FDR < 0.05; S3 Table). CTL6, CTL17, and LPBP2 were more highly expressed in mature SQs than mature PKs, but CTL9 showed the opposite pattern (FDR < 0.05; S3 Table). Also, CTL10 and CTL14 expression levels in female soldiers were more than 2.5 times as high as those male soldiers, and CTL3 showed male-biased expression in workers (FDR < 0.05; S3 Table). Among male reproductives (male alates and young and mature PKs), CTL12 expression showed the highest level in alates. However, the expression levels of 8 genes (CTL7, CTL9, CTL11, CTL13, CTL15, and LPSBP4–6) peaked in young PKs, and those of 5 genes (CTL1, CTL4, CTL16, LPBP2, and laminin1) peaked in mature PKs (FDR < 0.05; S4 Table). In female reproductives (female alates and young PQs), brevican 1 and CTLD4 expression levels in alates were more than 1.2 times as high as those in young PQs, but the expressions of 9 other genes (CTL11, CTL13, CTL15, CTL16, LPSBP1, LPSBP4, LPSBP5, LPSBP6, and laminin 1) in young PQs were more than three times as high as those in alates (FDR < 0.05; S4 Table).

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Table 1. Number of caste-, sex-, and age-specific immune-related genes.

https://doi.org/10.1371/journal.pone.0175417.t001

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Fig 1. Differential expressions of CTLDs, GNBPs and PGRPs among castes.

These heatmaps indicate the differential expression of 29 genes belonging to the CTLD superfamily, the GNBP family and PGRPs among termite castes (PQ: primary queen, PK: primary king, SQ: secondary queen). Relative expression level indicates the mean normalized count per million (CPM), ranging from black (scaled expression of 0) to white (scaled expression of 2.5). The tree at the left corresponds to hierarchical clustering of cluster-averaged expression. Numbers in parentheses after caste names refer to the numbers of biological replicates. Ten individuals were pooled for each sex of worker and soldier to obtain sufficient amounts of RNA, while single individuals were used for RNA extraction in the other castes. CTL: C-type lectin-like domain protein, LPSBP: lipopolysaccharide-binding protein, GNBP: Gram-negative binding protein, PGRP: peptidoglycan recognition protein.

https://doi.org/10.1371/journal.pone.0175417.g001

Seven PGRPs were annotated (Table 1 and S1 Table). Five genes (PGRP I-alpha, PGRP LB, PGRP LE1, PGRP SC1, PGRP) showed caste-specific expression patterns; for example, the expression levels of PGRP LB and PGRP SC1 in soldiers were more than twice as high as that in other castes (FDR < 0.001, Fig 1 and S2 Table). Comparing the expression level between female and male in each caste, only PGRP SC1 showed the king-biased expression pattern in mature reproductives (FDR < 0.001, S3 Table). Also, the expression levels of three genes (PGRP LB, PGRP LE1 and PGRP SC1) significantly differed between reproductive and neuter castes, and four genes (PGRP I-alpha, PGRP LE1, PGRP SC1 and PGRP SC2) showed sexually dimorphic expression regardless of reproductive status (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.05, S5 Table). In male reproductives, the expression levels of four genes (PGRP I-alpha, PGRP LB, PGRP SC2 and PGRP SD) rose through the king’s life (FDR < 0.001, S4 Table). In female reproducitves, the expressions of two genes (PGRP I-alpha and PGRP) rose through the queen’s life, but that of PGRP SC1 showed the opposite pattern (FDR < 0.001, S4 Table).

Three apolipophorin III genes were predicted for R. speratus (Table 1 and S1 Table), and apolipophorin III-1 expression was higher in both young and mature reproductives than in other castes. The remaining two apolipophorin III genes were expressed the highest in young reproductives (FDR< 0.05; S1 Fig and S2 Table). Apolipophorin III-1 and 3 showed differential expression levels between reproductive and neuter castes (FDR < 0.05, Table 1 and S5 Table). The expression level of apolipophorin III-3 in mature SQs was more than twice as high as that in mature PKs (FDR < 0.05, S3 Table). Also, in male reproductives, apolipophorin III-1 expression showed the highest level in mature PKs, and apolipophorin III-2 and 3 showed in young PKs (FDR < 0.05; S4 Table). In contrast, in female reproductives, apolipophorin III-1 and 2 expression levels in female alates were seven times as high as young PQs (FDR < 0.05; S4 Table).

An endo-β-1,4-glucanase was also annotated (Table 1), and it showed high sequence similarity to that of C. formosanus (Identities = 96%; S1 Table). Although this gene was expressed in alates, young reproductives, and soldiers (FDR < 0.05; S3 Fig and S2 Table), it was little expressed in mature reproductives and soldiers in comparison to other castes. Also, this gene showed expression differences between reproductive statuses and between sexes (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). This gene was expressed higher in young PQs than in young PKs (FDR < 0.05; S3 Table). The expression level decreased with age in male reproductives (FDR < 0.01; S4 Table), but increased with age in female reproductives (FDR < 0.05, S4 Table).

Three GNBP genes were annotated (Table 1 and S1 Table). The expression level of GNBP3 in soldiers was more than 1.5 times as high as those in other castes (FDR < 0.001; Fig 1 and S2 Table). Also, the expression levels of GNBP1 and GNBP3 differed between reproductive and neuter castes, and GNBP1 and GNBP2 showed sexually dimorphic expression patterns regardless of reproductive status (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). In male reproducitve castes, the expression of GNBP1 peaked in young PK, and those of GNBP2 and GNBP3 peaked in mature PK (FDR < 0.001, S4 Table). In female reproducitve castes, the expressions of GNBP1 and GNBP3 rose through the PQ’s life (FDR < 0.001, S4 Table).

Signalling proteins

Seventy SPs were annotated through a BLAST search against sequences from various insects (Table 1 and S1 Table). Fourteen SPs (SP2, SP9, SP10, SP13, SP21, SP22, SP33, SP36, SP44, SP50, SP51, SP53, SP56, and SP70) showed the highest expression levels in young reproductives, 11 SPs (SP3, SP7, SP8, SP26, SP28, SP30, SP37, SP38, SP43, SP57, and SP69) in soldiers, five SPs (SP4, SP14, SP18, SP58, and SP62) in mature reproductives, and five SPs (SP15, SP25, SP34, SP49, and SP55) in workers (FDR < 0.05; Fig 2 and S2 Table). Thirty-eight genes showed differential expression between reproductive statuses, and 23 genes showed sexually dimorphic expression (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). When comparing expression levels between sexes within each caste, significant sexually dimorphic expression was found for four SPs in alates (female > male: SP18, SP41, and SP62; male > female: SP66; FDR < 0.05; S3 Table), 10 SPs in young reproductives (PQ > PK: SP4, SP11, SP18, SP41, SP44, and SP62; PK > PQ: SP37, SP46, SP63, and SP66; FDR < 0.01; S3 Table), 12 SPs in mature reproductives (SQ > PK: SP4, SP14, SP18, SP41, SP48, SP58, and SP62; PK > SQ: SP2, SP20, SP32, SP63, and SP66; FDR < 0.05; S3 Table), and six SPs in soldiers (female > male: SP46 and SP69; male > female: SP1, SP3, SP10, and SP70; FDR < 0.05; S3 Table). In male reproductives, while SP21 expression peaked in male alates (FDR < 0.05; S4 Table), the expressions of 32 SPs (SP4, SP5, SP9, SP10, SP13, SP16, SP23, SP25, SP27, SP28, SP36, SP38, SP42–46, SP48–54, SP57, SP58, SP61, SP64–66, SP69, and SP70) peaked in young PKs, and 22 SPs (SP6, SP7, SP11, SP12, SP15, SP18–20, SP30–35, SP37, SP39, SP40, SP47, SP55, SP60, SP62, and SP68) peaked in mature PKs (FDR < 0.05; S4 Table). In female reproductives, the expression levels of SP14, SP62, and SP63 peaked in alates, but those of 38 other SPs (SP2, SP5–7, SP9, SP10, SP12, SP13, SP15, SP18–20, SP23, SP25, SP27, SP28, SP30, SP31, SP33–36, SP40, SP42, SP44, SP45, SP47–49, SP51–55, SP57, SP64, SP65, and SP68) peaked in young PQs (FDR < 0.05; S4 Table).

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Fig 2. Differential expression of serine proteases among castes.

The heatmap indicates the differential expression of 70 serine protease transcripts among castes. Abbreviations are as indicated in Fig 1. Relative expression level indicates the mean normalized CPM, ranging from black (scaled expression of 0) to white (scaled expression of 3.0). The tree at the left corresponds to hierarchical clustering of cluster-averaged expression.

https://doi.org/10.1371/journal.pone.0175417.g002

Seven SPIs were annotated (Table 1 and S1 Table). While Kazal-type SPI domain-containing protein 1 and SPI2 showed the highest expression in soldiers, SPI3, dipetalogastin 1, and dipetalogastin 2 showed in young reproductives, and SPI4 showed in young reproductives and soldiers (FDR < 0.05; S1 Fig and S2 Table). SPI1 was expressed in all castes except for alates (FDR < 0.01; S1 Fig and S2 Table). Four genes exhibited differential expression between reproductive and neuter castes, and one gene exhibited sexually dimorphic expression (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). Comparing between young reproductives, the expression of dipetalogastin 1 was slightly higher in PKs than that in PQs, but the expression of dipetalogastin 2 in PQs was twice as high as that in PKs (FDR < 0.01; S3 Table). In male reproductives, the expression level of SPI1 increased with age, but those of SPI3, SPI4, and dipetalogastin 1 peaked in young PKs (FDR < 0.01; S4 Table).

One prophenoloxidase activating factor was annotated (Table 1 and S1 Table) and was highly expressed in young reproductives and soldiers than other castes (FDR < 0.01; S3 Fig and S2 Table). The expression level of this gene differed between reproductive and neuter castes (FDR < 0.05, Table 1 and S5 Table). In both male and female reproductives, its expression exhibited the highest level in young reproductives among all age classes (FDR < 0.01; S4 Table).

Three 14-3-3 proteins were annotated (Table 1 and S1 Table), of which the expression of 14-3-3 protein 1 in young reproductives was 1.5 times as high as that in other castes, and the expression of 14-3-3 protein 2 in both young reproductives and soldiers was twice as high as that in other castes (FDR < 0.01; S2 Fig and S2 Table). The expression level of 14-3-3 protein 2 differed significantly between reproductive and neuter castes (FDR < 0.05, Table 1 and S5 Table). Comparing the expression level between sexes, 14-3-3 protein 3 expression was doubly higher in young PQs and mature SQs than young and mature PKs, respectively (FDR < 0.05; S3 Table). In male reproductives, all genes were the most highly expressed in young PKs among all age classes, but in female reproductives, 14-3-3 protein 1 and 14-3-3 protein 2 were expressed at the highest levels in young PQs (FDR < 0.01; S4 Table).

Five calpains were annotated (Table 1 and S1 Table). Calpain 1 showed the highest expression in young reproductives among all castes (FDR < 0.01; S1 Fig and S2 Table). While the expression levels of calpain 2 and calpain 3 were higher in young reproductives and soldiers than other castes, that of calpain 4 was higher in young and mature reproductives (FDR < 0.01; S1 Fig and S2 Table). Calpain 5 expression showed the highest level in mature reproductives (FDR < 0.01; S1 Fig and S2 Table). Calpains 2 and 4 showed significant expression differences between reproductive and neuter castes, and calpain 4 expression was sexually dimorphic (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). Only the expression level of calpain 3 in young PQs was three times as high as that in young PKs (FDR < 0.01; S3 Table). Among all age classes in male reproductives, the expression of calpain 1 peaked in young PKs, but the expressions of calpain 4 and calpain 5 peaked in mature PKs (FDR < 0.01; S4 Table). On the other hand, in female reproductives, the expressions of calpain 1 and calpain 2 increased with age (FDR < 0.05; S4 Table).

Two MHAs were annotated (Table 1) through a BLAST search against the MHA sequences of Z. nevadensis, and showed high sequence similarities (Identities ≥ 77%, Positives ≥ 85%; S1 Table). MHA1 was expressed particularly in young reproductives and soldiers, and MHA2 was remarkably expressed in soldiers (FDR < 0.01; S2 Fig and S2 Table). The expression level of MHA2 in mature PKs was 2.5 times as high as that in SQs (FDR < 0.01; S3 Table). MHA2 was also differentially expressed between reproductive and neuter castes (FDR < 0.001, Table 1 and S5 Table). In male reproductives, MHA2 expression showed the highest level in mature PKs among all age clsasses, and in female reproductives, the expressions of MHA1 and MHA2 showed the highest level in young PQs (FDR < 0.05; S4 Table).

Eight LRPs were annotated (Table 1) through a BLAST search against the LRP sequences of Z. nevadensis (S1 Table). Each LRP of R. speratus exhibited the caste-specific expression pattern (FDR < 0.05; S2 Fig and S2 Table). Five LRPs exhibited differential expression between reproductive and neuter castes, and four LRPs exhibited sexually dimorphic expression (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). Although the expression levels of LRP2 and LRP8 in mature PKs were more than twice as high as those in SQs, LRP3 and LRP6 showed the female-biased expression patterns in mature reproductives. Also, LRP5 was more highly expressed in young PQs and female soldiers than young PKs and male soldiers (FDR < 0.05; S3 Table). As for male reproductives, the expression of LRP8 decreased with age, but LRP1 and LRP2 showed the opposite pattern (FDR < 0.05; S4 Table). The expression levels of LRP3, LRP6, and LRP7 peaked in young PKs (FDR < 0.01; S4 Table).

One FHL was annotated in R. speratus (Table 1), and the sequence showed high similarity to that of A. mellifera (Identities = 93%; S1 Table). This gene was remarkably expressed in soldiers (FDR < 0.01; S2 Table), and its expression differed significantly between reproductive and neuter castes (FDR < 0.001, Table 1 and S5 Table). There was no sexually dimorphic expression among the castes (FDR > 0.05; S3 Table). In male reproductives, the expression of this gene showed the highest level in young PKs among all age classes (FDR < 0.01; S4 Table).

Effectors

Eleven CPases were annotated in R. speratus through a BLAST search against Z. nevadensis sequences (Table 1 and S1 Table). CPase1, CPase3, CPase4, and CPase11 were remarkably expressed in young reproductives than other castes, and CPase8 was higher in young and mature reproductives than other castes (FDR < 0.05; S3 Fig and S2 Table). Five genes were differentially expressed between reproductive and neuter castes (FDR < 0.05, Table 1 and S5 Table). Comparing expression levels between males and females in each caste, the expression of CPase2 in mature PKs was more than 2.5 times as high as that in SQs, but CPase3 showed the opposite pattern (FDR < 0.05; S3 Table). CPase8 was doubly expressed in female soldiers than males (FDR < 0.05; S3 Table). In male reproductives, the expression of seven genes (CPase1–4, CPase6, CPase10, and CPase11) peaked in young PKs among all PK’s ages, and those of three genes (CPase5, CPase7, and CPase8) peaked in mature PKs (FDR < 0.01; S4 Table). In female reproductives, the expression of CPase9 peaked in female alates, but those of seven other genes (CPase1–4, CPase6, CPase8, and CPase11) peaked in young PQs (FDR < 0.01; S4 Table).

Ten cathepsins were predicted (Table 1 and S1 Table). Cathepsin 4, cathepsin 6, and cathepsin 9 showed the mature reproductives-specific expressions, and cathepsin 8 showed the young reproductives-specific expressions (FDR < 0.05; Fig 3 and S2 Table). Cathepsin 10’s expression differed between reproductive and neuter castes, and cathepsin 1 and 2 showed sexually dimorphic expression (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). When comparing expression levels between sexes in each caste, cathepsin 1 and cathepsin 2 showed male-biased expression in soldiers, and the expression of cathepsin 3 in young PKs was four times as high as that in PQs (FDR < 0.05; S3 Table). In male reproductives, the expression levels of four genes (cathepsin 4, cathepsin 6, cathepsin 7, and cathepsin 9) rose with the king’s age, and those of two genes (cathepsin 8 and cathepsin 10) peaked in young PKs (FDR < 0.05; S4 Table). In female reproductives, the expression levels of five genes (cathepsin 4, cathepsin 6, cathepsin 7, cathepsin 8, and cathepsin 9) rose with the queen’s age (FDR < 0.05; S4 Table).

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Fig 3. Differential expression of cathepsins among castes.

The heatmap indicates the differential expression of 10 cathepsin transcripts among castes. Abbreviations are as indicated in Fig 1. Relative expression level indicates the mean normalized CPM, ranging from black (scaled expression of 0) to white (scaled expression of 3.0). The tree at the left corresponds to hierarchical clustering of cluster-averaged expression.

https://doi.org/10.1371/journal.pone.0175417.g003

Previous studies have discovered two lysozyme genes in R. speratus [32,48,49]. In this study, we annotated a total of nine lysozyme (or lysozyme-like protein) genes (Table 1 and S1 Table), which include three C-type lysozymes, one C-type-like lysozyme, three I-type lysozymes, one P-type, and one lysozyme-like protein. The phylogeny inferred from the amino acid sequences of lysozymes in Blattodea (termites and cockroaches) was consistent with the previously known topology of the lysozyme types (S4 Fig). C-type 1 expression showed the highest level in mature reproductives among all castes; P-type, C-type 2, I-type 1, and I-type 3 showed in soldiers; and C-type 3 showed in workers (FDR < 0.01; Fig 4 and S2 Table). The expression levels of six genes differed between reproductive and neuter castes, and, of those, four genes showed sexual dimorphism (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). Comparing expression levels between sexes in each caste, P-type lysozyme expression in young PKs was four times as high as that in PQs (FDR < 0.01; S3 Table). Although the expression levels of P-type, C-type 2, and I-type 2 were higher in mature PKs than in SQs, those of I-type 1 and C-type-like protein showed the opposite pattern (FDR < 0.05; S3 Table). In male reproductives, the expression levels of C-type 1, C-type 2, C-type-like protein, and I-type 2 increased with the PK’s age, but those of I-type 1 and I-type 3 peaked in young PKs (FDR < 0.05; S4 Table). In female reproductives, the expression levels of P-type, C-type 2, and C-type 3 decreased with the PQ’s age, but those of C-type 1 and I-type 3 showed the opposite pattern (FDR < 0.05; S4 Table).

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Fig 4. Differential expression of lysozymes among castes.

The heatmap indicates the differential expression of 9 lysozyme transcripts among castes. Abbreviations are as indicated in Fig 1. Relative expression level indicates the mean normalized CPM, ranging from black (scaled expression of 0) to white (scaled expression of 2.75). The tree at the left corresponds to hierarchical clustering of cluster-averaged expression.

https://doi.org/10.1371/journal.pone.0175417.g004

Two MCA-like CPs were predicted (Table 1 and S1 Table). MCA-like CP1 was remarkably expressed in young reproductives, soldiers, and workers, but MCA-like CP2 was remarkably expressed in alates, soldiers, and workers (FDR < 0.01; S3 Fig and S2 Table). MCA-like CP2 exhibited differential expression between reproductive and neuter castes (FDR < 0.001, S5 Table). Also, the MCA-like CP2’s expression in young PKs was seven times as high as that in PQs (FDR < 0.05; S3 Table). Among age classes in male reproductives, the expression of MCA-like CP1 peaked in young PKs, and that of MCA-like CP2 peaked in male alates (FDR < 0.05; S4 Table). On the other hand, in female reproductives, MCA-like CP1 was higher in young PQs than alates, but MCA-like CP2 showed the opposite pattern (FDR < 0.05; S4 Table).

One AE-like CP was annotated (Table 1) through a BLAST search against the AE-like CP sequence of C. formosanus (S1 Table). AE-like CP1 was more expressed in soldiers than other castes (FDR < 0.01; S2 Table), and its expression level differed between reproductive and neuter castes (FDR < 0.001, Table 1 and S5 Table). The expression of this gene in young PKs was three times as high as that in young PQs (FDR < 0.05; S3 Table). It was also higher in female alates than young PQs (FDR < 0.01; S4 Table).

One lysosomal Pro-X carboxypeptidase was annotated (Table 1) through a BLAST search against the Z. nevadensis sequence (S1 Table). This gene was highly expressed in soldiers, and young and mature reproductives (FDR < 0.01; S2 Table), and it was higher in young PQs than in female alates (FDR < 0.05; S4 Table).

One prolixicin antimicrobial protein was annotated (Table 1 and S1 Table) and was remarkably expressed in mature reproductives and soldiers than other castes (FDR < 0.01; S2 Table). Its expression level also differed between reproductive and neuter castes, and showed sexual dimorphism (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). The expression of this gene in SQs was tenfold as high as that in PKs (FDR < 0.01; S3 Table). Among all age classes in male reproductives, the expression level peaked in mature PKs (FDR < 0.01; S4 Table).

Among three predicted transferrins (Table 1 and S1 Table), transferrin 1 showed the highest expression in mature reproductives and workers among all castes, and transferrin 2 and 3 showed the highest expression in mature reproductives (FDR < 0.01; S3 Fig and S2 Table). The expression level of transferrin 1 differed between reproductive and neuter castes, and those of transferrin1 and 3 showed significant sexual dimorphism (reproductive status: FDR < 0.001, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). Comparing expression levels between sexes in each caste, the transferrin 2’s expression level was higher in young PKs than PQs, but transferrin 3 showed the opposite pattern (FDR < 0.05; S3 Table). Also, transferrin 1 expression was higher in mature PKs than SQs, but transferrin 3 was higher in mature SQs (FDR < 0.05; S3 Table). In male reproductives, transferrin 1 and transferrin 2 were highest in mature PKs, and in female reproductives, transferrin 1 and transferrin 3 were higher in young PQs than female alates (FDR < 0.05; S4 Table).

One termicin was also annotated (Table 1 and S1 Table) and was higher in mature reproductives (FDR < 0.01; S3 Fig and S2 Table). The expression levels of termicin 1 differed between reproductive and neuter castes, and it exhibited sexually dimorphic expression (reproductive status: FDR < 0.01, sex nested by reproductive status: FDR < 0.001, Table 1 and S5 Table). Termicin 1 expression was higher in mature SQs than PKs (FDR < 0.01; S3 Table). In male reproductives, the expression level of termicin 1 peaked in mature PKs (FDR < 0.01; S4 Table). In female reproductives, termicin 1 increased with age (FDR < 0.05; S4 Table).

We found 14 CRPs (Table 1 and S1 Table). CRP3 and CRP10 showed lower expression level only in alates, and CRP8 and CRP12 showed the highest expression level in mature reproductives among all castes (FDR < 0.01; S3 Fig and S2 Table). Eight genes showed significant expression differences between reproductive and neuter castes, and five genes showed sexually dimorphic expression (reproductive status: FDR < 0.05, sex nested by reproductive status: FDR < 0.05, Table 1 and S5 Table). Although CRP5 expression was higher in young PKs than PQs, CRP8, CRP11, and CRP12 showed the opposite pattern (FDR < 0.05; S3 Table). The expression levels of CRP1, CRP8, CRP9, and CRP12 in SQs were more than twice as high as those in mature PKs (FDR < 0.05; S3 Table). Additionally, CRP14 showed female-biased expression in soldiers (FDR < 0.01; S3 Table). Among all age classes in male reproductives, the expression of CRP1, CRP3, CRP5, CRP7, CRP11, and CRP14 peaked in mature PKs, and those of CRP9 and CRP10 peaked in young PKs (FDR < 0.05; S4 Table). CRP6, CRP12, and CRP13 showed the highest expression levels in alates (FDR < 0.05; S4 Table). In female reproductives, CRP6, CRP13, and CRP14 were expressed higher in female alates than young PQs, but CRP3 and CRP10 showed the opposite pattern (FDR < 0.01; S4 Table).

Among four annotated ferritins (Table 1 and S1 Table), ferritin 2 showed the highest expression in young PQs, and ferritin 3 and ferritin 4 showed in SQs (FDR < 0.05; S3 Fig and S2 Table). Ferritin 1 exhibited sexually dimorphic expression (FDR < 0.001, Table 1 and S5 Table), and it showed male-biased expression in soldiers (FDR < 0.01; S3 Table). In female reproductives, the expression of ferritin 3 and ferritin 4 increased with age (FDR < 0.05; S4 Table).

One melanotransferrin, one venom allergen, one termicin, and one thaumatin-like protein were also annotated (Table 1 and S1 Table). Melanotransferrin 1 and venom allergen 1 were more expressed in young reproductives and soldiers than other castes (FDR < 0.01; S3 Fig and S2 Table). The expression level of thaumatin-like protein 1 was low only in alates (FDR < 0.01; S2 Table). The expression levels of venom allergen 1 and thaumatin-like protein 1 differed between reproductive and neuter castes (reproductive status: FDR < 0.01, Table 1 and S5 Table). Comparing expression levels between sexes in each caste, venom allergen 1 showed female-biased expression in young reproductives and king-biased expression in mature reproductives (FDR < 0.05; S3 Table). Among age classes in male reproductives, the expression of melanotransferrin 1 peaked in young PKs (FDR < 0.01; S4 Table). In female reproductives, the expression levels of melanotransferrin 1 and venom allergen 1 increased with age (FDR < 0.05; S4 Table).