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Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus

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ABSTRACT

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

No MeSH data available.


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.
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pone.0175417.g004: 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.

Mentions: 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).


Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus
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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5391962&req=5

pone.0175417.g004: 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.
Mentions: 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).

View Article: PubMed Central - PubMed

ABSTRACT

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

No MeSH data available.