Gene expression changes during caste-specific neuronal development in the damp-wood termite Hodotermopsis sjostedti

Abstract

Background: One of the key characters of social insects is the division of labor, in which different tasks are allocated to various castes. In termites, one of the representative groups of social insects, morphological differences as well as behavioral differences can be recognized among castes. However, very little is known about the neuronal and molecular bases of caste differentiation and caste-specific behavior. In almost all termite species, soldiers play defensive roles in their colonies, and their morphology and behavior are largely different from workers (or pseudergates). Therefore, we predicted that some genes linked to defensive behavior and/or those required for neuronal changes are differentially expressed between workers and soldiers, or during the soldier differentiation, respectively.

Results: Using the brain and suboesophageal ganglion (SOG) of the damp-wood termite Hodotermopsis sjostedti, we first screened genes specifically expressed in soldiers or during soldier differentiation by the differential display method, followed by quantitative real-time polymerase chain reaction. No distinctive differences in expression patterns were detected between pseudergates and soldiers. In the course of soldier differentiation, however, five genes were found to be up-regulated in brain and/or SOG: 14-3-3 epsilon, fibrillin2, beta-tubulin, ciboulot, and a hypothetical protein containing a SAP motif. Some of these genes are thought to be associated with cytoskeletal structure or motor-associated proteins in neuronal tissues.

Conclusion: The identified five genes could be involved in soldier-specific neuronal modifications, resulting in defensive behaviors in termite soldiers. The temporal expression patterns of these genes were consistent with the neuronal changes during soldier differentiation, suggesting that molecular machineries, in which the identified factors would participate, play important roles in behavioral differentiation of termite soldiers.

Figure 1

Soldier differentiation and stationary molt of H. sjostedti. Soldier differentiation of the focal termite species can be artificially induced by JHA (pyriproxyfen) application. In this species, 6-7th instar larvae function as workers, i.e., pseudergates (PE). At 2 weeks after the JHA application, abdomens of pseudergates before the presoldier molt turn white because of gut purge (2wkPE). In 2 to 3 weeks after the application, the JHA-applied pseudergates molt into presoldiers (PS). Almost all pseudergates without artificial induction underwent stationary molt (SM).

Figure 2

Screening of soldier- or differentiation-specific genes. We performed differential display for the first screening and obtained 40 differentiation-specific and 6 soldier-specific gene candidates. Among these, the expressions of 23 candidates were screened by qRT-PCR (second screening, first qRT-PCR). Thirteen of these were confirmed to be up-regulated during soldier differentiation. Based on Gene Ontology and the BLAST-X algorithm, molecular function terms and biological process terms were assigned to six sequences, which were subsequently quantified by qRT-PCR ("Quantification, 2nd qRT-PCR" in Figure 2).

Figure 3

Gene expression patterns of candidate genes quantified by qRT-PCR. As a result of the first qRT-PCR, sequences of 23 candidate genes were classified by gene expression patterns. Up-regulated gene candidates were categorized into six groups based on the expression patterns. A: up-regulated during soldier differentiation (2wkPE, PS, or nPS) in both of brain and SOG; B: up-regulated during soldier differentiation in brain and during molt (2wkPE, PS, or nPS, and also SM) in SOG; C: up-regulated during molt in brain and during soldier differentiation in SOG; D: up-regulated during soldier differentiation only in SOG; E: up-regulated during molt in both brain and SOG; F: no specific up-regulation, i.e., false positive.

Figure 4

Quantifications of candidate genes in different castes and stages. Relative expression levels of HsjCib, Hsj14-3-3e, HsjTubb, HsjFib2, HsjSAP, and HsjUPL in different castes and stages were analyzed by qRT-PCR (second qRT-PCR). Most of the examined genes showed significantly higher levels of expressions during soldier differentiation. Y-axes indicate gene expression levels normalized based on the GAPDH expression. Experimental triplications were performed for all samples. Error bars indicate 95% confidence intervals. Statistical analysis used ANOVA with post hoc Tukey's multiple comparison tests.