The homolog of Ciboulot in the termite (Hodotermopsis sjostedti): a multimeric beta-thymosin involved in soldier-specific morphogenesis

Abstract

Background: Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail.

Results: To examine the HsjCib gene, full-length cDNAs were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) and sequencing. Multiple isoforms were identified, and on the basis of the results of northern and Southern hybridization analyses, these isoforms were considered to be transcriptional variants from a single gene. On the basis of their sequence similarity to homologous genes of other organisms, functions in actin assembly were assumed to be different among isoforms. Expression analysis revealed high expression in the head during soldier differentiation, which was consistent with their allometric growth. Although isoform expression was observed in various tissues, different expression levels were observed among tissues, suggesting the possibility of tissue-specific morphogenetic regulation by HsjCib isoforms.

Conclusion: This study revealed the characteristics and dynamics of the HsjCib gene during soldier differentiation as a potential representative of downstream effector genes in caste-specific morphogenesis. From the expression patterns observed, this gene is considered to be involved in cephalic morphogenesis and neural reorganization, resulting in the establishment of caste-specific morphology and behavior.

Figure 1

Castes of the damp-wood termite Hodotermopsis sjostedti. A: Alate. B: Soldier. C: Pseudergates. D: Diagram of the caste-differentiation pathway, modified from Miura et al. [41]. The highlighted text denotes the castes used in the present study, and the non-highlighted, parenthesized letters are the short transitional stages used in the study. In the "JHA-Treated Pseudergate" category, we prepared several time stages after JHA application. Gray arrows indicate transitions with molting, white arrows indicate those without molting, and the black arrow indicates hatching.

Figure 2

cDNA and putative amino acid sequences for the longest isoform of HsjCib. The 1245-bp cDNA, obtained by 5' and 3'-RACE, contains a putative ORF that encodes a polypeptide of 202 amino acid residues. Boxed sequences indicate putative WH2 domains with a different color for each domain. The gray vertical bars indicate positions of introns. The gray shadow indicates a fragment from the original differential screening [28]. A potential polyadenylation signal is underlined.

Figure 3

Isoform structure of HsjCib gene. A: The gene structure of HsjCib inferred from PCR amplification of genomic DNA. Coding regions are represented in black. Numbers indicate the lengths (bp) of regions. B: Structure of obtained isoforms. Smaller isoforms were thought to skip some exons. The gray bar indicates the fragment used as probes in Southern, northern, and in situ hybridization. D1 to D5 indicates putative WH2 domains. C: Southern hybridization indicates that HsjCib exists as a single-copy gene on the genome. D: Northern hybridization for HsjCib. Several isoforms seem to overlap between the 0.96- and 1.38-kbp markers. The longer fragment around 2.5 kb may be a paralog or an isoform that has not been cloned. E: Alignment of putative amino acid sequences of HsjCib with Drosophila Cib and human Thymosin-β4.

Figure 4

Western blot and in situ hybridization to detect HsjCib expression. A: Western blot analysis detected by anti-Drosophila Cib antibody against head homogenates of the termite. The antibody detected strong bands in the 14 d (14 days after JHA application) lane and weak bands in the PE (normal pseudergate) lane, which were presumed to correspond to isoforms of HsjCib. B-E: In situ hybridization of the HsjCib mRNA in newly formed mandible (14 d stage). Transverse paraffin sections (6 μm) were subjected to in situ hybridization with antisense (B and D) and sense (C and E) DIG-labeled RNA probes. Bars indicate 200 μm (B and C) and 20 μm (D and E), respectively. The expression of HsjCib was observed in the mandibular epidermis.

Figure 5

Expression analyses of HsjCib in various stages and tissues. A: Alignment of WH2 domains in various proteins. Black arrows indicate structurally important amino acid residues as suggested by Hertzog et al. [55]. According to the Hertzog's model, HsjCib isoforms 2 and 5 are supposed to act as assembly-promoting proteins, whereas isoforms 1, 3, and 4 resemble G-actin sequestering proteins. B: Temporal expression pattern of HsjCib during caste differentiation. Exon 1 was contained in all isoforms; consequently, the expression levels of exon 1 indicate the sum of the expressions of all isoforms. The expression levels of exon 2 were thought to indicate G-actin sequestering isoforms. PE: Pseudergate; 6 h-14 d: pseudergate of 6 h to 14 days after JHA application; PS: Presoldier; S: Soldier; SM: Pseudergate before stationary molt; N: Nymph; LN: Late nymph before imaginal molt; A: Alate. White arrows indicate two comparable stages in which the times until the next molt are approximately the same. C: Measurement of expression levels of exon 1 and exon 2 in the indicated tissues at 14 d after JHA application.