Expression analysis of <i>RsDsx</i> after siRNA injection.

Quantitative real-time PCR expression analysis in siRNA-injected male nymphs of R. speratus. Expression levels (number of biological replications = 9) of RsDsx were compared between GFP and RsDsx siRNA-injected individuals one and three days after the injection. Asterisks indicate significant differences (Mann–Whitney U test, P (TIFF)</p

<i>RsDsx</i> dsRNA injection affects the expression of genes associated with spermatogenesis and lifespan regulation.

Quantitative real-time PCR expression analysis in dsRNA-injected male nymphs of R. speratus. Expression levels (number of biological replications = 15) of RsDsx (a), piwi-like protein (b), and B-box type zinc finger protein (c) were compared between GFP and RsDsx dsRNA-injected individuals one, three, and six days after the injection. Asterisks indicate significant differences (Mann–Whitney U test, P < 0.01**, P < 0.05*). n.s. means no significant differences.</p

Dsx binding motif sequences used in this study.

Eusocial insects such as termites, ants, bees, and wasps exhibit a reproductive division of labor. The developmental regulation of reproductive organ (ovaries and testes) is crucial for distinguishing between reproductive and sterile castes. The development of reproductive organ in insects is regulated by sex-determination pathways. The sex determination gene Doublesex (Dsx), encoding transcription factors, plays an important role in this pathway. Therefore, clarifying the function of Dsx in the developmental regulation of sexual traits is important to understand the social evolution of eusocial insects. However, no studies have reported the function of Dsx in hemimetabolous eusocial group termites. In this study, we searched for binding sites and candidate target genes of Dsx in species with available genome information as the first step in clarifying the function of Dsx in termites. First, we focused on the Reticulitermes speratus genome and identified 101 candidate target genes of Dsx. Using a similar method, we obtained 112, 39, and 76 candidate Dsx target genes in Reticulitermes lucifugus, Coptotermes formosanus, and Macrotermes natalensis, respectively. Second, we compared the candidate Dsx target genes between species and identified 37 common genes between R. speratus and R. lucifugus. These included several genes probably involved in spermatogenesis and longevity. However, only a few common target genes were identified between R. speratus and the other two species. Finally, Dsx dsRNA injection resulted in the differential expression of several target genes, including piwi-like protein and B-box type zinc finger protein ncl-1 in R. speratus. These results provide valuable resource data for future functional analyses of Dsx in termites.</div

Target genes of <i>Coptotermes formosanus</i> Dsx (CfDsx) identified by HOMER v4.11.

Target genes of Coptotermes formosanus Dsx (CfDsx) identified by HOMER v4.11.</p

Target genes of <i>Reticulitermes lucifugus</i> Dsx (RlDsx) identified by HOMER v4.11.

Target genes of Reticulitermes lucifugus Dsx (RlDsx) identified by HOMER v4.11.</p

Comparison of target genes between termite species.

Venn diagrams showing the numbers of candidate Dsx target genes of R. speratus and R. lucifugus (a), R. speratus and C. formosanus (b), and R. speratus and M. natalensis (c).</p

Phylogenetic relationships of termites with genome information and related cockroaches.

Phylogenetic relationships of termites with genome information and related cockroaches.</p

Stability values of reference genes in real-time qPCR analysis after dsRNA injection.

Stability values of reference genes in real-time qPCR analysis after dsRNA injection.</p

Expression analysis of other candidate target genes after <i>RsDsx</i> dsRNA injection.

Quantitative real-time PCR expression analysis in dsRNA-injected male nymphs of R. speratus. Expression levels (number of biological replications = 13–15) of vitellogenin (a), cilia- and flagella-associated protein (b), proteoglycan 4-like (c), hypothetical protein (RS006266) (d), hypothetical protein (RS009322) (e), and beta-glucosidase (f) were compared between GFP and RsDsx dsRNA-injected individuals three and six days after the injection. The numerals in parentheses represent the number of biological replications. Asterisks indicate significant differences (Mann–Whitney U test, P (TIFF)</p

Target genes of <i>Macrotermes natalensis</i> Dsx (MnDsx) identified by HOMER v4.11.

Target genes of Macrotermes natalensis Dsx (MnDsx) identified by HOMER v4.11.</p