Downregulation of Orco and 5-HTT Alters Nestmate Discrimination in the Subterranean Termite Odontotermes formosanus (Shiraki)

Abstract

Nestmate discrimination allows social insects to recognize nestmates from non-nestmates using colony-specific chemosensory cues, which typically evoke aggressive behavior toward non-nestmates. Functional analysis of genes associated with nestmate discrimination has been primarily focused on inter-colonial discrimination in Hymenopterans, and parallel studies in termites, however, are grossly lacking. To fill this gap, we investigated the role of two genes, Orco and 5-HTT, associated with chemosensation and neurotransmission respectively, in nestmate discrimination in a highly eusocial subterranean termite, Odontotermes formosanus (Shiraki). We hypothesized that knocking down of these genes will compromise the nestmate recognition and lead to the antagonistic behavior. To test this hypothesis, we carried out (1) an in vivo RNAi to suppress the expression of Orco and 5-HTT, respectively, (2) a validation study to examine the knockdown efficiency, and finally, (3) a behavioral assay to document the phenotypic impacts/behavioral consequences. As expected, the suppression of either of these two genes elevated stress level (e.g., vibrations and retreats), and led to aggressive behaviors (e.g., biting) in O. formosanus workers toward their nestmates, suggesting both Orco and 5-HTT can modulate nestmate discrimination in termites. This research links chemosensation and neurotransmission with nestmate discrimination at the genetic basis, and lays the foundation for functional analyses of nestmate discrimination in termites.

Keywords: chemosensation; in vivo RNAi; nestmate discrimination; neurotransmission; termites.

Figure 1

Social behaviors associated with nestmate discrimination in O. formosanus.

Figure 2

The structure, phylogeny and spatial expression of O. formosanus Orco. (A) Protein alignment of the Orco gene; the transmembrane domain is marked with a blue line, and the 7tm_6 domain is marked with a red line. (B) Phylogenetic tree of Orco in O. formosanus with homologs in other 10 species. (C) Expression patterns of Orco in antenna, leg, head, and abdomen-thorax tissue of workers (Tukey's HSD test, P < 0.05).

Figure 3

Phenotypic impact of Orco knockdown on nestmate discrimination. (A) The expression level of Orco after RNAi treatment. (B) Biting, (C) vibrating, (D) retreat, (E) grooming, and (F) trophallaxis behaviors modulated by silencing of Orco. The red columns represent social behaviors triggered by non-nestmate discrimination, including biting, vibrating, and retreat; the green columns represent social behaviors triggered by nestmate discrimination, including grooming and trophallaxis. Treatment group 1 consisted of 1 termite treated with dsOrco and 5 termites treated with dsGFP, treatment group 2 consisted of 1 termite treated with dsOrco and 5 termites treated with dsOrco, and the control group consisted of 1 termite treated with dsGFP and 5 additional termites treated with dsGFP. Data are shown as the means ± S.E.M. (paired t-test; *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4

The structure, phylogeny, and spatial expression of O. formosanus 5-HTT. (A) Protein alignment of the 5-HTT gene, the coiled coil domain is marked with a blue line, and the SNF domain is marked with a red line. (B) Phylogenetic tree of 5-HTT in O. formosanus with homologs in 10 other species. (C) Expression patterns of 5-HTT in antenna, leg, head and abdomen-thorax tissue of workers (Tukey's HSD test, P < 0.05).

Figure 5

Phenotypic impact of 5-HTT knockdown on nestmate discrimination. (A) The expression level of 5-HTT after RNAi treatment. (B) Biting, (C) vibrating, (D) retreat, (E) grooming, and (F) trophallaxis behaviors modulated by silencing 5-HTT. The red columns represent social behaviors triggered by non-nestmate discrimination, including biting, vibrating, and retreat; the green columns represent social behaviors triggered by nestmate discrimination, including grooming and trophallaxis. Treatment group 1 consisted of 1 termite treated with ds5-HTT and 5 termites treated with dsGFP, treatment group 2 consisted of 1 termite treated with ds5-HTT and 5 termites treated with ds5-HTT, and control group consisted of 1 termite treated with dsGFP and 5 additional termites treated with dsGFP. Data are shown as the means ± S.E.M. (paired t-test; *P < 0.05; **P < 0.01; ***P < 0.001).