Expression of a symbiosis-specific gene in Symbiodinium type A1 associated with coral, nudibranch and giant clam larvae

Abstract

Symbiodinium are responsible for the majority of primary production in coral reefs and found in a mutualistic symbiosis with multiple animal phyla. However, little is known about the molecular signals involved in the establishment of this symbiosis and whether it initiates during host larval development. To address this question, we monitored the expression of a putative symbiosis-specific gene (H⁺-ATPase) in Symbiodinium A1 ex hospite and in association with larvae of a scleractinian coral (Mussismilia hispida), a nudibranch (Berghia stephanieae) and a giant clam (Tridacna crocea). We acquired broodstock for each host, induced spawning and cultured the larvae. Symbiodinium cells were offered and larval samples taken for each host during the first 72 h after symbiont addition. In addition, control samples including free-living Symbiodinium and broodstock tissue containing symbionts for each host were collected. RNA extraction and RT-PCR were performed and amplified products cloned and sequenced. Our results show that H⁺-ATPase was expressed in Symbiodinium associated with coral and giant clam larvae, but not with nudibranch larvae, which digested the symbionts. Broodstock tissue for coral and giant clam also expressed H⁺-ATPase, but not the nudibranch tissue sample. Our results of the expression of H⁺-ATPase as a marker gene suggest that symbiosis between Symbiodinium and M. hispida and T. crocea is established during host larval development. Conversely, in the case of B. stephanieae larvae, evidence does not support a mutualistic relationship. Our study supports the utilization of H⁺-ATPase expression as a marker for assessing Symbiodinium-invertebrate relationships with applications for the differentiation of symbiotic and non-symbiotic associations. At the same time, insights from a single marker gene approach are limited and future studies should direct the identification of additional symbiosis-specific genes, ideally from both symbiont and host.

Keywords: ATPase; Tridacna; larval ecology; scleractinia; sea slug; zooxanthellae.

Figure 1.

Host broodstock and larvae used in the experiment. (a) Mussismilia hispida colonies collected at Recife de Fora, (b) Berghia stephanieae spawning individuals (note brownish area in the cerata, harbouring Symbiodinium cells captured from the anemone Aiptasia sp.), (c) Tridacna crocea broodstock clam, (d) Mussismilia hispida planula after acquiring multiple Symbiodinium A1 cells, (e) Berghia stephanieae veliger larvae immediately before hatching and (f) Tridacna crocea veliger larva with Symbiodinium A1 cells inside the digestive tract.

Figure 2.

Phylogeny of (a) H⁺-ATPase and (b) RuBisCO genes of Symbiodinium A1 in this experiment. Trees were constructed using maximum-likelihood analysis and 1000 bootstrap replicates; only values above 70 are shown. Accession numbers are from the NCBI database.