Long-term Cultivation of the Deep-Sea Clam Calyptogena okutanii: Changes in the Abundance of Chemoautotrophic Symbiont, Elemental Sulfur, and Mucus

Abstract

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

Effects of a long-term rearing system for deep-sea vesicomyid clams on host survival and endosymbiont retention

“…In the FISH analysis, intense hybridization signals were detected in the inner area of the gill filament of each freshly collected clam, as described previously [19,6] ( Fig. 2a).…”

“…1) [5,6] 65 days before the onset of rearing. After the cruise, 29 individual clams were transferred to the artificial chemosynthetic tank (tank A).…”

“…However, it is difficult to maintain Phreagena clams in rearing tanks [5]. Recently, Ohishi et al reared P. okutanii and Phregena soyoae for over 150 days in a tank system at the Enoshima Aquarium that mimicked the deep-sea chemoautotrophic ecosystem [6], using decaying dog food as a sulfide source [5]. However, they reported that more than half of the clams died within 57 days after the onset of the rearing period [6].…”

“…Recently, Ohishi et al reared P. okutanii and Phregena soyoae for over 150 days in a tank system at the Enoshima Aquarium that mimicked the deep-sea chemoautotrophic ecosystem [6], using decaying dog food as a sulfide source [5]. However, they reported that more than half of the clams died within 57 days after the onset of the rearing period [6]. Thus, there are still problems that must be addressed before the stable long-term rearing of Phreagena clams can be achieved.…”

“…Thus, there are still problems that must be addressed before the stable long-term rearing of Phreagena clams can be achieved. For example, the researchers observed a decrease in symbiotic bacteria in the gill epithelia during long-term rearing using the artificial chemosynthetic aquarium [6]. Because the symbionts are required by the host clams, it can be assumed that the observed decrease in symbiotic bacteria resulted in the deaths of the reared hosts.…”