NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host

Abstract

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

Sulfur-Oxidizing Symbionts without Canonical Genes for Autotrophic CO 2 Fixation

“…Furthermore, the previous study (21) also observed that adding sulfide or thiosulfate decreased the CO 2 fixation rates, although sufficient electron acceptor was available. A lithoautotroph should instead fix more CO 2 under such conditions, as observed in other thioautotrophic symbioses (47). This decrease is, however, consistent with our interpretation of heterotrophic CO 2 fixation during PHA mobilization, because PHA conversion to biomass is a net oxidation reaction (see Text S3 in the supplemental material), which would not be favored under reducing conditions.…”

Inorganic carbon and nitrogen assimilation in cellular compartments of a benthic kleptoplastic foraminifer

“…5E ). Other NanoSIMS studies looking at 13 C-bicarbonate assimilation in cyanobacteria, anaerobic photosynthetic bacteria or chemotrophic bacteria have shown strong bacterial 13 C-enrichments 49 – 52 . Therefore, even if the multi-vacuolar structures observed in our study were bacteria, the absence of 13 C incorporation into their structure suggests that they are not photosynthesizing or assimilating inorganic carbon and that thus they do not play any role in the inorganic 13 C assimilation in Haynesina germanica .…”

Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging

“…Most of these studies have focused on nutrient transfer and metabolic processes (for review, see Berry & Loy, ). For example, in characterization of symbiont to host transfer of biomolecules, NanoSIMS has been used to (a) localize 15 N‐labelled nitrogen‐fixing bacteria inside of coral larvae (Lema et al, ); (b) visualise the transfer of fixed nitrogen molecules from the bacteria symbionts to the marine shipworm and cockroach hosts (Tai et al, ; Volland et al, ); (c) trace the incorporation of labelled C‐ and N‐containing photosynthates translocated from symbiotic algae to their coral host (Kopp et al, ; Kopp et al, ); and (d) describe the nutritional interaction between the coral host and its symbiont under pathogenic conditions (Gibbin et al, ). In addition, several studies have examined transfer of molecules from the host to its symbionts.…”

“…(b) visualise the transfer of fixed nitrogen molecules from the bacteria symbionts to the marine shipworm and cockroach hosts (Tai et al, 2016;Volland et al, 2018); (c) trace the incorporation of labelled C-and N-containing photosynthates translocated from symbiotic algae to their coral host (Kopp et al, 2013;Kopp et al, 2015); and (d) describe the nutritional interaction between the coral host and its symbiont under pathogenic conditions (Gibbin et al, 2019). In addition, several studies have examined transfer of molecules from the host to its symbionts.…”