Characterization of the population of the sulfur-oxidizing symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses

Abstract

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 mum in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.

FIG. 1.

TEM of ultrathin sections of C. orbicularis gill and bacteriocyte. (A) Low magnification of gill filaments from adult individuals dissected immediately upon recovery. Each gill filament was characterized by a ciliated zone (CZ) separated from the lateral zone (LZ) by several nonciliated intermediary cells (NCI). Bacteriocytes (BC) filled with chemoautotrophic symbionts were part of the lateral zone with numerous intercalary cells characterized by their nucleus in an apical position (asterisks). (B) Bacteriocytes (BC), which were the most prevalent cells in the gill filament, had a basal nucleus (N) and a rounded apical pole developing broad contact with pallial seawater (asterisks). The cytoplasm was crowded by envacuolated bacteria (b), which were usually individually enclosed inside bacteriocyte vacuoles. IC, intercalary cells; BL, blood lacuna. Sulfur globules appeared as electron-lucent bodies within the symbiont cells.

FIG. 2.

FCM analyses of a sulfur-oxidizing population hosted by C. orbicularis (A and C) and of seawater (B and D) sampled above the sampling site of C. orbicularis. (A and B) Contour plot presentations. Gray lines represent the contour plots that are isopleths of the number of events and are scaled proportionally to the maximum values. Note that the innermost contour plots describe the peaks of the different subpopulations. Black lines delimit the identified subpopulations. (C and D) Three-dimensional projections of the A and B cytograms. The cells were stained with SYBR green I. Fluorescent beads (1 μm and 2 μm) were added to the samples as an internal standard. Note the difference in scale for both variables due to different gain settings. True-count (TC) beads were used to determine total cell concentrations. Seven subpopulations (P1 to P7) were discriminated according to SSC and FL1 (green fluorescence) signals in the symbiotic population (A and C). Three different subpopulations, HNA and LNA cells and virus-like particles (VLP), were identified according to SSC and FL1 signals in the seawater sample (B and D).

FIG. 3.

Analysis of the percentages of the different subpopulations (P1 to P7) (Fig. 2A and C) (see Table S1 in supplemental material) among the five different individual bivalves collected in August 2003 (gills G1 to G5).

FIG. 4.

SSC and FL1 mean normalized values for the different subpopulations discriminated by FCM analysis of the populations of symbionts (•) and the seawater samples (□) (A). FL1 and SSC values were normalized with respect to 1-μm and 2-μm green fluorescent cytometry beads. A focus of low SSC and FL1 values in A is shown (B). Horizontal and vertical bars represent 95% confidence intervals of the means. The mean values were calculated from the five endosymbiotic populations (G1 to G5) and from the three seawater samples (SW1 to SW3).

FIG. 5.

Frequency distribution of symbiont cell abundances and sulfur amounts among the different size classes (see Materials and Methods and Results). The average cell-specific sulfur content is also given for each size class.

FIG. 6.

Contour plot representation of FCM analysis of the total population of the initial nonfractionated pooled sample W (A) and of the cell-size-fractioned populations after successive filtrations through 3-μm (B)-, 2-μm (C)-, and 1-μm (D)-pore-size membranes of symbionts in C. orbicularis. Gray lines represent the contour plots that are isopleths of number of events and are scaled proportionally to the maximum values. Also see the legend of Fig. 2. Black lines delimit the subpopulations identified in the nonfractionated sample. Bacterial cells were stained with SYBR green I, and fluorescent beads (1 μm and 2 μm) were added to the sample as an internal standard. True-count (TC) beads were used to determine the total cell concentration, which is mentioned at the bottom within each cytogram (see the legend of Fig. 2).