Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis, Lucinidae) and Its Symbiont Population

Collection of bivalves.A set of 60 individuals of Codakia orbicularis was collected by hand at a depth of 5 to 10 cm in the sediment of a seagrass bed of Thalassia testudinum at “îlet Cochon” (16°12′53″N; 61°32′05″W, close to Pointe-à-Pitre, Guadeloupe, French West Indies). At the sampling site, water column depths varied from 0.3 to 1 m. Over the year, the water temperature varied between 25°C and 30°C.

Starvation experiment.The freshly collected specimens of Codakia orbicularis were divided into several 50-liter plastic containers filled with artificial seawater, prepared by dissolving sea salt (Sigma) in distilled water (salinity, ∼35 ppt). The bivalves were starved for 4 months. The experiment was conducted at room temperature, between 25°C and 30°C. The artificial seawater was highly oxygenated using an aquarium pump and renewed twice a week to avoid any toxic effect from the excretion of nitrogen waste products, such as ammonia, released by the clams. At regular intervals (14 and 28 days and 3 and 4 months), three clams were selected at random for analysis. One gill was dissected and used to extract and purify the endosymbionts; the other gill was fixed and embedded as described below for CARD-FISH experiments.

Extraction and purification of gill endosymbionts.Gill endosymbionts were extracted and purified using the Percoll cushion method (19), with some modifications. To extract the endosymbiotic bacteria from the gill tissue, one gill was homogenized in 8 ml of sterile seawater (35 ppt) using a handheld Dounce homogenizer. The homogenized tissue was centrifuged (30 × g for 1 min). Four ml of the supernatant was then centrifuged at 400 × g for 2 min to collect the bacteria in the pellet, which was resuspended in 1 ml of filtered (0.2 μm) seawater. The suspension was gently layered on a Percoll (Sigma) cushion (3 ml) diluted with imidazole-buffered saline (490 mM NaCl, 30 mM MgSO₄, 11 mM CaCl₂, 3 mM KCl, and 50 mM imidazole) and centrifuged at 1,000 × g for 8 min at 4°C. Since the Percoll cushion method (19) is based on differences in density between the symbiont and the host organelles, the loss of elemental sulfur globules (S°) in the periplasmic space of the symbionts over a period of long-term starvation prevents the separation of the endosymbionts from the host debris. The final Percoll concentration, therefore, had to be adjusted from 50% to 30% throughout this study. The symbionts were finally collected under the cushion, washed once, and finally suspended in 1 ml of filtered (0.2 μm) artificial seawater (= purified symbiont suspension). The purification was performed at 4°C to avoid the growth of marine bacteria, initially present on the gills or between the gill filaments as contaminants (35). An aliquot of the purified symbiont suspension was fixed with formaldehyde (1%, final concentration) and immediately stored in liquid nitrogen for nucleic acid content analysis by FCM.

TEM preparation of the gill.Individuals were prefixed for 1 h at 4°C in 2.5% glutaraldehyde in 0.1 M pH 7.2 cacodylate buffer adjusted to 900 mosM with NaCl and CaCl₂ in order to improve membrane preservation. After a brief rinse, they were stored in the same buffer at 4°C until fixed. The gills were dissected, fixed for 45 min at room temperature in 1% osmium tetroxide in the same buffer, and then rinsed in distilled water and postfixed with 2% aqueous uranyl acetate for one more hour before embedding and observation as described already (33).

Histological preparation.An overall view and histological information were obtained from paraffin sections. The gill dissected from one individual was fixed in Bouin's fluid (29) for 24 h at room temperature and then embedded in Paraplast. Sections (7 μm thick) were stained by various techniques as described by Gabe (29). Goldner's trichrome staining was used for morphological information and periodic acid-Schiff staining for identifying glycoconjugates.

Fluorescence in situ hybridization.The gills were fixed for 1 to 3 h at 4°C in 2% paraformaldehyde in seawater filtered through a 0.2-μm-pore-size membrane. The specimens were then washed in seawater three times for 10 min each at room temperature and then dehydrated through an ascending series of ethanol concentrations and stored at −20°C before being embedded in Paraplast. Four-μm-thick sections were placed on precoated slides from Sigma before hybridization. Paraffin was removed prior to hybridization experiments using toluene, and sections were rehydrated in decreasing series of ethanol concentrations, finishing with distilled water. CARD-FISH experiments were performed as described by Pernthaler et al. (48, 49). Bacterial cell membranes were permeabilized using HCl (0.2 M, room temperature), Tris-HCl (20 mM, pH 8, room temperature), and proteinase K (0.5 μg/ml, 37°C). The C. orbicularis symbiont-specific probe Symco 2 (5′-ATGTCTCCCAGCGGTTGGGCAC-3′) (derived from reference 31) was used as a horseradish peroxidase-labeled probe. After cell membrane permeabilization, peroxidases present in the tissue were inhibited using HCl (0.01 M, room temperature). Hybridizations were performed using 50% formamide, and signals were amplified using a buffer containing carboxyfluorescein (fluorescein isothiocyanate). Slides were mounted with Cytomation fluorescent mounting medium (Dako, France) and viewed under an epifluorescence microscope, Eclipse 80i (Nikon, France).

Image recording.Images were acquired using a charge-coupled-device camera, DXM 1200F (Nikon, France), and stored as .jpg files. ImageJ for Windows software, a public-domain image analysis program (http://rsb.info.nih.gov/ij/ ), was used to process the images. The procedure involved a series of operations common to most image analysis software. The first step converted color images into 8-bit greyscale images. The greyscale image was then converted to binary format by defining a greyscale cutoff point. Greyscale values below the cutoff became black, and those above became white. The system manually adjusted to delimit clearly the zone with intracellular bacteria in bacteriocytes in order to optimize the computer detection and highlight objects below the machine threshold. Adjustment and background corrections were maintained constant during the operations. The regions of interest were then delimited for each image before processing. The analysis procedure calculated the relative area occupied by symbionts (percentage of fluorescent surface versus total surface) with selected objects which were outlined and numbered in a new window.

FCM analysis of relative nucleic acid content and cell size of symbionts in the whole population.Nucleic acid content analysis of the symbiont cells purified from C. orbicularis was performed using a FacsCalibur flow cytometer (Becton Dickinson) equipped with an air-cooled argon laser (488 nm). The purified symbiont suspensions in 1% formaldehyde, stored in liquid nitrogen, were thawed and diluted (1/100) in saline water (30 ppt NaCl). Nucleic acids were stained for 15 min in the dark at 4°C with SYBR green I (Molecular Probes, Eugene, OR) according to the method described by Marie et al. (45) (1:10,000 [vol/vol]). Two-μm yellow-green fluorescent cytometry beads (Polysciences, Inc.) were added to the samples as an internal standard to normalize the symbiont fluorescence emission. The sheath fluid (NaCl solution, 30 ppt) was filtered through a 0.2-μm-pore-size membrane. Analyses were run at low speed (around 18 μl min⁻¹) and acquisition was performed for 2 min, corresponding to a total of 25,000 to 35,000 detected cells. Fluorescence from SYBR green-stained symbionts was collected in the green fluorescence channel FL1 (530 nm). Side-scattered light (SSC) was used as a proxy of cell size (7, 58). Both parameters were collected on a logarithmic scale. The relative nucleic acid content and cell size of symbionts were analyzed using samples from freshly collected bivalves (T₀) and starved bivalves.

Enumeration of respiring symbionts (CTC⁺) by epifluorescence microscopy.The respiratory activity of the symbionts was measured using the CTC method, described by Rodriguez et al. (54). The purified symbiont suspension, adjusted to ∼10⁷ total cells ml⁻¹, was incubated with CTC at a final concentration of 4 mM in the dark at room temperature for 4 h. Incubation was stopped by addition of formaldehyde (4%, final concentration). The samples were stored at 4°C until they were examined. By microscopic observation, we checked that insoluble CTC formazan crystals had been formed inside the cells. A suspension of symbionts killed by using sodium azide (0.1%, final concentration) was also incubated as an abiotic control. Fixed samples, first incubated with CTC, were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) at a final concentration of 2.5 μg ml⁻¹ in Tris-HCl buffer (0.1 M, pH 7.1) for 10 min in the dark. CTC-DAPI-doubly stained symbionts were filtered through 0.2-μm-pore-size black polycarbonate membranes (Nuclepore) and counted using an epifluorescence microscope (Provis; Olympus). About 200 to 400 total DAPI-stained cells were examined to calculate the percentage of respiring cells (CTC⁺).

Histological and cytomorphological changes.Each gill filament was composed of a ciliated zone and an extremely short intermediary zone, both without bacterial symbionts, and a lateral zone hosting the intracellular bacteria (Fig. 1A and 2A). The intermediary zone was so small in this lucinid species that it could not be detected using a light microscope in histological sections (Fig. 2A), although it can be seen in TEM images (Fig. 1A and B). Four different types of cells were observed in the lateral zone, such as mucocytes, granule cells, intercalary cells, and bacteriocytes (Fig. 1A and C and 2A). CARD-FISH and TEM data showed that the whole lateral zone of each gill filament seemed to be occupied with bacteriocytes full of bacteria (Fig. 1A and C and 2B). Each bacteriocyte corresponded to a large cell (up to 35 μm in length) characterized by a rounded apical pole in contact with circulating seawater and a cytoplasm filled with individually enclosed bacteria (Fig. 1C). Bacterial endosymbionts were characterized by numerous sulfur granules, which appeared as electron lucent vesicles after conventional TEM preparation (Fig. 1C). After 1 month of starvation, changes were noted in the gills, with a decrease of intracellular bacteria throughout the lateral zone shown by CARD-FISH hybridization (Fig. 2C). The intensity of the green fluorescent signal, proportional to the abundance of symbionts, decreased noticeably compared to that of individual signals at the beginning of the experiment (Fig. 2B). Each bacteriocyte in the lateral zone of the gill filament was hybridized, but the signal intensity from each bacteriocyte was weaker (Fig. 2C).

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FIG. 1.

Ultrastructural modifications in the C. orbicularis gills during starvation, as determined with ultrathin sections (TEM analysis). (A) Low magnification of gill filaments from adults dissected immediately after recovery. Each gill filament is characterized by a ciliated zone (CZ) separated from the lateral zone (LZ) by several nonciliated intermediary cells (NCI). Bacteriocytes (BC) filled with chemoautotrophic symbionts are part of the lateral zone, with numerous intercalary cells characterized by their nucleus in an apical position (asterisks). (B) Low magnification of gill filaments from adults dissected after 4 months of starvation. While the organization of the ciliated zone (CZ) is fairly similar to that observed in field specimens, the lateral zone (LZ) is significantly different. The most prevalent cells are granule cells (GC), with no bacteria and few intercalary cells (asterisks). There are a few undifferentiated cells (stars) without bacterial endosymbionts evenly distributed through the lateral zone. (C) Bacteriocytes (BC), which are the most prevalent cells in the gill filament, have a basal nucleus (N) near the blood lacuna (BL) of the filament axis and a rounded apical pole developing broad contact with pallial seawater. The cytoplasm is crowded with envacuolated bacteria (b) which are individually enclosed inside the bacteriocyte vacuole. Intercalary cells (IC), characterized by a trumpet shape and an apical nucleus, are interspersed uniformly among the bacteriocytes. (D) The bacteriocytes (BC) within the lateral zone of a C. orbicularis specimen kept in starvation for 56 days (2 months). The larger symbionts have disappeared, and only small symbionts, located mainly at the periphery of the cell, can be observed. BL, blood lacuna; GC, granule cell; H, hemocyte; IC, intercalary cells. (E) In specimens that have been starved for 3 months, a few residual bacteriocytes (BC) with a small apical pole in contact with seawater (arrow) can be observed hosting a very few bacteria (b) and large lysosomes (Ly) in their cytoplasm. Most of the cells in the lateral zone are devoid of bacteria. The small number of symbionts is below the detection limit of the other techniques used in this study (i.e., FISH and FCM). GC, granule cell; IC, intercalary cells; N, nucleus. (F) In specimens starved for 4 months, undifferentiated cells without any bacteria in their cytoplasm can be observed throughout the lateral zone. Such cells, with a rounded apical pole (arrow) in contact with the pallial seawater, could be considered putative bacteriocytes, as previously described for aposymbiotic juveniles of C. orbicularis. Some contain lysosome-like structures (Ly). GC, granule cells; H, hemocyte in the blood lacuna; N, nucleus.

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FIG. 2.

Changes in the bacterial population in C. orbicularis gills during starvation, shown by histological gill sections. (A) Goldner staining of gill filaments from a freshly collected Codakia orbicularis clam. The ciliated zone (CZ), characterized by numerous cilia, is devoid of bacteria. The lateral zone (LZ) contains mostly bacteriocytes (BC) hosting the bacteria, as well as mucocytes in blue (asterisks) and granule cells (GC) stained in orange. (B) In situ hybridization (CARD-FISH) with the C. orbicularis symbiont-specific probe, showing that bacterial symbionts fill the bacteriocytes (BC) found all along the lateral zone of gill filaments in a freshly collected C. orbicularis clam. The ciliated zone (CZ) is not hybridized by the C. orbicularis-specific probe owing to the lack of symbionts in this part of the gill filament. Asterisk, mucocyte; GC, granule cell. (C) In situ hybridization (CARD-FISH) with the C. orbicularis-specific probe of gill filaments from an individual starved for 30 days. CZ, ciliated zone; GC, granule cell. (D) In situ hybridization (CARD-FISH) with the specific probe Symco 2 for gill filaments in a C. orbicularis clam starved for 56 days. Only a few poorly hybridized bacteriocytes can be observed. GC, granule cell; CZ, ciliated zone. (E) In specimens starved for 3 months, a few residual bacteriocytes (arrows) can be observed, while large lysosomes, appearing in orange owing to their autofluorescence, can be observed throughout the lateral zone. Most of the cells in the lateral zone are not positively hybridized by the specific probe. The remaining bacteriocytes occupying the lateral zone are scattered and small (compared to the control in panel 2B), with a weak signal since there are very few gill endosymbionts per cell. (F) Higher-magnification focusing on the abfrontal zone of a gill filament of an individual clam starved for 3 months. At this magnification, only two to four bacteria (arrows) can be observed in each sectioned bacteriocyte. The large lysosome-like structures (orange dots) are widely distributed throughout the abfrontal zone, indicating strong cellular digestion of the bacteria inside the bacteriocytes or of the bacteriocytes themselves.

The structural changes in the gill filaments noticed after 1 month appeared more pronounced after 2 months of starvation. The CARD-FISH signal intensity, i.e., symbiont abundance, markedly decreased compared to that for gills from T₀ individuals (Fig. 2B to D) and was even more pronounced after 3 months of starvation, when only a few remaining bacteria could be observed (Fig. 2E). At an ultrastructural level, bacteriocytes contained fewer bacteria after 2 months of starvation, mostly located in the upper part of the bacteriocyte and which were smaller than at the beginning of the experiment (Fig. 1D). After 3 months of starvation, lysosome-like structures and numerous mitochondria could be seen in the bacteriocyte cytoplasm of the lateral zone, whereas only a very few intracellular bacteria, mostly without sulfur granules, could be observed on thin sections from TEM observations (Fig. 1E). Granule cells appeared in the lateral zone, while the gill filament did not change in length but became thinner (not shown), suggesting that the gill structure had been significantly modified. This was confirmed after 4 months (115 days) of starvation. The cell organization of each filament had clearly changed, since granule cells then occupied the greater part of the lateral zone (Fig. 1B) and were in contact with the intermediary zone just below the ciliated zone (Fig. 1B). No bacteria could be detected using TEM (Fig. 1F). Small undifferentiated cells (up to 12 μm), similar in shape to the undifferentiated cells found in aposymbiotic juveniles of C. orbicularis (32) without any bacteria, were regularly interspersed between the granule cells throughout the lateral zone (Fig. 1F). Bacteria could not be detected in those bivalves by the techniques used in this study.

Quantifying the decrease in abundance of symbionts.To estimate the gradual disappearance of symbionts hosted in C. orbicularis gills through starvation, ImageJ software was used to measure the proportion of fluorescent area on the photos presented in Fig. 2B to E from histological sections (4 μm). The results are presented in Table 1. Fluorescence covered 32.4% of the gill at T₀. If this fluorescent area is considered to be a good proxy of the number of symbionts, then approximately one-third of the gill volume of freshly collected C. orbicularis (T₀) was occupied by symbionts. This fluorescent area regularly decreased with starvation (Table 1), confirming the decrease in the abundance of symbionts already revealed by TEM observations (Fig. 1). The host appeared to lose one-third of its symbionts in each month of starvation.

Percentage of respiring symbionts hosted by starved bivalves.The percentage of respiring symbionts (CTC⁺) was measured for purified symbionts from freshly collected clams (T₀) and from clams kept in starvation for 1 and 2 months. The percentage of actively respiring symbionts (CTC⁺) versus total symbionts remained high and constant throughout the experiment (Table 1). At T₀, 80.1% ± 6.6% of symbionts hosted in gills could be considered to be metabolically active, which supported the importance of symbionts in association with the host. After 2 months of starvation, the percentage of CTC⁺ symbionts was still high (83.9% ± 10.5%) despite the decrease in the abundance of symbionts shown by CARD-FISH. No data were available after 3 months of starvation because the decrease in the abundance of symbionts in the gills led to a very low symbiont concentration in the purified suspension, undetectable by epifluorescence microscopy.

Physiological changes in the symbiont population investigated by FCM.The physiological changes undergone by symbionts through host starvation were investigated, first considering the SSC and FL1 (fluorescence in the green channel) values of the whole populations (Fig. 3); a second and more detailed analysis was performed, considering the SSC and FL1 values of each subpopulation discriminated by FCM along with its relative proportion of the whole population (Fig. 4).

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FIG. 3.

FCM analyses of the sulfur-oxidizing population hosted by Codakia orbicularis during host starvation. The cells were stained with SYBR green I, and green fluorescent cytometry beads (2 μm) were added to the samples as an internal standard. For each period of starvation, one plot represents the whole population of symbionts, characterized by SSC and FL1 mean values normalized with respect to 2-μm beads. Horizontal and vertical bars represent 95% confidence intervals for the means, calculated for three distinct symbiotic populations, with the exception of the 3-month starvation period owing to the high clam mortality rate (30%).

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FIG. 4.

SSC and FL1 mean normalized values for the different symbiotic subpopulations, discriminated by FCM after SYBR green I staining. Analyses of the subpopulations of symbionts were performed in triplicate at T₀ (A) or after host starvation for 14 days (B) or 28 days (C). For the 3-month starvation period (D), only one clam was analyzed. SSC and FL1 values were normalized with respect to 2-μm green fluorescent cytometry beads. Horizontal and vertical bars represent 95% confidence intervals of the SSC and FL1 means. Each plot corresponds to a subpopulation discriminated by FCM analysis. The percentage of each subpopulation is shown beside the plots. Contour plot presentations of the subpopulations discriminated by FCM are shown for T₀ (E) and for the 3-month starvation period (F).

Figure 3 shows that the first consequences of host starvation (14 days) were a decrease of the SSC value calculated for the whole population and an increase in the FL1 signal of the symbiotic population. The decrease in the SSC signal of the entire population (half that at T₀) might be caused by a reduction in the sulfur content and/or in the mean cell size, since the SSC signal was influenced by both parameters (11), or by the disappearance of larger cells. In both cases, there was an apparent reduction of the mean cell size for the whole population. At the same time, FL1 modifications showed an increase in the mean nucleic acid content of the population (twice as high as at T₀). Between 14 and 28 days of starvation, the SSC signal of the whole population continued to decrease whereas the FL1 signal (nucleic acid content) remained constant. After 3 months of host starvation, the population of symbionts was characterized by an FL1 signal similar to that at T₀ but with a dramatically reduced SSC (10 times lower).

Figure 4 shows the number of subpopulations and their relative importance as a percentage for each starvation time. In the initial population (Fig. 4A), four subpopulations of symbionts were discriminated (Fig. 4A and E), with a majority of cells (62.1%) with high SSC (1.14) and FL1 (0.088) values. If the lower FL1 value (0.014) is artificially converted into one equivalent genome (1n), cells at this stage were characterized by six equivalent genomes (6n). These cells were previously described as large cells with multiple copies of their genome, rich in sulfur granules (11). A few cells (6.5%) were characterized by a very low SSC value (<0.1) and FL1 signal, i.e., very small cells, and approximately one-third of the population had an SSC value close to 0.15. After 14 days of starvation, four subpopulations of symbionts were still discriminated but with modifications in their SSC and FL1 values. For all subpopulations, the SSC signal decreased and the FL1 value increased, resulting in 3n and up to 12n for the lowest and highest FL1 signals, respectively. Moreover, the relative percentages of the subpopulations changed compared to those at T₀. The proportion of “large” cells (high SSC) decreased from 62.1% to 34.5%, leading to an increase in the proportion of smaller cells. Over a longer starvation period, there was a reduction in the cytometric pattern, with three and two subpopulations remaining after 28 days (up to 15n) and 3 months (up to 5n) of starvation, respectively (Fig. 4C and D). Larger cells progressively disappeared during host starvation, and after 3 months of starvation, taking the two remaining subpopulations together (Fig. 4F), the symbiotic population was totally (100%) composed of very small cells (with an SSC value of <0.1) as opposed to 6.5% at T₀ (Fig. 4D).