Field and laboratory observations of the mass mortality of the yellow clam Mesodesma mactroides in South America: the case of Isla del Jabali, Argentina.

ABSTRACT Since the mid-1990s, the yellow clam Mesodesma maetroides (Deshayes, 1854) has experienced mass mortalities throughout its biogeographic range (23 [degrees]S to 41[degrees]S) along Atlantic exposed sandy beaches of South America. However, there is no information about the potential causes underlying these large-scale events. In the summer of 2002, a sudden massive mortality almost decimated the yellow clam population located to the south of Isla del Jabali (Argentina), at the southern edge of its geographical range. Field observations showed a drastic decrease in abundance front 2000 to 2002, without recovering since then. Mortality sequentially occurred at the beach in a north-south direction, following the same trend as in the large-scale event. Significant differences in individual size were found between dead and live clams with the largest individuals of the population being the most affected. Histologic analysis revealed the presence of a meront stage of an unidentified coccidian parasite in the epithelium of the middle intestine of 3 of the 14 clams examined. Necrosis was found in gills and stomach in 13 of the 14 clams examined. The results suggest that these parasites could play a role in the massive mortality events of the yellow clam populations all along South American sandy beaches since 1993. These findings provide an alternative explanation to the widely held notion that mass mortalities in sandy beach macrofauna are due to harmful algae blooms.

KEY WORDS: mass mortalities, bivalves. Mesodesma mactroides, coccidian parasite, sandy beaches, Argentina

INTRODUCTION

The yellow clam Mesodesma mactroides is an intertidal bivalve distributed along the warm-temperate Atlantic coast of South America, from San Paulo State, Brazil (24 [degrees]S) to the south of the Buenos Aires province, Argentina (41 [degrees]S; Fig. 1a). Yellow clam populations prosper primarily in the intertidal zone of microtidal dissipative beaches with gentle slope, fine sand, heavy wave action, and a wide surf zone often characterized by high primary production by surf diatoms (Defeo & Searabino 1990). The yellow clam has supported recreational and artisanal fisheries of high socio-economic importance (Defeo et al. 1993, Castilla & Defeo 2001).

[FIGURE 1 OMITTED]

Since 1993, episodic mass mortalities have decimated yellow clam populations throughout its entire geographic range. These events occurred mainly between late spring and early summer, following a sequential north-south direction (see Fig. 1a). Mortalities occurred first in Brazil in March 1993 (Odebrecht et al. 1995), then in Uruguay in December 1994 (Mendez 1995) and last in Argentina from September to November 1995 (Fiori 1996, Fiori & Cazzaniga 1999). These mass mortalities have prevented the rehabilitation of yellow clam populations throughout its range (Fiori 2002, Defeo 2003). These large-scale events have not been successfully explained, mainly because of the lack of sampling opportunities concurrent with the occurrence of these sudden phenomena. Odebrecht et al. (1995) conjectured that high abundance of dinoflagellates was a source of yellow clam mortality in Brazil, but when the mass mortality occurred in Uruguay, bioassays indicated that paralytic shellfish toxins were not present (Mendez 1995). The same held true for Argentinean coasts, where metal contamination, phytotoxins, abnormal phytoplankton composition, and protozoan tissue-parasites were discarded as causative factors (Fiori & Cazzaniga 1999).

In January 2002 (austral summer), a mass mortality occurred in the southernmost and isolated yellow clam population located at the south of Isla del Jabali, Argentina. During this event, living organisms were recovered for histologic study. Here we describe the mass mortality event at Isla del Jabali. First, we quantify the magnitude of the event in terms of population density and structure. Second, we perform a histologic examination of the visceral mass of clams, including gills, digestive gland and stomach. Finally, we discuss the large-scale implications of these findings.

MATERIALS AND METHODS

The study area located to the south of Isla del Jabali (Argentina: 40 [degrees]33'S; 62 [degrees]14'W) is a continuous sandy beach, ca. 15 km long, with gentle slope (1.64 [+ or -] 1.16[degrees]), fine sand (mean grain size = 0.20 [+ or -] 0.91 mm) strong wave action and high salinity (34.3 [+ or -] 0.17 ppm) (Fiori 2002). The mean air temperature fluctuates between 6 [degrees]C (winter) and 19 [degrees]C (summer). During the mortality event, a stratified random sampling was carried out in 3 beach sites separated by 4 km and located in the north south direction, thereafter mentioned as sites 1, 2 and 3 (see Fig. 1b). A total of 18 random parcels were located in the mesolittoral area inhabited by the yellow clam, using 50 x 50 cm frames and digging up to a depth of 50 cm. The sediment from each parcel was sieved through a 1-cm mesh and all clams retained were counted, measured with calipers (0.1-mm precision) and discriminated as dead or alive. All clams collected were used to estimate the length frequency distribution (LFD) of the population discriminated by site and clam status: (1) dead clams, in life position into the sediment with the foot and siphons everted and the shell paired open, or with decomposed flesh; (2) alive clams, with closed shell and ability to burrow themselves in the substrate. We did not consider empty valves placed on the sand because we cannel attribute incontestably if these valves came from the mortality event. Two-way ANOVAs were used to test for differences between density and clam length, with status and beach site as main factors. A simple length based survival ratio SR was applied as SR = 1 - [D.sub.i]/([D.sub.I] + [L.sub.i]), where D is the number of dead clams and L is the number of live clams for each length class i. The analysis was compared with density estimates obtained in summer of 2000, following the same stratified random sampling detailed above.

A total of 14 clams collected in sites 2 and 3 (we did not find clams in site 1) were fixed after shucked in 10% buffered formaldehyde in seawater immediately after collection and transported to CINVESTAV-Merida (Mexico) for histologic examination. After arrival, formaldehyde was washed out and replaced with Davison's AFA solution for 4 days. Then, a section of the visceral mass of each preserved clam that contained gills, digestive gland and. stomach was embedded in paraffin and cut at 5 [micro]m thickness. The tissue sections were affixed to glass slides and stained with hematoxylin and eosin (Howard & Smith 1983). A frequently causal agent of mass mortality in oysters, clams, cockles, scallops, and abalones documented worldwide is the Apicomplexan protozoan Perkinsus spp. (Bower et al 1994). Unfortunately, thioglycollate fluid or other culture media to determine the presence of these Apicomplexan parasites were not available during the morality event. In this case, we had to rely on the histologic findings. Samples of clam tissue embedded in paraffin were also sent to Dr. Eugene Burreson (Virginia Institute of Marine Science) for detecting Perkinsus spp. using a genus-specific DNA probe. A Perkinsus marinus-specific PCR test based on the primers of Robledo et al. (1998) was also undertaken at CINVESTAV Merida. Finally, digital photographs of the parasites were sent to other experts in coccidian parasites (Dr. Donald Duszinsky, University of New Mexico. USA: Dr, Iva Dykova. Institute of Parasitology of the Academy of Sciences of the Czech Republic) for identification.

RESULTS

Dramatic declines in abundance of yellow clam were observed in two of the three sites sampled at Isla del Jabali. About 15 km of the beach was covered by an accumulation of empty shell, particularly on the supralittoral zone (Fig. 2) The moribund yellow clam remained buried into the sand, even though they weakly retracted the siphons after a mechanical stimulus. Table 1 shows the clams density before and after the mass mortality event. Density significantly differed between sites ([F.sub.2,30] = 18.31: P < 0.001), but did not differ between clam status ([F.sub.1,30] = 0.77: P < 0.973). The 'site x status' interaction factor was significant ([F.sub.2,30] = 3.95: P = 0.03), as a result of the contrasting patterns in clam status between sites 2 and 3 (LSD test, P < 0.01: see Fig. 3a).

[FIGURES 2-3 OMITTED]

In site 2, individual size of live clams were in the range 45-67 mm (mean [+ or -] SD: 61.8 [+ or -] 5.9 mm), whereas dead clams varied between 51 and 76 mm (65.3 [+ or -] 4.1 mm). In site 3, dead yellow clam varied between 40 and 77 mm (mean [+ or -] SD: 65.2 [+ or -] 8.3 mm), whereas live clams were in the range of 54-80 mm (67.3 [+ or -] 6.1 mm). The survival ratio SR markedly differed between sites (see Fig. 3b): in site 2, SR exponentially decreased ([R.sup.2] = 0.69; P < 0.01) towards larger clam sizes, with 0% survival at sizes >70 mm; in site 3, size specific SR were significantly higher than in site 2. and linearly decreased ([R.sup.2] = 0.40; P = 0.02) towards oldest length classes, reaching values as high as 67% at 76 mm. The two-way ANOVA revealed significant differences in mean size between beach sites ([F.sub.1,272] = 5.74: P < 0.017) and clam status ([F.sub.1,272] = 5.06; P < 0.025), while the 'site x status' interaction factor was not ([F.sub.1.272] = 0.53; P = 0.468). This means independence of the effect of "site" on the level of the factor "status", reflected in highest sizes at the northern site and for dead clams (LSD test: P < 0.01).

Eleven of the 14 (78%) clams examined presented necrosis in the stomach (Fig. 4a), which was characterized by necrotic hemocytes and pycnotic cells (see Fig. 4a). Thirteen of the 14 (93%) clams also presented necrosis in gills (see Fig. 4b). The ciliate Trichodina sp. was found in one of the 14 (7%) clams. Samples did not react with the Perkinsus genus-specific DNA probe, nor were positive for the PCR test for Perkinsus marinus either. The parasite in Figure 4c was a meront stage of an unidentified coccidia 21-16 [micro]n long and 13-17 [micro]m wide (n = 10), with 16-21 merozoites per meront. The merozoites were elongated with 4-5 [micro]m long and 2-3 [micro]m wide (n = 10). This parasite was present in 3 of the 14 (21%) clams examined. There was necrosis of the tubules of digestive diverticula in the infected host (see Fig. 4d).

[FIGURE 4 OMITTED]

DISCUSSION

We provide the first observations for the mass mortality that decimated the yellow clam population at the southernmost edge of its range. Sites 1 and 2 showed a dramatic decrease in density, whereas site 3, situated southwards, appeared to be unaffected in the short term during the mortality event. This gives support to the directional event from north to south, as occurred on a biogeographic scale. However, it is worth mentioning that, after that date, all clams in the three sites died and up to now (June 2004) the recovery of the population has been negligible (Fiori, personal observation). Analysis of the LFDs and the size-based SR showed that larger clams were more susceptible than smaller ones. Survival significantly decreased towards larger sizes, and this was consistent for the two main beach sites analyzed, even though the declining ratio was exponential in site 2 and linear in site 3, suggesting again a north-south direction of the mortality event.

The histologic analysis revealed the presence of gill, stomach. and intestine necrosis. It is difficult to link this tissue damage to the coccidian parasite found, because it was present only in 3 of the 14 clams examined. However, it is not possible to discard the possibility either, because the collected clams were survivors of the massive mortality. In these circumstances, it would be reasonable to expect that collected specimens should have low infection levels. In addition, Cremonte and Figueras (2003) reported, for the first time for this species, coccidian and gregarines in a sample of yellow clam from Isla del Jabali, which was taken 2 years before the mass mortality. Coccidians were found in 93% of the 32 clams processed. Furthermore, several authors have found that mass mortalities of other mollusks (scallops) in the Atlantic coast of America and Spain have been related to coccidian parasites, especially those of the genera Pseudoklossia and Margolisiella. These parasites produced extensive destruction of kidney and digestive gland of their hosts (Leibovitz et al. 1984, Desser & Bower 1997, Carballal et al. 2001). Unfortunately, it was not possible to examine kidney because this tissue did not appear in the fixed material. However, it is not possible to attribute the necrosis to bad fixation of the biologic material because all clams were properly shucked and fixed. Clearly, more research is needed to determine the relationship between this coccidian parasite and the tissue damage observed.

The mass mortality at Isla del Jabali occurred in summer, in agreement with the other yellow clam mortality events that have occurred throughout the species range (Fiori & Cazzaniga 1999). This indicates high vulnerability of the yellow clams during this season, concurrently with high temperature, salinity and species density (Fiori 2002). A possible explanation for this pattern could be an increase in the vulnerability of the parasitized clams with respect to the unparasitized ones. For example, Perkinsus-associated mass mortalities have been observed with high values of temperature, salinity, and species density (Choi et al. 2002). Mass mortalities tend to occur in summer (Chu et al. 1996, Choi et al. 2002) or even as a result of increasing winter water temperatures (Cook et al. 1998). However, to the best of our knowledge, there are not published studies on the seasonal variation of the infection parameters (prevalence, mean intensity) or mortality produced by coccidian parasites of bivalves, and its association with environmental factors such as temperature or salinity remains unknown.

One of the most important characteristics of the mass mortalities affecting the yellow clam throughout South America is its strong host specificity, as other members of the macrofauna, including the very abundant sympatric suspension feeders in Uruguay and Brazil, Donax hanleyanus (Defeo & de Alava 1995) and Emerita brasiliensis (Defeo & Cardoso 2002) were not affected (Defeo 2003). As transplanting of clams has not been performed, it seems that the mortality agent is transported by littoral currents, suggesting a strong dispersal capability and tolerance to surpass zoogeographic barriers like the estuarine environments Rio de la Plata, Bahia Blanca, and Bahia Anegada, which present highly variable physico-chemical conditions, especially in salinity.

Our results, even preliminary, provide the first potential explication to the mass mortality event in this species. Further studies involving immunologic and molecular biology techniques are needed to provide conclusive results about the role of this parasite in the massive mortality of this endangered species (Fiori & Cazzaniga 1999).

TABLE 1.
Mean ([+ or -] SD) density of Mesodesma mactroides (ind/[m.sup.2])
per site at Isla del Jabali beach.

SITE             2000                    2002

1       189.33 [+ or -] 140.93             0
2        68.00 [+ or -] 76.00     12.00 [+ or -] 9.12
3        94.67 [+ or -] 18.90    110.00 [+ or -] 90.54
Total   102.77 [+ or -] 89.79     40.67 [+ or -] 70.75

ACKNOWLEDGMENTS

This paper was written during the post-doctoral stage of Sandra Fiori at CINVESTAV Merida, with a fellowship granted by CONACYT and Secretaria de Relaciones Exteriores (Mexico). The authors thank Susana Castelnuovo and Alberto Alzugaray for field support. We are indebted to Prof. Eugene Burreson (VIMS, USA), Dr. Donald Duszinsky (University of New Mexico, USA) and Dr. Iva Dykova (Institute of Parasitology of the Academy of Sciences of the Czech Republic) for their help in the identification of the coccidian parasite.

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SANDRA FIORI, * VICTOR M. VIDAL-MARTINEZ, (2) RAUL SIMA-ALVAREZ, (2) ROSSANNA RODRIGUEZ-CANUL, (2) MA. LEOPOLDINA AGUIRRE-MACEDO (2) AND OMAR DEFEO (2) *

(1) Universidad Nacional del Sur San Juan 670, 8000 Bahia Blanca, Buenos Aires, Argentina; (2) CINVESTAV Unidad Merida, A.P. 73 Cordemex, 97310 Merida, Yucatan, Mexico

* Corresponding author. E-mail: [e-mail]