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A review and new analysis of trophic interactions between Prorocentrum minimum and clams, scallops, and oysters
Harmful Algae 4 (2005) 585–592 www.elsevier.com/locate/hal
A review and new analysis of trophic interactions between Prorocentrum minimum and clams, scallops, and oysters Gary H. Wikfors* NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA. Received 1 March 2004; received in revised form 1 June 2004; accepted 1 August 2004
Abstract There has been no consensus on whether Prorocentrum minimum is ‘‘toxic,’’ despite sporadic reports suggesting possible shellfish toxicity and laboratory studies showing harmful effects of this dinoflagellate on molluscan shellfish. Shellfish toxicity outbreaks associated with natural blooms of P. minimum have been confounded by co-occurrence of other toxic phytoplankton. Laboratory studies have demonstrated unequivocally that some P. minimum isolates can produce toxins that kill mice on injection, but the bioactive compound or compounds remain unidentified, and accumulation of toxin in grazing mollusks has not been demonstrated. Laboratory experiments testing the responses of grazing mollusks to P. minimum cultures have yielded variable results, ranging from mortality in scallops and oysters to normal growth of oysters. Effects observed in the laboratory include rejection as pseudofeces by clams, poor larval development in oysters, tissue pathologies (sometimes transient) in oysters and scallops, and systemic immune responses in oysters and scallops. Several recent studies have provided evidence that variation in toxicity of P. minimum is dependent on environmental conditions and their effects on the physiology of this dinoflagellate. Accordingly, seemingly conflicting observations from field and laboratory studies may be explained by transient toxin expression in P. minimum. # 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve mollusk; Dinoflagellate; HAB; Harmful algae
1. Introduction Spring and summer blooms of P. minimum have been recorded as a fairly regular feature of annual phytoplankton successions in estuarine waters worldwide (Dodge, 1975; Hajdu et al., 2005; Heil et al., 2005). Where filter-feeding bivalves exert strong * Tel.: +1 203 882 6525; fax: +1 203 882 6517. E-mail address: [email protected].
grazing pressure on estuarine phytoplankton, biomass accumulation of a specific alga may be a consequence of population growth more rapid than normal grazing removal or of grazing repression by the algae. Some microalgal biotoxins are thought to have a grazingdeterrent genesis in evolutionary time (Landsberg, 2002) and examples of chemical and physical deterrents to grazing are common in land plants (Bell and Woodcock, 1968; Muenscher, 1975; Smith, 1992; Simmons and Ekarius, 2001). Different bivalve
1568-9883/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2004.08.008
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species accumulate and tolerate different quantities of toxin from dinoflagellates such as Alexandrium (Fernandez et al., 2003) and selection for tolerant grazers in regions experiencing recurrent toxic algal blooms has been demonstrated (Bricelj et al., 2000; Colin and Dam, 2002; He´ garet and Wikfors, 2005b). Co-occurrence of bivalve populations and P. minimum blooms thus provides the potential for various trophic interactions, including nutritional utilization, grazing repression through a variety of physiological effects, and toxin accumulation. Until recently, most evidence for harmful effects of the dinoflagellate P. minimum on molluscan shellfish was based on anecdotal observations of shellfish mortalities coincident with blooms in coastal waters and several laboratory experiments in which cultured strains of P. minimum were fed to shellfish. Two recent reports by He´ garet and Wikfors, 2005a,b), showing the same changes in immune-system status associated with laboratory exposures and field measurements of oysters feeding on natural blooms of P. minimum, provide confidence that ‘‘artificial’’ exposures of grazing shellfish to P. minimum in the laboratory can be good proxies for natural trophic interactions. The present re-analysis of laboratory experiments was undertaken to explore possible reasons for inconsistent findings and to interpret these findings in an environmental context.
2. Field observations The first suggestion that P. minimum be included in the list of harmful algae arose from a trophic interaction with shellfish. A shellfish-poisoning outbreak in Japan was associated initially with consumption of Venerupus clams harvested from a bay with a P. minimum bloom (Akiba and Hattori, 1949). This shellfish poisoning outbreak could not be attributed directly to P. minimum and subsequent associations between shellfish toxicity and natural blooms of P. minimum have been inconclusive because of cooccurrence of known toxin-forming dinoflagellates (see discussion in Landsberg, 2002). Environmental observations suggesting that P. minimum blooms are harmful to other marine species are not consistent for different species, or even for a given species in different instances. A positive
correlation between P. minimum and presence of the planktivorous fish, Brevoortia tyrannus, was noted by Friedland et al. (1989), but kills of planktivorous fish, sometimes but not always attributed to hypoxia during P. minimum blooms, have been recorded as well (Rabbani et al., 1990; Yaquin-Li, Connecticut Department of Environmental Protection, personal communication). Responses of grazing zooplankton, crustaceans, and protozoans to P. minimum are discussed elsewhere in this volume, but the consensus seems to be that this dinoflagellate may be nutritionally deficient–or excellent food–but generally is not toxic to these pelagic grazers. However, repeated, but not consistent, observations of detrimental effects have been recorded for several species of bivalve mollusks, including scallops, clams, and oysters. The first account in the scientific literature of specific harmful effects of Prorocentrum on a bivalve mollusk was that of Leibovitz et al. (1984) noting intact Prorocentrum cells in the open vascular system of bay scallops (Argopecten irradians irradians) feeding in a natural bloom. It was hypothesized that high scallop mortalities during this bloom were caused by physical damage from the apical tooth, a sharp anterior spine on some Prorocentrum taxa. This hypothesis has not been confirmed and follow-up studies of scallops in natural P. minimum blooms were not conducted until very recently (see below). In another bivalve species, the northern quahog clam Mercenaria mercenaria, evidence that a natural bloom of P. minimum interfered with growth was obtained during an aquaculture site-comparison study in Long Island Sound (Connecticut, USA) (Wikfors and Smolowitz, 1993). In two sites experiencing a bloom of several weeks duration, food availability was very high, but clam growth ceased. No mortalities were seen and normal growth resumed after the bloom. This also led to laboratory experiments to determine if arrested clam growth could be attributed to either of two Prorocentrum taxa co-occurring in this bloom, P. minimum or P. micans (see below). In addition to scallops and clams, oysters may be affected by P. minimum blooms. Mortality and poor growth in wild populations of eastern oysters Crassostrea virginica ingesting Chesapeake Bay P. minimum blooms have also been noted (Luckenbach et al., 1993; Sellner et al., 1995). Empirical observations of shellfish mortalities during P. minimum blooms are also recorded in
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the literature (Yongjia et al., 1995) and monitoring reports (shellfish rearing records, NMFS Milford Laboratory), but a number of system parameters could produce mortality, including changes in water chemistry associated with the bloom (e.g., low dissolved oxygen) or other factors coincident with the bloom. Field observations have provided evidence, but not proof, that P. minimum can be toxic to bivalve mollusks or make them toxic to human consumers.
3. Laboratory observations There is little doubt that bivalves ingest P. minimum. Various life-history stages of several bivalve species are able to filter and ingest cultured P. minimum cells at environmentally-realistic concentrations: larval mussels, Mytilus galloprovincialis, (Jeong et al., 2004); adult Glauconome chinensis (Lee and Chung, 2001; Lee et al., 2003); adult sea scallops, Placopecten magellanicus (Brilliant and MacDonald, 2002); and juvenile eastern oysters, C. virginica (Sellner et al., 1995). 3.1. Northern quahog clams, Mercenaria mercenaria The observations of poor clam growth during a Prorocentrum (P. micans and P. minimum co-dominant) bloom in Long Island Sound were explored in an experiment where juveniles were fed cultured P. micans, alone or in combination with a control diet of Isochrysis sp. (T-ISO), or P. minimum in combination with T-ISO (Wikfors and Smolowitz, 1993). Survival and growth of clams on the mixed diet of P. minimum and T-ISO were no different than for unfed clams and undigested P. minimum cells were seen in clam biodeposits. Thus, direct evidence of toxicity of P. minimum to clams was not apparent and it is more likely that clams minimized their exposure to P. minimum by reducing feeding and rejecting cells as pseudofeces. Results in the field might also have been a result of P. micans having poor nutritional value for clams. This was assessed using the Jonasdottir et al. (1998) model (Fig. 1) where growth would be linearly dependent upon percentages of ‘‘good’’ and ‘‘bad’’ food (the better food is simply ‘diluted’ by the poorer). Growth lower than predicted by dilution suggests toxicity of the poorer; wheras, higher growth indicates that the
Fig. 1. Growth of northern quahogs, Mercenaria mercenaria (grams per clam in 8 weeks), on cultures of Prorocentrum micans and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1993). Maximum cell densities were 5 104 and 5 105 ml 1, respectively. A straight line represents nutritional deficiency of the poorer diet, while observations above or below the line represent nutritional enhancement of food quality or toxicity, respectively.
two foods complement each other’s nutritional deficiencies. The linear decline in clam growth with P. micans indicates simply poor nutritional value of this species to clams. P. minimum could not be assessed with this model because it was fed to clams in one mixture only. Therefore, in the P. minimum and P. micans bloom, Prorocentrum spp. apparently interfered with clam growth through combined effects of poor assimilation of P. minimum and poor nutritional value of P. micans. 3.2. Northern bay scallops, Argopecten irradians irradians Juveniles were exposed to the same mixed algal diet of EXUV and T-ISO as detailed above for clams. In one trial, all scallops fed a mixed diet of Isochrysis sp. (T-ISO) and P. minimum were dead in one week; whereas, no changes in shellfish survival or health in other feeding treatments were apparent. On re-starting with new scallops and the same EXUV+T-ISO mix, mortality occurred slowly over several weeks. Affected scallops showed heavily-impacted digestive diverticula, with necrosis of absorptive and some basal cells. In addition, accumulations of hemocytes were observed throughout the open vascular system consistent with tissue damage and/or effects of a chemical toxin (Wikfors and Smolowitz, 1993). Further, mortalities of bay scallops, but not clams, exposed to P. minimum in the laboratory suggested that the responses of bivalves to P. minimum could be species-specific.
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P. minimum blooms are common in oyster habitats, occasionally seasonally coincident with oyster spawning. Earlier work with C. virginica included laboratory studies of the dinoflagellate’s trophic interactions with several life history stages of the eastern oyster, including spat and juveniles (Luckenbach et al., 1993; Sellner et al., 1995), with dramatic impacts, including pseudofeces production in short term exposures and high mortalities after several weeks. Some of these studies subsequently were repeated and applied to younger oysters using the strain of P. minimum (EXUV) used in previous clam and scallop exposures (Wikfors and Smolowitz, 1993). With the EXUV strain, embryo development was not affected by live cultures or culture extracts, suggesting the absence of a water-soluble toxin (Wikfors and Smolowitz, 1995). Larvae-fed EXUV showed a number of developmental and histopathological abnormalities and did not metamorphose. Reanalysis of oyster larval growth data using the Jonasdottir et al. (1998) model shows no evidence of toxicity (Fig. 2). It appears that P. minimum somewhat compensates for nutritional deficiencies in the alternative food T-ISO, as growth on mixed diets was better than predicted by the dilution line, while itself being deficient in an essential nutrient present in T-ISO, noted by poor growth on P. minimum alone (far right of Fig. 2). Failure to metamorphose, however, suggested a fairly specific effect of P. minimum upon larval development.
In juveniles, transient digestive gland and systemic pathologies, followed by good growth, were seen (Wikfors and Smolowitz, 1995). After 2 weeks of no growth and copious pseudofeces production, juvenile oysters fed P. minimum grew more rapidly than those fed T-ISO alone, and combined diets were clearly superior to either alga alone (Fig. 3). A form of ‘‘cellular constipation,’’ in which digestive gland epithelial cells showed accumulations of undigested food vacuoles, was seen in juvenile oysters upon initial exposure to the dinoflagellate. Normal digestion eventually resumed, however, after approximately 2 weeks of continuous exposure. Results of oyster experiments conducted previously (Luckenbach et al., 1993) were far more direct. Young adult oysters were exposed to unialgal and mixed diets of cultured P. minimum and Thalassiosira weissflogii at bloom ((0.89 25) 104 cells/ml) and sub-bloom (33 and 5% of bloom) densities for several weeks. In this study, oyster mortalities were directly related to percentage of P. minimum in a mixed diet and to algal cell density. Results from the Jonasdottir et al. (1998) model reveal clear evidence of P. minimum toxicity, as a 50:50 mixed diet supported much less oyster growth than predicted by dilution (Fig. 4). Initial attempts to reconcile findings from the two studies suggested the possibility that oyster condition prior to exposure to P. minimum may have been responsible for the different results (Wikfors and Smolowitz, 1995); perhaps ‘‘healthier’’ oysters were better able to defend against tissue damage caused by initial exposure to P. minimum. This hypothesis is supported by the recent
Fig. 2. Growth (shell length in mm) of larvae of the eastern oyster, Crassostrea virginica, on cultures of Prorocentrum minimum and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1995). Maximum cell densities were 4 103 and 6 104 ml 1, respectively.
Fig. 3. Growth of juvenile oysters, Crassostrea virginica (shell height in mm), on cultures of Prorocentrum minimum and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1995). Maximum cell densities were 5 104 and 5 105 ml 1, respectively.
3.3. Eastern oysters, Crassostrea virginica
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Fig. 4. Growth of adult oysters, Crassostrea virginica (mm2 per day shell area), on cultures of Prorocentrum minimum and Thalassiosira weissflogii (replotted data from Luckenbach et al., 1993). Maximum cell densities were 2.5 105 ml 1.
demonstration of differences in immune response of oysters from different populations to P. minimum (He´ garet and Wikfors, 2005b). The additional findings described below, however, suggest that differences in ‘‘health’’ of the P. minimum cultures in these two studies may have contributed to differences in oyster response as well. The P. minimum cultures used in the Wikfors and Smolowitz (1993, 1995) studies were bacteria-free and were produced under highly-controlled environmental conditions (Ukeles, 1973) whereas the algal cultures used in the Luckenbach et al. (1993) study were produced in large (2500l) tanks where control of environmental conditions and microbial contamination was not possible.
4. Variability in P. minimum toxicity It appears that P. minimum is toxic only sporadically. P. minimum populations ‘‘in decline’’ appear to be more toxic than those growing actively. Grzebyk et al. (1997) found mouse-bioassay toxicity in methanol extracts from some, but not all, cultured P. minimum isolates, and only in cultures in the ‘‘decline’’ phase of the growth cycle. Highest toxicity was associated with increases in bacteria within the algal cultures, but bacteria-free cultures produced toxic effects in mice as well. Thus, it appears that P. minimum is capable of producing some uncharacterized toxins under some conditions. However, accumulation of the toxins in shellfish tissues, and subsequent effects in mammals after consuming the shellfish, have not been demonstrated. Further, it is not
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at all clear if the compound implicated as a mammalian toxin (Akiba and Hattori, 1949) is responsible for toxic effects seen in mollusks. Recent studies have been undertaken to determine the immune responses of oysters and scallops to HAB exposure. Bay scallops had been more sensitive than oysters in previous experiments; therefore, post-set scallop ‘‘bioassays’’ were conducted with cultures of several microalgal strains, including P. minimum strain JA-98-01, isolated from a Chesapeake Bay bloom associated with oyster mortalities (Glibert, personal communication). This strain was acutely toxic to bay scallops in 24 h experiments conducted two years previously (Rosetta and McManus, 2003). However, several 24 and 48 h scallop exposures ended with no mortalities, but heavy production of pseudofeces (Wikfors, unpublished observation). Carboy cultures from which algae were harvested for these exposures were unusually dense and deeply-pigmented. To cause a ‘‘decline’’ in one of two identical carboy cultures, carbon, supplied by an airstone dispersing an airstream enriched with 5% carbon dioxide gas, was withheld. After five days of carbon deprivation, the culture began to appear achlorotic, while the second culture with carbon supply remained well pigmented. In a subsequent scallop bioassay, the carbon-deprived P. minimum culture was acutely toxic (100% mortality in 24 h), while the healthy culture killed scallops only after four days of continuous exposure (He´ garet, 2003; He´ garet and Wikfors, 2005a). In subsequent studies of oyster immune response (He´ garet and Wikfors, 2005a,b), oysters produced pseudofeces containing intact P. minimum when the dinoflagellate was present, alone or in a mix, in laboratory exposures and in a natural bloom. Thus, it appears that oysters detect P. minimum and adjust feeding and biodeposit production within hours to minimize exposure. This observation is in contrast to relatively high initial clearance and ingestion rates measured in 4 h exposures of another bivalve species, G. chinensis (Lee and Chung, 2001; Lee et al., 2003). Despite limited ingestion, consistent changes in hemocyte numbers and functional characteristics were shown in oysters and scallops exposed to P. minimum in natural and simulated blooms; decreased numbers of hyalinocytes and increased numbers of dead hemocytes were among the most extreme effects (He´ garet and Wikfors, 2005a,b).
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The observation that toxicity of one strain of P. minimum was dependent upon carbon availability and previous observations that cultures ‘‘in decline’’ produce more extractable toxin (Grzebyk et al., 1997) suggest that differences between laboratory experiments previously ascribed to shellfish or algal strain differences may, in fact, have a basis in the physiological status of P. minimum cultures tested (Luckenbach et al., 1993; Wikfors and Smolowitz, 1995). Changes in per-cell toxin content dependent upon physiological status of a culture have been reported in P. lima (Quilliam et al., 1996) and other harmful algal species (Graneli et al., 1998). Specific environmental conditions could induce toxin production in P. minimum. Carbon deprivation is likely during an intense bloom on a sunny day (Hinga, 1992). Changes in pH and nutrient availability during periods of high photosynthesis could also cause physiological stress in P. minimum leading to toxin production. Such physiological stress could result in a portion of the population undergoing apoptotic cell degeneration (Veldhuis et al., 1997). This is consistent with previously observed associations of dinoflagellate autolysosomes with digestive pathologies in oysters (Wikfors and Smolowitz, 1995). Alternatively, it is possible that an unusual fatty acid, 18:5n3, present in P. minimum (Leblond and Chapman, 2000) may be the toxic agent in this species as it is in another dinoflagellate, Gymnodinium mikimotoi (Sola et al., 1999). At present, the chemical compound or compounds responsible for P. minimum effects on mollusks and mice remain unknown. Current evidence, however, indicates that this dinoflagellate has the capability to affect marine ecosystems by stressing or killing grazing mollusks and to produce toxins affecting mammals. The environmental conditions under which P. minimum expresses its toxic nature, and the possible effects of chronic, sub-lethal exposure of marine life to this dinoflagellate, remain to be clarified.
5. Mollusk grazing importance in the natural environment The role of bivalve mollusks in top-down control on P. minimum bloom initiation and termination appears to be weak because:
(a) The first and most consistent response of three bivalve species tested to long-term P. minimum exposure is production of pseudofeces containing intact cells; these cells, presumably, could be remixed into the water column. (b) ‘‘Pelagic’’ grazers, including micro- and mesozooplankton and some planktivorous fishes, appear to be more effective predators than bivalves (Friedland et al., 1989; Dam and Colin, 2005). (c) Harmful effects of P. minimum upon bivalves are most severe only after a bloom has been established and some portion of the population is in physiological decline. Thus, one possible condition for bloom initiation and termination could be failure of the pelagic grazing community to maintain top-down control on population growth (Turner and Tester, 1997; Johnson et al., 2003). In this scenario, bivalves ultimately suffer from changes in trophic interactions elsewhere in the food web. As poor larval development (Wikfors and Smolowitz, 1995) and altered immune-system competence (hence, disease resistance) in oysters (He´ garet and Wikfors, 2005a,b), are among the effects of P. minimum blooms, recovery of oyster populations in waters experiencing recurring P. minimum blooms could require a more holistic program of ecosystem restoration than simply planting more oysters. Acknowledgements I thank Pat Glibert for the invitation to contribute this review. Many ideas expressed herein arose from conversations with collaborators on primary research on HAB-shellfish interactions: Roxanna Smolowitz, He´ le`ne He´ garet, Hans Dam, George McManus, and Sandra Shumway. Elizabeth Wikfors provided many helpful comments on an initial draft. Primary research summarized here was supported by the NOAA Oyster Disease Research Initiative, ECOHAB, Connecticut Sea Grant, OAR International Programs, and the National Marine Fisheries Service.
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A review and new analysis of trophic interactions between Prorocentrum minimum and clams, scallops, and oysters Gary H. Wikfors* NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA. Received 1 March 2004; received in revised form 1 June 2004; accepted 1 August 2004
Abstract There has been no consensus on whether Prorocentrum minimum is ‘‘toxic,’’ despite sporadic reports suggesting possible shellfish toxicity and laboratory studies showing harmful effects of this dinoflagellate on molluscan shellfish. Shellfish toxicity outbreaks associated with natural blooms of P. minimum have been confounded by co-occurrence of other toxic phytoplankton. Laboratory studies have demonstrated unequivocally that some P. minimum isolates can produce toxins that kill mice on injection, but the bioactive compound or compounds remain unidentified, and accumulation of toxin in grazing mollusks has not been demonstrated. Laboratory experiments testing the responses of grazing mollusks to P. minimum cultures have yielded variable results, ranging from mortality in scallops and oysters to normal growth of oysters. Effects observed in the laboratory include rejection as pseudofeces by clams, poor larval development in oysters, tissue pathologies (sometimes transient) in oysters and scallops, and systemic immune responses in oysters and scallops. Several recent studies have provided evidence that variation in toxicity of P. minimum is dependent on environmental conditions and their effects on the physiology of this dinoflagellate. Accordingly, seemingly conflicting observations from field and laboratory studies may be explained by transient toxin expression in P. minimum. # 2004 Elsevier B.V. All rights reserved. Keywords: Bivalve mollusk; Dinoflagellate; HAB; Harmful algae
1. Introduction Spring and summer blooms of P. minimum have been recorded as a fairly regular feature of annual phytoplankton successions in estuarine waters worldwide (Dodge, 1975; Hajdu et al., 2005; Heil et al., 2005). Where filter-feeding bivalves exert strong * Tel.: +1 203 882 6525; fax: +1 203 882 6517. E-mail address: [email protected].
grazing pressure on estuarine phytoplankton, biomass accumulation of a specific alga may be a consequence of population growth more rapid than normal grazing removal or of grazing repression by the algae. Some microalgal biotoxins are thought to have a grazingdeterrent genesis in evolutionary time (Landsberg, 2002) and examples of chemical and physical deterrents to grazing are common in land plants (Bell and Woodcock, 1968; Muenscher, 1975; Smith, 1992; Simmons and Ekarius, 2001). Different bivalve
1568-9883/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2004.08.008
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species accumulate and tolerate different quantities of toxin from dinoflagellates such as Alexandrium (Fernandez et al., 2003) and selection for tolerant grazers in regions experiencing recurrent toxic algal blooms has been demonstrated (Bricelj et al., 2000; Colin and Dam, 2002; He´ garet and Wikfors, 2005b). Co-occurrence of bivalve populations and P. minimum blooms thus provides the potential for various trophic interactions, including nutritional utilization, grazing repression through a variety of physiological effects, and toxin accumulation. Until recently, most evidence for harmful effects of the dinoflagellate P. minimum on molluscan shellfish was based on anecdotal observations of shellfish mortalities coincident with blooms in coastal waters and several laboratory experiments in which cultured strains of P. minimum were fed to shellfish. Two recent reports by He´ garet and Wikfors, 2005a,b), showing the same changes in immune-system status associated with laboratory exposures and field measurements of oysters feeding on natural blooms of P. minimum, provide confidence that ‘‘artificial’’ exposures of grazing shellfish to P. minimum in the laboratory can be good proxies for natural trophic interactions. The present re-analysis of laboratory experiments was undertaken to explore possible reasons for inconsistent findings and to interpret these findings in an environmental context.
2. Field observations The first suggestion that P. minimum be included in the list of harmful algae arose from a trophic interaction with shellfish. A shellfish-poisoning outbreak in Japan was associated initially with consumption of Venerupus clams harvested from a bay with a P. minimum bloom (Akiba and Hattori, 1949). This shellfish poisoning outbreak could not be attributed directly to P. minimum and subsequent associations between shellfish toxicity and natural blooms of P. minimum have been inconclusive because of cooccurrence of known toxin-forming dinoflagellates (see discussion in Landsberg, 2002). Environmental observations suggesting that P. minimum blooms are harmful to other marine species are not consistent for different species, or even for a given species in different instances. A positive
correlation between P. minimum and presence of the planktivorous fish, Brevoortia tyrannus, was noted by Friedland et al. (1989), but kills of planktivorous fish, sometimes but not always attributed to hypoxia during P. minimum blooms, have been recorded as well (Rabbani et al., 1990; Yaquin-Li, Connecticut Department of Environmental Protection, personal communication). Responses of grazing zooplankton, crustaceans, and protozoans to P. minimum are discussed elsewhere in this volume, but the consensus seems to be that this dinoflagellate may be nutritionally deficient–or excellent food–but generally is not toxic to these pelagic grazers. However, repeated, but not consistent, observations of detrimental effects have been recorded for several species of bivalve mollusks, including scallops, clams, and oysters. The first account in the scientific literature of specific harmful effects of Prorocentrum on a bivalve mollusk was that of Leibovitz et al. (1984) noting intact Prorocentrum cells in the open vascular system of bay scallops (Argopecten irradians irradians) feeding in a natural bloom. It was hypothesized that high scallop mortalities during this bloom were caused by physical damage from the apical tooth, a sharp anterior spine on some Prorocentrum taxa. This hypothesis has not been confirmed and follow-up studies of scallops in natural P. minimum blooms were not conducted until very recently (see below). In another bivalve species, the northern quahog clam Mercenaria mercenaria, evidence that a natural bloom of P. minimum interfered with growth was obtained during an aquaculture site-comparison study in Long Island Sound (Connecticut, USA) (Wikfors and Smolowitz, 1993). In two sites experiencing a bloom of several weeks duration, food availability was very high, but clam growth ceased. No mortalities were seen and normal growth resumed after the bloom. This also led to laboratory experiments to determine if arrested clam growth could be attributed to either of two Prorocentrum taxa co-occurring in this bloom, P. minimum or P. micans (see below). In addition to scallops and clams, oysters may be affected by P. minimum blooms. Mortality and poor growth in wild populations of eastern oysters Crassostrea virginica ingesting Chesapeake Bay P. minimum blooms have also been noted (Luckenbach et al., 1993; Sellner et al., 1995). Empirical observations of shellfish mortalities during P. minimum blooms are also recorded in
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the literature (Yongjia et al., 1995) and monitoring reports (shellfish rearing records, NMFS Milford Laboratory), but a number of system parameters could produce mortality, including changes in water chemistry associated with the bloom (e.g., low dissolved oxygen) or other factors coincident with the bloom. Field observations have provided evidence, but not proof, that P. minimum can be toxic to bivalve mollusks or make them toxic to human consumers.
3. Laboratory observations There is little doubt that bivalves ingest P. minimum. Various life-history stages of several bivalve species are able to filter and ingest cultured P. minimum cells at environmentally-realistic concentrations: larval mussels, Mytilus galloprovincialis, (Jeong et al., 2004); adult Glauconome chinensis (Lee and Chung, 2001; Lee et al., 2003); adult sea scallops, Placopecten magellanicus (Brilliant and MacDonald, 2002); and juvenile eastern oysters, C. virginica (Sellner et al., 1995). 3.1. Northern quahog clams, Mercenaria mercenaria The observations of poor clam growth during a Prorocentrum (P. micans and P. minimum co-dominant) bloom in Long Island Sound were explored in an experiment where juveniles were fed cultured P. micans, alone or in combination with a control diet of Isochrysis sp. (T-ISO), or P. minimum in combination with T-ISO (Wikfors and Smolowitz, 1993). Survival and growth of clams on the mixed diet of P. minimum and T-ISO were no different than for unfed clams and undigested P. minimum cells were seen in clam biodeposits. Thus, direct evidence of toxicity of P. minimum to clams was not apparent and it is more likely that clams minimized their exposure to P. minimum by reducing feeding and rejecting cells as pseudofeces. Results in the field might also have been a result of P. micans having poor nutritional value for clams. This was assessed using the Jonasdottir et al. (1998) model (Fig. 1) where growth would be linearly dependent upon percentages of ‘‘good’’ and ‘‘bad’’ food (the better food is simply ‘diluted’ by the poorer). Growth lower than predicted by dilution suggests toxicity of the poorer; wheras, higher growth indicates that the
Fig. 1. Growth of northern quahogs, Mercenaria mercenaria (grams per clam in 8 weeks), on cultures of Prorocentrum micans and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1993). Maximum cell densities were 5 104 and 5 105 ml 1, respectively. A straight line represents nutritional deficiency of the poorer diet, while observations above or below the line represent nutritional enhancement of food quality or toxicity, respectively.
two foods complement each other’s nutritional deficiencies. The linear decline in clam growth with P. micans indicates simply poor nutritional value of this species to clams. P. minimum could not be assessed with this model because it was fed to clams in one mixture only. Therefore, in the P. minimum and P. micans bloom, Prorocentrum spp. apparently interfered with clam growth through combined effects of poor assimilation of P. minimum and poor nutritional value of P. micans. 3.2. Northern bay scallops, Argopecten irradians irradians Juveniles were exposed to the same mixed algal diet of EXUV and T-ISO as detailed above for clams. In one trial, all scallops fed a mixed diet of Isochrysis sp. (T-ISO) and P. minimum were dead in one week; whereas, no changes in shellfish survival or health in other feeding treatments were apparent. On re-starting with new scallops and the same EXUV+T-ISO mix, mortality occurred slowly over several weeks. Affected scallops showed heavily-impacted digestive diverticula, with necrosis of absorptive and some basal cells. In addition, accumulations of hemocytes were observed throughout the open vascular system consistent with tissue damage and/or effects of a chemical toxin (Wikfors and Smolowitz, 1993). Further, mortalities of bay scallops, but not clams, exposed to P. minimum in the laboratory suggested that the responses of bivalves to P. minimum could be species-specific.
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P. minimum blooms are common in oyster habitats, occasionally seasonally coincident with oyster spawning. Earlier work with C. virginica included laboratory studies of the dinoflagellate’s trophic interactions with several life history stages of the eastern oyster, including spat and juveniles (Luckenbach et al., 1993; Sellner et al., 1995), with dramatic impacts, including pseudofeces production in short term exposures and high mortalities after several weeks. Some of these studies subsequently were repeated and applied to younger oysters using the strain of P. minimum (EXUV) used in previous clam and scallop exposures (Wikfors and Smolowitz, 1993). With the EXUV strain, embryo development was not affected by live cultures or culture extracts, suggesting the absence of a water-soluble toxin (Wikfors and Smolowitz, 1995). Larvae-fed EXUV showed a number of developmental and histopathological abnormalities and did not metamorphose. Reanalysis of oyster larval growth data using the Jonasdottir et al. (1998) model shows no evidence of toxicity (Fig. 2). It appears that P. minimum somewhat compensates for nutritional deficiencies in the alternative food T-ISO, as growth on mixed diets was better than predicted by the dilution line, while itself being deficient in an essential nutrient present in T-ISO, noted by poor growth on P. minimum alone (far right of Fig. 2). Failure to metamorphose, however, suggested a fairly specific effect of P. minimum upon larval development.
In juveniles, transient digestive gland and systemic pathologies, followed by good growth, were seen (Wikfors and Smolowitz, 1995). After 2 weeks of no growth and copious pseudofeces production, juvenile oysters fed P. minimum grew more rapidly than those fed T-ISO alone, and combined diets were clearly superior to either alga alone (Fig. 3). A form of ‘‘cellular constipation,’’ in which digestive gland epithelial cells showed accumulations of undigested food vacuoles, was seen in juvenile oysters upon initial exposure to the dinoflagellate. Normal digestion eventually resumed, however, after approximately 2 weeks of continuous exposure. Results of oyster experiments conducted previously (Luckenbach et al., 1993) were far more direct. Young adult oysters were exposed to unialgal and mixed diets of cultured P. minimum and Thalassiosira weissflogii at bloom ((0.89 25) 104 cells/ml) and sub-bloom (33 and 5% of bloom) densities for several weeks. In this study, oyster mortalities were directly related to percentage of P. minimum in a mixed diet and to algal cell density. Results from the Jonasdottir et al. (1998) model reveal clear evidence of P. minimum toxicity, as a 50:50 mixed diet supported much less oyster growth than predicted by dilution (Fig. 4). Initial attempts to reconcile findings from the two studies suggested the possibility that oyster condition prior to exposure to P. minimum may have been responsible for the different results (Wikfors and Smolowitz, 1995); perhaps ‘‘healthier’’ oysters were better able to defend against tissue damage caused by initial exposure to P. minimum. This hypothesis is supported by the recent
Fig. 2. Growth (shell length in mm) of larvae of the eastern oyster, Crassostrea virginica, on cultures of Prorocentrum minimum and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1995). Maximum cell densities were 4 103 and 6 104 ml 1, respectively.
Fig. 3. Growth of juvenile oysters, Crassostrea virginica (shell height in mm), on cultures of Prorocentrum minimum and Isochrysis sp. (T-ISO) (replotted data from Wikfors and Smolowitz, 1995). Maximum cell densities were 5 104 and 5 105 ml 1, respectively.
3.3. Eastern oysters, Crassostrea virginica
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Fig. 4. Growth of adult oysters, Crassostrea virginica (mm2 per day shell area), on cultures of Prorocentrum minimum and Thalassiosira weissflogii (replotted data from Luckenbach et al., 1993). Maximum cell densities were 2.5 105 ml 1.
demonstration of differences in immune response of oysters from different populations to P. minimum (He´ garet and Wikfors, 2005b). The additional findings described below, however, suggest that differences in ‘‘health’’ of the P. minimum cultures in these two studies may have contributed to differences in oyster response as well. The P. minimum cultures used in the Wikfors and Smolowitz (1993, 1995) studies were bacteria-free and were produced under highly-controlled environmental conditions (Ukeles, 1973) whereas the algal cultures used in the Luckenbach et al. (1993) study were produced in large (2500l) tanks where control of environmental conditions and microbial contamination was not possible.
4. Variability in P. minimum toxicity It appears that P. minimum is toxic only sporadically. P. minimum populations ‘‘in decline’’ appear to be more toxic than those growing actively. Grzebyk et al. (1997) found mouse-bioassay toxicity in methanol extracts from some, but not all, cultured P. minimum isolates, and only in cultures in the ‘‘decline’’ phase of the growth cycle. Highest toxicity was associated with increases in bacteria within the algal cultures, but bacteria-free cultures produced toxic effects in mice as well. Thus, it appears that P. minimum is capable of producing some uncharacterized toxins under some conditions. However, accumulation of the toxins in shellfish tissues, and subsequent effects in mammals after consuming the shellfish, have not been demonstrated. Further, it is not
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at all clear if the compound implicated as a mammalian toxin (Akiba and Hattori, 1949) is responsible for toxic effects seen in mollusks. Recent studies have been undertaken to determine the immune responses of oysters and scallops to HAB exposure. Bay scallops had been more sensitive than oysters in previous experiments; therefore, post-set scallop ‘‘bioassays’’ were conducted with cultures of several microalgal strains, including P. minimum strain JA-98-01, isolated from a Chesapeake Bay bloom associated with oyster mortalities (Glibert, personal communication). This strain was acutely toxic to bay scallops in 24 h experiments conducted two years previously (Rosetta and McManus, 2003). However, several 24 and 48 h scallop exposures ended with no mortalities, but heavy production of pseudofeces (Wikfors, unpublished observation). Carboy cultures from which algae were harvested for these exposures were unusually dense and deeply-pigmented. To cause a ‘‘decline’’ in one of two identical carboy cultures, carbon, supplied by an airstone dispersing an airstream enriched with 5% carbon dioxide gas, was withheld. After five days of carbon deprivation, the culture began to appear achlorotic, while the second culture with carbon supply remained well pigmented. In a subsequent scallop bioassay, the carbon-deprived P. minimum culture was acutely toxic (100% mortality in 24 h), while the healthy culture killed scallops only after four days of continuous exposure (He´ garet, 2003; He´ garet and Wikfors, 2005a). In subsequent studies of oyster immune response (He´ garet and Wikfors, 2005a,b), oysters produced pseudofeces containing intact P. minimum when the dinoflagellate was present, alone or in a mix, in laboratory exposures and in a natural bloom. Thus, it appears that oysters detect P. minimum and adjust feeding and biodeposit production within hours to minimize exposure. This observation is in contrast to relatively high initial clearance and ingestion rates measured in 4 h exposures of another bivalve species, G. chinensis (Lee and Chung, 2001; Lee et al., 2003). Despite limited ingestion, consistent changes in hemocyte numbers and functional characteristics were shown in oysters and scallops exposed to P. minimum in natural and simulated blooms; decreased numbers of hyalinocytes and increased numbers of dead hemocytes were among the most extreme effects (He´ garet and Wikfors, 2005a,b).
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The observation that toxicity of one strain of P. minimum was dependent upon carbon availability and previous observations that cultures ‘‘in decline’’ produce more extractable toxin (Grzebyk et al., 1997) suggest that differences between laboratory experiments previously ascribed to shellfish or algal strain differences may, in fact, have a basis in the physiological status of P. minimum cultures tested (Luckenbach et al., 1993; Wikfors and Smolowitz, 1995). Changes in per-cell toxin content dependent upon physiological status of a culture have been reported in P. lima (Quilliam et al., 1996) and other harmful algal species (Graneli et al., 1998). Specific environmental conditions could induce toxin production in P. minimum. Carbon deprivation is likely during an intense bloom on a sunny day (Hinga, 1992). Changes in pH and nutrient availability during periods of high photosynthesis could also cause physiological stress in P. minimum leading to toxin production. Such physiological stress could result in a portion of the population undergoing apoptotic cell degeneration (Veldhuis et al., 1997). This is consistent with previously observed associations of dinoflagellate autolysosomes with digestive pathologies in oysters (Wikfors and Smolowitz, 1995). Alternatively, it is possible that an unusual fatty acid, 18:5n3, present in P. minimum (Leblond and Chapman, 2000) may be the toxic agent in this species as it is in another dinoflagellate, Gymnodinium mikimotoi (Sola et al., 1999). At present, the chemical compound or compounds responsible for P. minimum effects on mollusks and mice remain unknown. Current evidence, however, indicates that this dinoflagellate has the capability to affect marine ecosystems by stressing or killing grazing mollusks and to produce toxins affecting mammals. The environmental conditions under which P. minimum expresses its toxic nature, and the possible effects of chronic, sub-lethal exposure of marine life to this dinoflagellate, remain to be clarified.
5. Mollusk grazing importance in the natural environment The role of bivalve mollusks in top-down control on P. minimum bloom initiation and termination appears to be weak because:
(a) The first and most consistent response of three bivalve species tested to long-term P. minimum exposure is production of pseudofeces containing intact cells; these cells, presumably, could be remixed into the water column. (b) ‘‘Pelagic’’ grazers, including micro- and mesozooplankton and some planktivorous fishes, appear to be more effective predators than bivalves (Friedland et al., 1989; Dam and Colin, 2005). (c) Harmful effects of P. minimum upon bivalves are most severe only after a bloom has been established and some portion of the population is in physiological decline. Thus, one possible condition for bloom initiation and termination could be failure of the pelagic grazing community to maintain top-down control on population growth (Turner and Tester, 1997; Johnson et al., 2003). In this scenario, bivalves ultimately suffer from changes in trophic interactions elsewhere in the food web. As poor larval development (Wikfors and Smolowitz, 1995) and altered immune-system competence (hence, disease resistance) in oysters (He´ garet and Wikfors, 2005a,b), are among the effects of P. minimum blooms, recovery of oyster populations in waters experiencing recurring P. minimum blooms could require a more holistic program of ecosystem restoration than simply planting more oysters. Acknowledgements I thank Pat Glibert for the invitation to contribute this review. Many ideas expressed herein arose from conversations with collaborators on primary research on HAB-shellfish interactions: Roxanna Smolowitz, He´ le`ne He´ garet, Hans Dam, George McManus, and Sandra Shumway. Elizabeth Wikfors provided many helpful comments on an initial draft. Primary research summarized here was supported by the NOAA Oyster Disease Research Initiative, ECOHAB, Connecticut Sea Grant, OAR International Programs, and the National Marine Fisheries Service.
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