Botanica Marina 55 (2012): 19–29 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/BOT.2011.123

Bioactive effects of Prorocentrum minimum on juvenile bay scallops ( Argopecten irradians irradians ) are dependent upon algal physiological status

Yaqin Li 1, *, Inke Sunila 2 and Gary H. Wikfors 1

1 Milford Laboratory , NOAA National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460 , USA , e-mail: judy.yaqin.li@noaa.gov 2 State of Connecticut , Department of Agriculture, Bureau of Aquaculture, P.O. Box 97, Milford, CT 06460 , USA

* Corresponding author

Abstract

The harmful dinofl agellate Prorocentrum minimum report-edly has variable toxicity to grazing animals. We used the bay scallop Argopecten irradians irradians as a bioassay organ-ism to compare expression of harmful effects of P. minimum cultures in growth or senescent phases. The non-toxic alga Rhodomonas sp. was used as a control. Exposure to both types of P. minimum cultures decreased the degree of shell opening, amount of biodeposits produced, motility and byssal-thread attachment. As P. minimum cultures approached senescence, effects became more severe, and mortality increased to 15 % in scallops exposed to senescent P. minimum . Pathological effects of P. minimum on scallops included derangements of scallop digestive tubules and the adductor muscles, and abnormal hemocyte distributions, which were more severe in scallops exposed to senescent cultures. These fi ndings help to explain the variable toxicity of P. minimum to scallops and other bivalves reported in the literature. Further, these fi ndings demonstrate that the defi nition of a “ harmful algal bloom ” should probably be expanded to include physiologi-cal status along with identity and abundance of a phytoplank-ton species.

Keywords: growth stage; Prorocentrum minimum ; scallop; toxicity.

Introduction

In 1997, Smayda did the fi eld of biological oceanography and the specialized fi eld of harmful algal bloom (HAB) research an enormous favor by evaluating both the language and con-cepts involved in defi ning phytoplankton blooms in general and HABs in particular (Smayda 1997 ). In this Commentary, Smayda differentiated the temperate, spring diatom bloom from population cycles of harmful species, thereby allow-ing the diversity of phytoplankton successional events and

trophic consequences to be considered independently of biases imposed by the spring bloom paradigm. In addressing directly the question, “ What is a harmful bloom ? ” Smayda ( 1997 ) concluded: “ Research on harmful algal blooms should contribute signifi cantly to the quantifi cation of both the common and divergent properties of the different bloom events occurring in the sea, beyond resolving many of the puzzling features of this particular class of blooms. ” In the present report, we address one of the “ divergent properties ” beyond the species and biomass issues discussed in Smayda ’ s Commentary, i.e., variable expression of toxicity in a species that may be benign or minimally harmful in one physiological state but possessing the potential to suddenly become toxic to grazing animals.

Laboratory studies have shown that harmful algae affect bivalve shellfi sh in a number of ways. Responses of bivalves to harmful algae can include pathological changes in tissues (Wikfors and Smolowitz 1993, 1995 , Galimany et al. 2008 ), immunomodulation ( H é garet and Wikfors 2005a,b , Galimany et al. 2008 ), or rejection as pseudofaeces ( H é garet et al. 2007 ). These effects may lead to mortality and poor larval develop-ment (Luckenbach et al. 1993 , Wikfors and Smolowitz 1995 , Brownlee et al. 2008 ). The effects of particular harmful algal species upon bivalves appear to be species specifi c; thus, responses and effects from one species cannot be generalized to another (Wikfors 2005 , Galimany et al. 2008 ).

The dinofl agellate Prorocentrum minimum (Pavillard) J. Schiller is listed among the HAB phytoplankton species, because it is sometimes associated with harmful effects upon shellfi sh and other marine life in coastal waters (Heil et al. 2005 ). Studies with cultured isolates of this species have revealed a diverse array of bioactive effects, including inhibi-tion of bacterial growth (Trick et al. 1984a ) and allelopathic effects on other phytoplankton species (Iwasaki 1979 ). In suspension-feeding bivalve mollusks, P. minimum causes altered feeding behavior (Shumway et al. 1985 , H é garet et al. 2007 ), pathological changes in tissues (Wikfors and Smolowitz 1993, 1995 ), immunomodulation ( H é garet and Wikfors 2005a,b , Galimany et al. 2008 ), and mortality (Luckenbach et al. 1993 ). Toxicity of cell extracts injected into mice has been demonstrated (Grzebyk et al. 1997 ), but accumulation of toxin in feeding shellfi sh and subsequent transfer to mammalian consumers of the shellfi sh has not been demonstrated unequivocally.

Chemical identities of toxic agents in P. minimum remain poorly known, although effects upon bacteria have been attributed to a diketone believed to be a product of carote-noid degradation (Trick et al. 1981 ). Other studies have

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20 Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops

shown no bioactive effects of P. minimum cultures (Sellner et al. 1995 ), leading to speculation that strains may dif-fer in toxicity, or expression of toxicity is dependent upon physiological status of the population (Grzebyk et al. 1997 , Wikfors 2005 ). In HAB species whose specifi c toxins can be quantifi ed, dependence of toxin content upon growth status of the species is observed often, e.g., in the diatom Pseudo-nitzschia seriata (Fehling et al. 2004 ), and the dinofl agellates Karlodinium venefi cum (Adolf et al. 2009 ), Dinophysis acum-inata (Tong et al. 2011 ), and Prorocentrum lima (Varkitzi et al. 2010 ). Recently Saba and co-workers reported higher mortalities of juvenile oysters exposed to late-stationary cultures of P. minimum , compared to log-phase cultures of the same strain in a bioassay experiment (Saba et al. 2011 ). No study, however, has explicitly and adequately addressed whether or not the specifi c effects of P. minimum on bivalves are dependent upon this dinofl agellate ’ s physiological status.

Among bivalve species with distributions sympat-ric with the dinofl agellate P. minimum is the northern bay scallop Argopecten irradians irradians Lamarck, a commercially-important shellfi sh species for both wild fi sh-eries and aquaculture and enhancement efforts (Blake and Shumway 2006 ). This scallop is native to the northwestern Atlantic coast, with a distribution extending from the north shore of Cape Cod, Massachusetts, to the area between New Jersey and Maryland (Brand 2006 ). The region frequently experiences summer P. minimum blooms (Heil et al. 2005 ). The mere presence of P. minimum does not indicate subse-quent adverse impact upon the scallops, because P. minimum is variably toxic to bivalves. Thus, revealing the conditions and physiological status under which P. minimum expresses toxicity to scallops, and the range of responses of scallops to P. minimum, has practical implications, such as providing early warning to aquaculture or wild-harvest fi sheries.

The present study was designed to investigate whether or not bioactivity of P. minimum leading to adverse effects upon northern bay scallops depends upon the physiological status of the alga. In the laboratory, we measured different physi-ological and pathological parameters of hatchery-raised bay scallops after exposure to cultured P. minimum in different physiological states.

Materials and methods

Algal cultures

The JA-98-01 strain of Prorocentrum minimum used in this study was obtained from the Milford Microalgal Culture Collection. It is not a bacteria-free culture. The strain was isolated from the Choptank River Estuary in Maryland (USA) in 1998. Cultures were grown in EDL7 medium, modifi ed from the E formulation of Ukeles (Ukeles 1973 ) as follows: substituting L-1 trace met-als (Guillard and Hargraves 1993 ) for E metals mix, substitute 46 mg l -1 sodium glycerophosphate for inorganic phosphate, replacing sodium phosphate with potassium phosphate, doubling EDTA (ethylene disodium diamine tetraacetate) to 4.3 mg l -1 , and adding 100 mg l -1 calcium chloride heptahydrate.

The non-toxic alga used as a control, Rhodomonas sp. (strain RHODO), was also from the Milford Microalgal Culture Collection. E-medium was used for Rhodomonas sp. cultures. Both microalgae were cultured in 20-l glass carboy assemblies using aseptic techniques (Ukeles 1973 ). Culture conditions were 18 ° C, 24-h light at 300 μ mol photons m -2 s -1 with a continuous stream of air enriched with 5 % CO 2 .

Three identical, replicate carboys were established for each of two P. minimum culture strategies. In one set of rep-licate cultures, half of the culture was harvested and replaced with fresh culture medium weekly; we refer to this culture management routinely as “ semi-continuous ” (Ukeles 1973 ), although the term “ serially-diluted batch culture ” could also be applied. Three other identical, replicate P. minimum cultures were maintained under batch culture mode without adding fresh culture medium. Cell densities of P. minimum were determined weekly with a FACSAN fl ow cytometer. In addition to total cell density measurements, live vs dead cells were also differentiated and quantifi ed by applying Sytox ® Green (Invitrogen, Carlsbad, CA, USA) at a fi nal concentra-tion of 0.5 μ m followed by 15 min dark incubation before cytometer reading. Dead cells were identifi ed and quantifi ed by elevated green fl uorescence.

Bacterial counts

Densities of bacteria in Prorocentrum minimum cultures were quantifi ed following Neter (1970) . In short, P. minimum cultures were centrifuged at 1000 × g for 10 min to remove P. minimum cells. A tenfold serial dilution was performed on algal-cell-free culture media to 1:100,000 dilutions. Diluted culture medium (0.1 ml) was streaked onto a prepared plate with E agar medium. Inoculated plates were incubated at 22 ° C, and bacterial colo-nies growing on plates were counted after 1 and 2 weeks of incubation. Counts were adjusted for dilution and sample size streaked, and standard plate counts were reported as colony forming units per ml (cfu ml -1 ). Previous work had shown that only three bacterial morphotypes are present in the JA-98-01 P. minimum strain, and aseptic methods were used throughout this study; therefore, plate counts were used as a convenient way to compare cultures, on the understanding that these counts represented a portion of bacterial cells present.

Scallops

Northern bay scallops, Argopecten irradians irradians , were spawned in March 2008 in the Milford Laboratory, and larvae and post-set were grown in heated seawater (15 ± 1 ° C) pumped from the Milford Harbor. The scallops were 2 – 3 months old with a length of 8.06 ± 0.16 mm (means ± SE) when used in the experiment. Twenty-four hours before exposure, animals were transferred to 0.1- μ m-fi ltered seawater and gradually increased in temperature to the experimental temperature of 21 ± 1.5 ° C.

Experimental design

The experimental design of this study served two purposes: (1) to test the postulate that the bioactive effects of Prorocentrum

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Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops 21

minimum on bay scallops are different when this microalga is in a different physiological status, specifi cally growth phase or senescent phase, and (2) to study the pathological effects of P. minimum on bay scallops when the animals are exposed to P. minimum cultures of different physiological status. To monitor P. minimum bioactivity, a bay scallop bioassay (details below) was performed weekly, starting 1 week after the cultures were inoculated using P. minimum from both batch and continuous cultures. At the same time, growth of P. minimum was monitored. Bacteria in the cultures were also monitored weekly. Once scallop mortality was observed, the experiment was terminated and pathological analyses (detail below) were then conducted on sacrifi ced scallops.

Effects of Prorocentrum minimum on scallops –

scallop bioassay

The weekly scallop bioassay of Prorocentrum minimum bio-activity was done following methods modifi ed from Rosetta and McManus (2003) . Briefl y, for each treatment, three rep-licate, 1-l beakers with 500 ml of algal culture diluted with 1- μ m-fi ltered seawater were set up, each containing fi ve juve-nile bay scallops. The four experimental treatments were: (1) P. minimum cells from semi-continuous cultures, (2) P. mini-mum cells from batch cultures, (3) non-toxic Rhodomonas sp. as control, and (4) fi ltered sea water as another control to test if culture medium had an effect. In the cases of microalgal treatments, one-fi fth of the volume was culture fi ltrate, with the remainder fi ltered seawater. The initial cell densities of microalgae in the beakers were adjusted to 1 × 10 4 cell ml -1 , to approximate typical P. minimum bloom densities in nature. The cell densities of microalgae and amount of culture fi ltrate were achieved either by concentrating cells through centrifu-gation at 1000 × g for 10 min or by collecting extra fi ltrate by fi ltering culture through a 0.2- μ m polycarbonate membrane fi lter. Scallops exposed to these experimental treatments were monitored for movement, valve closure, and bio-deposit pro-duction every 10 min for the initial 2 h. Scallop mortality was determined after 24-h of exposure.

Effects of Prorocentrum minimum on scallops –

histopathological analyses

Histopathology of scallops was analyzed at three time points: (1) initial (before exposure), and after (2) 12-h and (3) 24-h exposures to the three different microalgal treatments ( Prorocentrum minimum batch culture, P. minimum semi-continuous culture, and the non-toxic Rhodomonas sp. Control). For each treatment and time point, 30 scallops were processed for histology for a statistically-signifi cant sample size (Post and Millest 1991 ).

Whole scallops were fi xed in Davidson ’ s fi xative for 5 days. Immediately before being placed in the fi xative, the shell of each scallop was notched to allow fi xative to pene-trate. Samples were washed in artifi cial sea water (salinity, 20) and decalcifi ed in 10 % EDTA. Samples were washed again in artifi cial sea water (salinity, 20), then in 50 % etha-nol in artifi cial seawater (salinity, 20), and transferred to 70 %

ethanol. Samples were dehydrated, infi ltrated, and embed-ded in paraffi n. After processing, 5- μ m sections were stained using a hematoxylin-eosin staining procedure (Howard et al. 2004 ) and examined under a light microscope.

To test if prevalences of pathological conditions were sig-nifi cantly different between different treatments, a G-test of independence with the Williams and the Yates corrections was applied (Sokal and Rohlf 1997 ).

Results

Prorocentrum minimum growth and physiological

status

Cell densities of Prorocentrum minimum followed those of typical batch cultures and semi-continuous cultures (Figure 1 A). In the batch culture, cell densities did not change signifi cantly during the fi rst week after inoculation. The cell number then increased at the exponential rate from 3.9 × 10 4 to 2.0 × 10 5 cell ml -1 in the second week (0.32 divisions day -1 ). Cell densities remained at this constant, maximal level for 1 week before decreasing to 9.6 × 10 3 cell ml -1 when the culture

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Figure 1 Prorocentrum minimum growth curves under batch (P.m batch) and semi-continuous (P.m cont) culture modes. (A) Cell densities of P. minimum determined weekly with a fl ow cytometer. (B) Percentage of live P. minimum cells in the cultures determined weekly with a fl ow cytometer using Sytox ® Green. Values are means ± SE (n = 3).

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22 Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops

entered stationary and then declining, senescent phase for the following 4 weeks. The percentages of live cells in the cultures were highest during the initial 3 weeks and then decreased as the cultures became senescent (Figure 1 B). In contrast to the batch cultures, semi-continuous cultures had relatively con-stant cell densities and percentages of live cells during the entire 7-week period (Figure 1 A, B). Assuming a division rate similar to the batch cultures ( ∼ 0.3 divisions day -1 ), the semi-continuous cultures with half-volume replacement weekly would be in late-log or early stationary phase.

Bacteria were present in both batch and semi-continuous cultures throughout the 7-week experiment. Bacterial counts in the two types of cultures were not statistically different. Total culturable bacteria, including white, pink and yellow colonies in the Prorocentrum minimum cultures, increased over time. Initially (1 week after the cultures were inocu-lated) bacterial counts were in the range of 2 – 3 × 10 3 cfu ml -1 , increasing to 1 – 2 × 10 5 cfu ml -1 on week 3. From week 4 to 5, bacterial densities exceeded the maximum detection limit of 3.0 × 10 5 cfu ml -1 . In week 6, bacterial counts in all cultures were above 1.0 × 10 7 cfu ml -1 when the maximum detection limit was increased.

Bioactive effects of Prorocentrum minimum cultures

on bay scallops

Behavioral responses Weekly scallop bioassays showed that both batch and continuous cultures of Prorocentrum had some degree of negative, physiological effect upon scallops compared to both of the controls (fi ltered sea water and Rhodomonas sp. culture). The effects included a decrease in the degree of shell opening (an indication of pumping), decrease in the amount of biodeposits produced, decrease in motility, and decrease in ability to attach by byssal threads to a hard surface. During the fi rst 3 weeks, the effects of both P. minimum cultures upon scallops were similar in severity. Over time (from week 4 to 7), however, the effects of batch cultures became more severe. In contrast, semi-continuous cultures showed a constant effect upon scallops over the entire 7-week experiment. At week 7, 15 % mortality in scallops exposed to the P. minimum batch cultures was observed, with no mortality in scallops exposed to semi-continuous cultures or in the control groups (p < 0.05).

Histopathological responses Prorocentrum minimum cells were observed in the alimentary tract (intestine and stomach) of the scallops after 12 and 24 h exposure, indicating ingestion. Most cells in the alimentary tract appeared to be intact (Figure 2 A, B). The number of scallops with P. minimum cells present was signifi cantly higher with exposure to batch culture than for semi-continuous cultures (after 12 h G = 3.73, p < 0.1, after 24 h G = 6.70, p < 0.01). After 12 and 24 h, P. minimum cells were present in 43 % and 47 % of the scallops exposed to batch culture, respectively, and in 18 % and 13 % of the scallops exposed to semi-continuous culture, respectively. There were no recognizable, intact Rhodomonas cells visible in the alimentary tract at any time point.

There were histophysiological changes in the digestive diver-ticula and adductor muscles of Prorocentrum minimum -exposed scallops. Digestive diverticula of Rhodomona s-fed scallops consisted of regular digestive tubules with columnar absorptive cells showing small, vacuolar inclusions. Basophilic basal cells were present. Scallops exposed to P. minimum batch culture had polymorphic digestive tubules consisting of abnormally-vacuolated absorptive cells that varied from columnar to cuboi-dal to squamous (Figure 2 C, D). These cells had detached in large numbers, and detached cells were observed in the lumina of the digestive tubules. Basal cells were apparently intact. Large numbers of detached, absorptive cells were also observed in the digestive ducts and in the stomach, where they had lost their integrity but remained recognizable as “ ghostly fi gures ” (Figure 2 E, F). Proportions of scallops with detached digestive cells at different time points are depicted in Figure 3 A. The mean num-ber of scallops with detached digestive cells was signifi cantly higher on exposure to P. minimum batch culture than on expo-sure to Rhodomonas sp. (after 12 h G = 15.64, p < 0.01, after 24 h G = 7.29, p < 0.01). On the contrary, there were no statistically-signifi cant differences in prevalences of scallops with detached cells on exposure to semi-continuous culture of P. minimum in comparison to scallops feeding on Rhodomonas sp. [after 12 h G = 0.05, NS (not signifi cant), after 24 h G = 0.09, NS].

Muscle fi bers of the adductor muscles were at a different physiological stage in scallops feeding on the dinofl agel-late in batch culture in comparison to scallops feeding on Rhodomonas or Prorocentrum minimum semi-continuous culture (Figure 3 B). In these conditions, muscle fi bers were long and spindle shaped with a general, wavy appearance of the muscle (Figure 4 A). This probably corresponded to the relaxed stage of the muscle fi bers. In the scallops feeding on P. minimum batch culture, the muscle fi bers were usually shortened, rounded-up, and with a clear halo surrounding each fi ber (Figure 4 B). No apparent myodegeneration was detected, and the appearance was probably related to the con-tracted stage of the muscle fi bers. A signifi cantly-higher por-tion of scallops was in the contracted stage after exposure to P. minimum batch culture than after exposure to Rhodomonas sp. (after 12 h G = 19.83, p < 0.01, after 24 h G = 7.54, p < 0.01), but there were no statistically signifi cant differences between the number of scallops in the contracted stage after being exposed to P. minimum semi-continuous culture or Rhodomonas sp. (after 12 h, G = 1.76, NS, after 24 h G = 2.18, NS).

Exposure to Prorocentrum minimum batch culture induced a transient migration of hemocytes into the tissues. There were some isolated hemocytes in the tissues of some scallops fed with Rhodomonas sp. (3, 3 and 4 % after 0, 12 and 24 h, respec-tively , Figure 4 C). After 12 h of exposure to P. minimum batch culture, 17 % of the scallops had aggregations of hemocytes in the connective tissues underlying the stomach and intestine epithelia (Figure 4 D), signifi cantly more than in specimens fed with Rhodomonas sp. (G = 2.96, p < 0.1), whereas, scallops exposed to P. minimum continuous culture did not differ from Rhodomonas -fed specimens (after 12 h G = 0.60, NS, after 24 h G = 0.00, NS). After 24 h, 13 % of scallops exposed to either batch or continuous culture of P. minimum had aggregations of

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Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops 23

hemocytes in tissues, and the difference between P. minimum batch culture or Rhodomonas sp. fed was no longer signifi cant at this time point (G = 0.00, NS).

Exposure of scallops to Prorocentrum minimum continu-ous cultures also induced migration of hemocytes into the stomach and intestine of the alimentary tract (Figure 4 E, F). Proportions of specimens with hemocytes in the alimentary tract are depicted in Figure 5 A. After 24 h, there was a sig-nifi cantly higher proportion of scallops with hemocytes in the alimentary tract with P. minimum semi-continuous cul-ture than scallops fed with Rhodomonas sp. (G = 3.31, p < 0.1). Hemocytes were observed crossing the epithelia by means

of diapedesis toward the gastric shield of the stomach where P. minimum cells were trapped (Figure 4 F). In contrast to scallops exposed to the P. minimum semi-continuous culture, exposure to neither P. minimum batch culture nor Rhodomonas sp. induced migration of hemocytes into the alimentary tract (after 12 h G = 0.15, NS, after 24 h G = 1.90, NS).

There were no infl ammatory responses or histological changes in the kidneys, gills, eyes, shells, ganglia, byssus glands, or the gonads. The prevalence of pathological obser-vations was higher in scallops exposed to Prorocentrum minimum batch than in scallops exposed to P. minimum semi-continuous culture, and the prevalence of pathological

Figure 2 Histological changes in bay scallop, Argopecten irradians irradians , after exposure to the toxic alga Prorocentrum minimum . Paraffi n sections, hematoxylin-eosin stain. (A) Cross-section of the intestine of an animal fed with Rhodomonas sp. No algal cells visible in the lumen. Scale bar 50 μ m. (B) Cross-section of the intestine of an animal fed with P. minimum , algal cells present in the intestine lumen. Scale bar 50 μ m. Inset: Higher magnifi cation of P. minimum cells in the intestine. Scale bar 10 μ m. (C) Digestive tubules of an animal fed with Rhodomonas sp. Notice regular-shaped tubules with absorbing digestive cells. Scale bar 30 μ m. (D) Digestive tubules of an animal fed with P. minimum . Notice irregular digestive tubules with disintegrating digestive cells. Scale bar 30 μ m. (E) Stomach of an animal fed with Rhodomonas sp. No contents visible in the lumen. Scale bar 50 μ m. (F) Stomach of an animal fed with P. minimum . Remnants of digestive cells present in the lumen, sloughed from the digestive tubules. Scale bar 50 μ m. Inset: Higher magnifi cation of the remnants of the digestive cells. Scale bar 10 μ m.

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24 Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops

observations was higher in scallops feeding on either cul-ture of P. minimum than in scallops fed with Rhodomonas (Figure 5 B).

Discussion

The postulate tested in this laboratory experiment was that the intensities of the bioactive effects of Prorocentrum minimum upon northern bay scallops are different between algal popu-lations in different physiological states. Our results showed that P. minimum culture in the senescent phase had the most severe physiological effects upon bay scallops, including decreased feeding activities, as indicated by the reduced opening of shells and decreased production of biodeposits, decreased mobility, and increased mortality in 24 h exposure. In contrast to senescent P. minimum , the cultures given new medium weekly under the semi-continuous culture mode pro-duced less severe, but similar, effects with no scallop mortal-ity at the end of a 24-h exposure. The fact that bacterial counts

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Figure 3 Proportions of histological changes observed in bay scallops, Argopecten irradians irradians, at the beginning of the exposure, and after 12 and 24 h of exposure to either Prorocentrum minimum batch culture (P.m batch), semi-continuous P. minimum (P.m cont), or Rhodomonas sp. (Rhodo) expressed as percentage of specimens with the condition plotted against time. (A) Sloughed digestive cells in the lumina of digestive tubules or in the stomach. (B) Adductor muscles with contracted muscle fi bers.

increased in both batch and semi-continuous P. minimum cul-tures, yet the biological effects differed, provides evidence that effects seen in scallops exposed to late stationary P. mini-mum cultures were attributable to the algal cells themselves. Further, the time-scale of P. minimum biological effects upon scallops (hours) is far too short to attribute effects to nutri-tional defi ciencies; it takes several weeks for juvenile bay scallops to starve to death (Meseck et al. 2007 ).

The 24-h time period of the experiment for collecting spec-imens for histology was too short to induce irreversible patho-logical changes. The onset of scallop mortality at 24 h set the time limit for the experiment. The histological changes con-sisted of sloughing of digestive cells from the digestive tubule epithelia, different patterns in muscle fi bers, and migration of hemocytes. Sloughing of digestive cells and contraction of muscle fi bers represent normal physiological states in a bivalve ’ s feeding cycle, and migration of hemocytes was attributed to infl ammatory responses, the fi rst signs of acti-vation of the scallop defense mechanisms. All these changes were reversible, with the potential to return to normal when the pathological irritant was removed.

There were signifi cantly more Prorocentrum minimum cells in the stomachs of scallops exposed to the batch culture in comparison to the semi-continuous culture (Figure 2 B). Many cells were apparently intact. P. minimum cells from the continuous culture were probably digested more effec-tively, as demonstrated by absorptive status of the digestive tubules in this group, which did not differ from Rhodomonas -fed specimens (Figure 2 C), whereas the scallops feeding on P. minimum batch culture showed disintegrating digestive tubules (Figure 2 D). Also, migration of hemocytes into the stomachs in scallops exposed to the semi-continuous culture (Figure 4 F) may have facilitated elimination of the algal cells. There were large numbers of P. minimum cells embedded in the gastric shield (Figure 4 F). The purpose of the gastric shield is to protect the stomach epithelium from the grinding action of the crystalline style. It appeared that the hard theca of P. minimum resisted digestion and the cells were pushed into the lamellar matrix of the gastric shield.

Feeding in bivalves is associated with synchronized cyclic changes in the histophysiology of the digestive tubules, rang-ing from absorption to reconstituting, normal (or holding), and disintegrating (or breakdown) tubules (Langton 1975 , Robinson et al. 1981 ). Absorptive tubules are tall, columnar cells; whereas, reconstituting tubules have a low, cuboidal epithelium. In the present study, digestive tubules in absorp-tive or holding stages were most frequent in scallops feeding on semi-continuous Prorocentrum minimum or Rhodomonas sp., whereas, digestive tubules of scallops feeding on P. minimum batch culture showed most often the disintegrat-ing stage (Figures 2 C, D, 3 A). This most likely refl ected an actively-feeding status of the scallops exposed to semi-continuous P. minimum or Rhodomonas sp., whereas, disinte-grating tubules in P. minimum batch culture-exposed scallops suggested an aberration from the normal feeding cycle. The cross-sections of tubules in P. minimum batch culture-exposed specimens were irregular, and cell height varied, suggesting more serious pathological consequences. These observations

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Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops 25

are in agreement with those of Wikfors and Smolowitz (1993) after exposing juvenile bay scallops to another strain of P. minimum (CCMP1329). These observations are not consis-tent with those reported for several species of adult bivalves after P. minimum exposure: histopathological changes in digestive tubules were not present in mussels (Galimany et al. 2009 ), hard clams ( H é garet et al. 2010 , personal communica-tion), or Manila clams ( H é garet et al. 2009 ), suggesting dif-ferent responses in different species or higher susceptibility in juvenile bivalves compared to adults.

Gross observations during the study indicated that fewer scallops were open, and those open were opened less widely when exposed to Prorocentrum minimum batch culture in comparison to animals feeding on Rhodomonas sp. or P. minimum semi-continuous culture (Table 1 ). This was refl ected in the histophysiological status of the muscle fi bers (Figures 3B and 4A, 4B). Scallops exposed to P. minimum batch culture had rounded, short, contracted muscle fi bers, indicating that the scallops had their valves closed and were not feeding. Myodegeneration as a consequence of exposure

Figure 4 Histological changes in bay scallops, Argopecten irradians irradians , after exposure to the toxic alga Prorocentrum minimum . Paraffi n sections, hematoxylin-eosin stain. (A) Adductor muscle of an animal fed with Rhodomonas sp.; muscle fi bers relaxed. Scale bar 30 μ m. (B) Adductor muscle of an animal fed with P. minimum ; muscle fi bers contracted. Scale bar 30 μ m. (C) Connective tissue underneath stomach epithelium of an animal fed with Rhodomonas sp. Notice the empty lumen of the stomach on the right, and hemolymph canals and occasional hemocytes in the tissues on the left. Scale bar 50 μ m. (D) Connective tissue underneath stomach epithelium of an animal fed with P . minimum . Notice the lumen of the stomach with P. minimum cells on the right, and hemolymph canals and increased aggregations of hemocytes in the tissues on the left. Scale bar 50 μ m. Inset: Higher magnifi cation of hemocytes in tissues. Scale bar 10 μ m. (E) Stomach epithelium with gastric shield of an animal fed with Rhodomonas sp. Scale bar 30 μ m. (F) Stomach epithelium with gastric shield of an animal fed with P. minimum . Notice P. minimum cells trapped into the gastric shield, and hemocytes in diapedesis through the stomach epithelium. Scale bar 30 μ m. Inset: Higher magnifi cation of the stomach epithelium with a P. minimum cells within the gastric shield and a hemocytes in diapedesis. Scale bar 10 μ m.

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26 Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops

to harmful algae species was reported by H é garet et al. (2009) after exposing Manila clams to P. minimum and by Haberkorn et al. (2010) after exposing Pacifi c oysters to the toxigenic dinofl agellate Alexandrium minutum Halim. Myopathy in these previous studies was more severe than in the present experiment, but exposure times were longer, and the histophys-iological muscle anomaly described here may have developed into myopathy if the exposure had continued. The histological examinations showed that immunological responses of juve-nile scallops to the two cultures of P. minimum were quite different. Upon exposure to semi-continuous P. minimum cultures, scallop hemocytes migrated through the epithelia (diapedesis) into the alimentary tract, including both stom-ach and intestine (Figure 4 F). In contrast, stationary-phase P. minimum did not induce migration of hemocytes into the alimentary tract, although aggregations of hemocytes were seen in the connective tissues underlying stomach and intes-tine epithelia. Migration of hemocytes into the stomach and intestine was reported in blue mussels ( Mytilus edulis Linnaeus) after exposure to P. minimum (Galimany et al. 2009 ). Hemocytes encapsulated P. minimum cells, presumably

40

30

20%%

10

0

40

30

20

10

0

P.m batch P.m cont Rhodo

P.m batchA

B

P.m cont Rhodo

0 12H

24

0 12H

24

Figure 5 Prevalences of histological changes observed in bay scal-lops, Argopecten irradians irradians, at the beginning of the expo-sure, and 12 and 24 h of exposure to either Prorocentrum minimum batch culture (P.m batch), semi-continuous P. minimum (P.m cont), or Rhodomonas sp. (Rhodo) expressed as percentage of specimens with the condition plotted against time. (A) Hemocytes in the lumina of the alimentary tract. (B) Sum of histological changes (percentage of positive observations of all observations).

to protect the tissues from the harmful algal cells. The authors (Galimany et al. 2009) postulated that mussels recognized P. minimum cells as parasites and supported this view by cit-ing the close phylogenic relationship between P. minimum and Perkinsus marinus , a well-known parasite of bivalves. A similar response, migration of hemocytes into the alimentary tract, was reported by H é garet et al. (2009) in Manila clams exposed to P. minimum . In the present work, there was no migration of hemocytes into the alimentary tract after expo-sure to P. minimum batch culture. It is possible that the para-site-defense mechanism was disabled by toxicity in scallops exposed to the batch culture.

There was a transient, infl ammatory response in the form of aggregations of hemocytes after 12 h exposure to Prorocentrum minimum batch culture. Similar histological responses to P. minimum were reported by Wikfors and Smolowitz (1993) when juvenile scallops were exposed to P. minimum, and large hemocyte aggregations containing melanized algal cells were found in various tissues. Also, H é garet et al. (2010) reported hemocyte aggregations (granulomas) in kidney, gill, mantle, foot, and heart after exposing hard clams ( Mercenaria merce-naria ) to the dinofl agellate. Responses of juvenile bay scal-lops to P. minimum revealed what appeared to be a series of events following initial exposure to this harmful alga. First, scallops tried to avoid the algal cells by closing the shells, and then activated defense mechanisms by mobilizing hemocytes to the alimentary tract to encapsulate the algal cells. Scallops also eliminated the harmful cells through pseudofeces produc-tion. During this stage, scallops recognized algal cells as non-self and non-food. These defense mechanisms were stimulated by the less-toxic, semi-continuously-cultured P. minimum cells. The next stage was failure of the defense mechanisms dur-ing exposure to the senescent P. minimum cultures, leading to consequent histophysiological anomalies. Finally, mortality of scallops occurred after a short-term exposure to senescent P. minimum . We speculate that a biotoxin was produced when P. minimum cells became senescent.

The above physiological and histopathological fi nd-ings confi rmed that the strain of Prorocentrum minimum (JA-98-01) studied is harmful to bay scallops, as reported pre-viously for the same strain and blue mussels (Galimany et al. 2009 ), eastern oysters and bay scallops ( H é garet and Wikfors 2005a ), and another strain CCMP1329 upon hard clams and bay scallops (Wikfors and Smolowitz 1993 ). Present fi ndings of the changes in the intensity of bioactive effects based upon algal physiological status help to explain the variability in toxicity of P. minimum observed in the fi eld and laboratory.

It is well known from laboratory studies that toxin produc-tion by dinofl agellates in which a toxic chemical is defi ned and can be quantifi ed varies with growth phase and environ-mental conditions. Oshima et al. (1989) and Hwang and Lu (2000) reported that maximum production of PSP (paralytic shellfi sh poison) by Alexandrium minutum was during the stationary and post-stationary phase. Alexandrium exca-vate (Braarud) Balech & Tangen appeared also to produce more PSP toxin during the stationary phase (White 1978 ). Grzebyk et al. (1997) also suggested that Prorocentrum mini-mum was more toxic during the stationary phase. In contrast,

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Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops 27

Alexandrium fundyese Balech and Alexandrium tamarensis (Lebour) Balech produced maximum amounts of toxin during the exponential growth phase (Boyer et al. 1987 , Anderson et al. 1990 ).

To date, no toxin has been purifi ed or identifi ed for Prorocentrum minimum . A number of chemicals could be candidates for such toxins, including a diketone produced extracellularly by the breakdown of carotenoids, as reported by Trick et al. (1984b) and Trick et al. (1981) . Hu and co -workers (2010) reviewed existing knowledge concerning diketide compounds in the genus Prorocentrum , but the reviewed literature concentrates mainly on okadaic-acid-producing species, which P. minimum is not. An alternative, bioactive component of P. minimum could be metals, which could alter hemocyte mobility (e.g., Fisher et al. 2000 ). Amino acids or proteins could also be candidates, such as domoic acid which has been shown to alter the immune pro-fi le of blue mussels (Dizer et al. 2001 ). Reactive oxygen spe-cies have recently been implicated in the harmful effects of Chattonella marina (Subrahmanyan) Hara et Chihara upon the short-necked clam (Kim et al. 2004 ).

Regardless of the toxic compound or compounds, this study demonstrates clearly that a single isolate of the dino-fl agellate Prorocentrum minimum can exist in both toxic and non-toxic state, and that the state is related to the physi-ological condition of the population. It is understandable that some researchers have found P. minimum to be harm-less to bivalve mollusks (Brownlee et al. 2005 , Glibert et al. 2007 , Stoecker et al. 2008 ), but also now understand-able that the state of the culture, and indeed the population in nature, is critical in determining whether or not this commonly-

occurring dinofl agellate will have negative biological conse-quences to molluscan shellfi sh. Thus, variable expression of toxicity in a microalgal species belongs among the “ diver-gent properties of [the] different bloom events ” identifi ed in Smayda ’ s Commentary, “ What is a bloom ? ” (1997).

Acknowledgments

Personal note by Judy Yaqin Li: Ted was an incredible mentor to me. His extraordinary passion and ability to link and synthesis vast amounts of complex, sometimes seemingly irrelevant information to generate hypotheses or to solve problems in phytoplankton ecology will always inspire me.

The authors are grateful to the following individuals from the Milford Laboratory who assisted with the research in various ways: Dave Veilleux provided scallops for the experiments, Diane Kapareiko performed the bacterial counts, Jennifer Alix inoculated the Prorocentrum minimum cultures, Mark Dixon provided the mass cultures of P. minimum and Rhodomonas sp. during the experiments, and Goulwen de Kermoysan assisted with the scallop-bioassay tests.

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Table 1 Results of the weekly scallop bioassay during the 7-week experiment.

Parameters Treatments

Scallop + fi ltered seawater

Scallops + Rhodomonas Scallops + Prorocentrum minimum batch culture

Scallops + P. minimum semi-continuous culture

Degree of shell openness Week 1 – 3 100 100 30 – 50 30 – 50 Week 4 – 6 100 100 5 – 30 30 – 50 Week 7 100 100 0 30 – 50Amount of bio-deposits Week 1 – 3 + * + + + + + + + Week 4 – 6 + * + + + + + + Week 7 + * + + + + * + + Degree of mobility Week 1 – 3 100 100 50 50 Week 4 – 6 100 100 10 50 Week 7 100 100 0 50 % Animals attached to surfaces Week 1 – 3 80 80 50 50 Week 4 – 6 80 80 0 50 Week 7 80 80 0 50 % Mortality in 24 h Week 1 – 3 0 0 0 0 Week 4 – 6 0 0 0 0 Week 7 0 0 15 0

*Indicates that biodeposits were produced only during the initial hours of experiments. +, ++ and +++ are arbitrary units indicating the relative amount of bio-deposit. +++ indicates the highest amount.

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28 Y. Li et al.: Variable bioactive effect of Prorocentrum minimum on bay scallops

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Received 23 November, 2010; accepted 17 November, 2011; online fi rst 21 December, 2011

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