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Feeding preferences and grazing rates of Pfiesteria piscicida and a cryptoperidiniopsoid preying on fish blood cells and algal prey
Harmful Algae 5 (2006) 419–426 www.elsevier.com/locate/hal
Feeding preferences and grazing rates of Pfiesteria piscicida and a cryptoperidiniopsoid preying on fish blood cells and algal prey Todd A. Egerton *, Harold G. Marshall Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529-0266, USA Received 10 October 2005; received in revised form 29 March 2006; accepted 1 April 2006
Abstract The grazing rates and feeding preferences of the dinoflagellates Pfiesteria piscicida and a cryptoperidiniopsoid on the alga Rhodomonas sp. and fish blood cells were calculated at different ratios of the two food types and at different total food densities. Data from 6 h grazing periods within microcosms were used to calculate grazing rates. Grazing rates of both dinoflagellates increased linearly with an increased ratio of blood cells to Rhodomonas, and P. piscicida had a higher maximum grazing rate than the cryptoperidiniopsoid. The grazing rate of P. piscicida on Rhodomonas also increased with increased Rhodomonas densities relative to the blood cells, but increased densities of Rhodomonas did not increase the grazing rate of the cryptoperidiniopsoid, suggesting a lower feeding threshold for this species. Both dinoflagellates demonstrated a preference for fish blood cells over Rhodomonas cells, with no significant difference in the index of preference between the two species. Total food abundance affected the degree of preference differently for each dinoflagellate species. A higher index of feeding preference was attained by P. piscicida when resource levels were high, while the cryptoperidiniopsoid did not show this response. A preference for fish blood cells occurred at all food ratios for both dinoflagellates, including when blood cells were scarce relative to the alternate food type (15% of total available food). These results suggest that these strains of P. piscicida and the cryptoperidiniopsoid share similar feeding preferences for the prey types tested, although cryptoperidiniopsoids have not been associated with fish kills. # 2006 Elsevier B.V. All rights reserved. Keywords: Cryptoperidiniopsoid; Feeding preference; Heterotrophic dinoflagellates; Pfiesteria piscicida
1. Introduction Optimal foraging theory assumes that a predator will preferentially consume the resource which will maximize its fitness through maximum net energetic intake (MacArthur and Pianka, 1966; McNamara and Houston, 1985). Differences in resource availability will favor certain foraging behaviors (e.g. generalist, specialist and facultative). When a predator encounters changing levels of prey, a facultative strategy should provide maximal
* Corresponding author. Tel.: +1 757 683 3595; fax: +1 757 683 5283. E-mail address: [email protected] (T.A. Egerton). 1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2006.04.011
energy uptake (Glasser, 1984). Facultative predators would specialize on the most profitable food items when they are abundant, and expand their diet to less profitable prey when resources are scarce (Glasser, 1984). Feeding preference experiments of protists are relatively rare, although selective predation has been identified (Stoecker et al., 1981; Jacobson and Anderson, 1986; Sˇimek et al., 1995). Dinoflagellates have life histories with different stages that can respond to changing environmental situations (Rengefors and Anderson, 1998) and react to stimuli that include light, temperature, gravity, chemical and mechanical cues (Levandowsky and Kaneta, 1987; Cancellieri et al., 2001), making dinoflagellates a reasonable model to study a behavioral response to varying food resources.
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Due to toxin production and associated fish kills, Pfiesteria piscicida Steidinger et Burkholder and Pfiesteria shumwayae Glasgow et Burkholder (Marshall et al., 2006) have been studied extensively since their identification (Burkholder and Glasgow, 1997; Glasgow et al., 2001; Gordon et al., 2002; Vogelbein et al., 2002; Burkholder et al., 2005; Gordon and Dyer, 2005). Cryptoperidiniopsoids (Seaborn et al., 1999, 2001; Burkholder et al., 2001; Parrow and Burkholder, 2003) are closely related to Pfiesteria spp. based on morphological, ecological and genetic similarities (Marshall, 1999; Parrow and Burkholder, 2003; Seaborn et al., 2006). Both P. piscicida and cryptoperidiniopsoids feed by means of myzocytosis (Seaborn et al., 2001), a process where the predatory dinoflagellate inserts a feeding tube (peduncle) into the interior of the prey cell and ingests the cell contents (Schnepf and Deichgra¨ber, 1984). Pfiesteria spp. and cryptoperidiniopsoids can feed on a diverse assemblage of algae (Seaborn et al., 2001), bacteria (Burkholder and Glasgow, 1995), finfish (Burkholder and Glasgow, 1997; Gordon et al., 2002), shellfish (Burkholder and Glasgow, 1997) and mammalian red blood cells (Glasgow et al., 2001). In systems that include multiple omnivorous predators and multiple prey items, it is likely that competing predators have different feeding preferences both to maximize energy intake and avoid competitive exclusion. This may be the scenario with heterotrophic dinoflagellates, in particular P. piscicida and cryptoperidiniopsoid taxa due to the broad food selection available to these species. Previous studies to identify the optimal food source for these dinoflagellates relied on comparing growth curves (e.g. Seaborn et al., 1999) and did not measure food preference when given multiple food types. The objectives of this study were to determine whether a preference is shown by P. piscicida and a cryptoperidiniopsoid between fish blood cells and an algal food source, and if preference is influenced by the total and relative abundance of these resources. 2. Materials and methods 2.1. Culturing The clone of P. piscicida used in this study was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP1834), West Boothbay Harbor, ME. A cryptoperidiniopsoid dinoflagellate (00DEQ029; with identical SSU rRNA sequence as that submitted to GenBank as cryptoperidiniopsoid sp. brodyi AF080097 by Litaker et al.
(1999), and informally named as ‘Cryptoperidiniopsis brodyi’, e.g. Litaker et al., 2000) was established from samples collected from Nomini Creek, a tributary of the Potomac River, VA, USA. These dinoflagellates were identified with scanning electron microscope analysis following the suture swelling technique of Glasgow et al. (2001). Real-time PCR analysis was used to verify their species identification and test for cross-contamination (Bowers et al., 2000). The algal food source for the dinoflagellate cultures was the cryptophyte Rhodomonas sp. (CCMP 768). Cultures were grown in 200 ml Falcon tissue flasks using F/2-Si medium (Guillard, 1975) at 20 8C in a Precision incubator on a 12-h light:12-h dark cycle. The medium was made using filtered (0.2 mm pore size) Atlantic Ocean seawater diluted to 15 ppt. A 1 ml aliquot of Rhodomonas (103 cells ml1) was added to each flask at 2–3 day intervals, with media changes made monthly. 2.2. Prey preparation The Rhodomonas sp. in the study was the same strain used in maintaining the dinoflagellate cultures. Fish blood cells were obtained from freshly caught Atlantic croaker (Micropogonias undulatus), George’s Seafood, Norfolk, VA, USA. Fish blood cells were collected by dissecting the fish along the ventral section of the body and removing the internal organs. Blood was taken from the dorsal aorta with a syringe and filtered through a 100 mm aperture mesh. The blood was then mixed with F/2-Si medium at 9 ppt and diluted as necessary to obtain the desired cell density. 2.3. Grazing experiments The grazing experiments consisted of seven food treatments, each with a different ratio of fish blood cells to Rhodomonas cells. Total prey density in each treatment was 300 cells (Table 1). Additionally, one treatment consisted of equal densities of the two food types (1:1) with a total abundance of 100 cells. Dinoflagellate densities were determined by light microscopy using a Palmer–Maloney chamber and reduced as needed by dilution with F/2-Si medium to obtain equal initial cell densities (100 cells well1) in all treatments. Dinoflagellates and food treatments were added to a Nunclon# Microwell cell culture plate by micropipetting. The volume of each well was brought to 150 ml using F/2-Si medium. There were three well replicates per treatment for each species and three controls that contained no dinoflagellates. Three
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Table 1 Initial cell densities (ml1) of food treatments per 150 ml microwell (n = 3) Treatment
1 2 3 4 5 6 7 8
Total cell number
Fish blood cells
Rhodomonas cells
Mean
S.E.
Mean
S.E.
Mean
S.E.
325 334 364 347 326 302 308 90
26 28 16 9 4 16 19 15
325 305 307 210 121 46 0 51
26 30 15 5 1 6 0 7
0 29 57 138 206 255 308 39
0 3 4 5 5 18 19 8
Percentage of blood cells
100 91.3 84.3 60.4 37.0 15.4 0 56.5
Each well also contained 100 4 dinoflagellates (mean 1 standard error, S.E.) as either Pfiesteria piscicida or cryptoperidiniopsoids.
replicates for each treatment were added to a separate identical well plate and fixed immediately to represent the initial cell densities. After application of the food treatments, the covered well plates were kept in the dark at room temperature. Experiments were incubated for 6 h in darkness, after which 2 ml of 25% glutaraldehyde was added to each well. Each well was examined using an inverted light microscope at 200. All dinoflagellate and prey cells were identified and counted in each well. Numbers of cells grazed were calculated after 6 h by subtracting the abundance of prey cells per well from the cell numbers in the immediately fixed wells. Dividing this number by the number of dinoflagellates in each well gave the grazing rate over the 6 h period. Dinoflagellate feeding preference was measured as an index of preference (C) and was calculated using the following equation described by Murdoch (1969), where Nc1 and Nc2 are the numbers of prey types I and II consumed and N1 and N2 are the numbers of prey types I and II initially.
confounding factors such as algal growth or loss of cells to factors other than grazing. 3.2. Grazing rates P. piscicida and the cryptoperidiniopsoid fed on both Rhodomonas and fish blood cells. In all treatments, food levels of both types were significantly reduced by both dinoflagellates compared to the no-dinoflagellate controls. The maximum grazing rate of P. piscicida (3.49 cells dinoflagellate1 6 h1) on blood cells was significantly ( p < 0.001) higher than the cryptoperidiniopsoid (2.00 cells dinoflagellate1 6 h1). Overall, the grazing rates of the dinoflagellates on Rhodomonas cells were significantly lower ( p < 0.001) than grazing rates on fish blood cells. Grazing rates of P. piscicida (1.88 cells dinoflagellate1 6 h1) were significantly higher ( p < 0.001) than grazing rates of the cryptoperidiniopsoid (0.747 cells dinoflagellate1 6 h1).
Nc1 N1 ¼ C Nc2 N2 Analysis of variance (ANOVA) was used to compare the grazing rates and indices of preference. SPSS for Windows 10.0 was used for all statistical analyses. 3. Results 3.1. Controls Analysis of controls that were fixed immediately and those fixed after 6 h had no significant differences for blood cells ( p = 0.162), Rhodomonas ( p = 0.988) and dinoflagellates ( p = 0.326), indicating no differences in cell densities during the 6 h experiment due to potential
Fig. 1. Grazing rates of Pfiesteria piscicida (solid line, R2 0.980) and a cryptoperidiniopsoid (dashed line, R2 0.890) on fish blood cells at different prey abundances. Both taxa demonstrated a linear Type 1 functional response, with increased grazing activity at increased prey levels. The maximum grazing rate of P. piscicida was 3.49 blood cells dinoflagellate1 6 h1; the maximum grazing rate of the cryptoperidiniopsoids was 2.00 blood cells dinoflagellate1 6 h1. All treatments had a total of 300 food items.
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Fig. 2. Grazing rates of Pfiesteria piscicida (solid line, R2 = 0.974) and a cryptoperidiniopsoid (dashed line, R2 = 0.887) on Rhodomonas at different prey abundances. P. piscicida demonstrated a functional response to increasing Rhodomonas levels with a maximum rate of 1.88 Rhodomonas dinoflagellate1 6 h1. The cryptoperidiniopsoid demonstrated a Type 3 response, with grazing rates leveling off at a maximum of 0.747 Rhodomonas dinoflagellate1 6 h1. All treatments had a total of 300 food items.
Both dinoflagellates demonstrated a functional feeding response to different levels of fish blood cells (Fig. 1) and Rhodomonas cells (Fig. 2). P. piscicida and the cryptoperidiniopsoid had a linear Type 1 functional response with increases in grazing rate to available blood cells, with R2 values of 0.963 and 0.913, respectively. When feeding on Rhodomonas, the relationship between P. piscicida grazing rate and resource availability increased at all resource treatments (R2 = 0.974). Conversely, the grazing rates of the cryptoperidiniopsoid reached a maximum and did not increase when the food treatments exceeded 200 Rhodomonas cells well1, resulting in a sigmoid Type 3 functional response (R2 = 0.887). Total numbers of food cells eaten increased for both species at higher blood cell to Rhodomonas ratios as a response to the higher grazing rates on blood cells. 3.3. Feeding preferences Dinoflagellate feeding preference was calculated from their relative predation on Rhodomonas and fish blood cells compared to the initial abundance of the two food types. Feeding preference is shown in Fig. 3 as the percentage of blood cells grazed by the dinoflagellates in relation to the percentage of blood cells available. In all food treatments the percentage of blood cells grazed exceeded the percentage of blood cells available, indicating blood cells as the preferred food type by these strains of both dinoflagellates. The indices of preference were calculated and shown in Table 2, and compared using ANOVA. There was a significant treatment effect ( p = 0.006), with higher preference for blood cells when
Fig. 3. Grazing preference as shown by percentages of fish blood cells in the diets of Pfiesteria piscicida (white) and the cryptoperidiniopsoid (gray), compared to the percentage of blood cells available in each well (black). Both species had a significantly higher percentage of blood cells in their diet relative to their surroundings, indicating a preference for blood cells in all food treatments.
they made up a higher percentage of the diet. However, there was no significant difference in feeding preference between the two species ( p = 0.560) and no significant treatment–species interaction ( p = 0.476). Both species maintained a feeding preference for fish blood cells in all treatments even when blood cells represented only 15% of the total available prey. To test for an effect of total food abundance at equal prey ratios on the feeding preferences of the two dinoflagellates, data from treatments with equal relative levels of the two food resources (1:1), but different total abundances was compared (Table 2, treatments 4 and 8). Results of ANOVA indicated a significant ( p = 0.002) interaction effect on the feeding preferences between abundance and species. A significantly higher degree of preference was exhibited by P. piscicida at higher (3 prey:1 dinoflagellate) prey abundance than at lower prey abundance (1 prey:1 dinoflagellate). The cryptoperidiniopsoid showed the opposite relationship to prey concentrations. Table 2 Mean indices of preference (C) for fish blood cells over Rhodomonas by Pfiesteria piscicida and a cryptoperidiniopsoid for each food treatment Treatment
Pfiesteria piscicida
Cryptoperidiniopsoid
1 2 3 4 5 6 7 8
– 3.27 4.79 2.86 1.97 2.17 – 1.39
– 4.90 4.73 2.12 3.08 1.66 – 4.37
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4. Discussion 4.1. Preference for blood cells shown by both dinoflagellates The strain of P. piscicida and of the cryptoperidiniopsoid tested here preferred fish blood cells over Rhodomonas. This result was expected for P. piscicida and is consistent with previous studies showing fish predation and mortality caused by this species (Burkholder and Glasgow, 1997; Marshall et al., 2000; Gordon et al., 2002). While most heterotrophic dinoflagellates, including Pfiesteria-like species, feed on other algal taxa (Gaines and Elbra¨chter, 1987; Seaborn et al., 1999), predation on fish cells has not been widely observed in other species. Experimental studies with cryptoperidiniopsoids have not demonstrated toxin production leading to fish mortality (Marshall et al., 2000; Seaborn et al., 2001; Burkholder et al., 2005). Burkholder et al. (2001) observed larval fish attack by cryptoperidiniopsoids, but their attacks were only associated with low percentages of fish death, suggesting that fish are not as important in the diet of cryptoperidiniopsoids as in Pfiesteria spp. Although the apparent preference for fish blood cells by the cryptoperidiniopsoids in this study does not necessarily indicate evidence for these organisms as a cause of fish mortality. It does offer the possibility that fish may comprise some component of the cryptoperidiniopsoid diet. Several other as-yet-unnamed species of heterotrophic dinoflagellates are closely related to Pfiesteria and cryptoperidiniopsoids, based on their morphological and genetic similarities (Steidinger et al., 2001; Seaborn et al., 2006; Marshall et al., 2006). These results suggest that further studies are needed to investigate the behavioral traits of species similar to Pfiesteria and their potential predatory and/or toxic activity toward fish and other estuarine prey. 4.2. Intrinsic differences between fish blood cells and Rhodomonas cells There are several possible reasons for the observed differences in grazing rates between fish blood cells and Rhodomonas cells. Rhodomonas and other cryptophytes are common components of the plankton community within the Chesapeake Bay, often in subdominant densities (Marshall and Lacouture, 1986). Fish blood cells and Rhodomonas were chosen as food types because of their near uniform size and shape. However, the fish blood cells were slightly elliptical (10 mm 8 mm), with the Rhodomonas cells more
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elongated (12 mm 6 mm) in shape. While the size ranges of both food types were similar, small differences may have been a factor in the feeding preferences of the dinoflagellates. Dinoflagellates are rather unique among aquatic predators because often their preferred prey is equal in size or slightly larger than themselves (Hansen et al., 1994). If prey size had been an important factor influencing grazing by P. piscicida and the cryptoperidiniopsoids in this study, the slightly larger Rhodomonas cells might have been preferred over the smaller fish blood cells. Another difference between the two food types is that fish blood cells are non-motile and settle to the bottom of the well plates, while Rhodomonas cells are motile. Dinoflagellates may increase foraging efficiency by preferentially feeding on non-motile prey when it is available; thus, the blood cells may have been easier to capture than the Rhodomonas cells. However, it should be noted that the majority of the Rhodomonas cells in this study congregated on the well plate bottom in culture, and their swimming speed appeared to be much lower than that of the dinoflagellates. A third distinction between the two food types is the potential nutritional value of each. Cryptophyte cells have a varied composition of organelles that include a nucleus, chloroplasts, a pyrenoid, plus various storage products (Throndsen, 1997). A biochemical analysis of Rhodomonas salina indicated that the cells consisted of 48% proteins, 22% carbohydrates and 22% lipids (Brown et al., 1998). Fish blood cells have less complex structure, lacking the organelles present in Rhodomonas. Vertebrate blood cells also tend to have a lower percentage of carbohydrates, e.g. 52% proteins, 8% carbohydrates and 40% lipids composition (Wegen and Segen, 1992). Despite the apparent preference for fish blood cells shown by the dinoflagellates in this study, this food resource alone does not appear to be sufficient to sustain populations of P. piscicida or cryptoperidiniopsoids over long periods (e.g. Egerton, 2005; Parrow et al., 2005). While blood cells do not have a suitable mixture of nutrients to support the dinoflagellates independently, they may serve a supplemental role to an algal diet. Alternatively, the dinoflagellates may have been responding to a cue that is shared by fish blood cells and other fish tissues, including certain tissues that contain the nutrients needed to sustain the populations. Differences have been found between the two Pfiesteria species in this regard: P. shumwayae, but not P. piscicida, has been successfully maintained in culture on a diet of Chinook salmon epithelial cells. This distinction was observed even though both species were
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observed to feed on the fish cells (Parrow et al., 2005). It is also possible, as suggested by Parrow et al. (2005), that some species of fish may provide a certain nutritional requirement for the dinoflagellates that is lacking in other fish species. 4.3. Differences in feeding behavior between P. piscicida and cryptoperidiniopsoids Two similar predators that are found in the same habitat might be expected to have differing feeding behavior as a possible mode of coexistence (Boenigk and Arndt, 2000). In this study, two closely related heterotrophic dinoflagellates that co-occur in similar estuarine habitats were presented with different ratios and abundances of two prey types. Grazing data demonstrated both P. piscicida and the cryptoperidiniopsoid have the ability to feed preferentially. Multiple studies have been conducted on the diets of heterotrophic dinoflagellates (e.g. Jacobson and Anderson, 1986, 1996; Strom and Buskey, 1993), including those of Pfiesteria (e.g. Burkholder and Glasgow, 1995) and related species (Seaborn et al., 1999; Parrow and Burkholder, 2003). Heterotrophic dinoflagellates have been shown to be capable of feeding on various types of food, and some species may select prey based primarily on size (Hansen, 1992). The dinoflagellates in this study showed a feeding preference for fish blood cells over algal prey, even at low densities of the preferred food type relative to algal cells. Switching feeding preferences did not occur, and blood cells remained the preferred food type at all food ratios, indicating that both species actively searched for the preferred food type even when given a high abundance of alternative prey. Optimal foraging theory assumes that time spent on a specific aspect of an organism’s feeding behavior should be increased as long as the gain in energy exceeds the loss (MacArthur and Pianka, 1966). This concept is extended to assume that when there are plentiful prey items, a predator can ‘afford’ to specialize in its diet to include only the most profitable type of prey. In contrast, at low prey densities, a specialized feeding strategy might be expected to use more energy in searching for specific prey than is gained from its consumption. Thus, the predator must include a wider variety of prey in its diet to survive. P. piscicida exhibited classical optimal foraging behavior, with the degree of preference for fish blood cells higher at higher total prey availability than at lower prey levels (Table 2, treatments 4 and 8). This is consistent with what would be expected for an organism that can specialize on a
prey type while remaining capable of feeding on multiple prey items. The cryptoperidiniopsoids, however, did not display this behavior; instead, they showed less preference for fish blood cells when blood cells were more abundant. One hypothesis for the different response to prey availability between the two dinoflagellate taxa is a possible difference in their ability to discern between the experimental high and low treatments. The grazing rate of P. piscicida continued to increase as available Rhodomonas increased, while the grazing rate of the cryptoperidiniopsoids reached a maximum level that did not continue to increase when Rhodomonas densities exceeded 200 cells well1. The grazing of the cryptoperidiniopsoid both at the high and low treatments may have been limited by satiation, searching or digestion time. Another factor that may have influenced the outcome is how the two predators attacked the prey. Swarming behavior, in which multiple dinoflagellates feed on a single food item (e.g. Spero and More´e, 1981), was observed by both species on both prey types, but appeared to occur more in cryptoperidiniopsoids grazing on Rhodomonas. Often, dinoflagellates appeared to ignore the majority of the available Rhodomonas population and joined other dinoflagellates to actively feed on a single Rhodomonas cell. This aggregating behavior in P. piscicida and cryptoperidiniopsoids has previously been documented (e.g. Parrow and Burkholder, 2003). Often between 3 and 7 dinoflagellates were observed in contact with a single prey item, although occasionally there were in excess of 20 dinoflagellates. This multiple organism feeding strategy may explain why maximum grazing rates of these heterotrophic dinoflagellates can sometimes be low (e.g. for cryptoperidiniopsoids, less than 1 Rhodomonas dinoflagellate1 6 h). Peduncular feeding likely results in some release of the prey cell contents into the surrounding medium. Dinoflagellates are known to detect various chemical stimuli (e.g. Levandowsky and Kaneta, 1987). P. piscicida has been shown to detect and swim toward a gradient of fish tissue and excreta (Cancellieri et al., 2001). This behavior may indicate that P. piscicida prefers to feed on ‘leaky’ cells, as suggested by Parrow and Burkholder (2003), or that the dinoflagellates fail to detect intact cells as potential prey in the presence of leaky cells. If leaky cells are preferred, a higher level of undamaged/intact Rhodomonas cells would not necessarily promote increased feeding. The above scenario would suggest that intraspecific competition for food may be limiting the grazing rates of these species, especially cryptoperidiniopsoids, where swarming behavior appeared to occur more commonly than
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expected, considering that available prey were abundant. Thus, possible differences in chemosensory ability between the two dinoflagellate species may partly explain the difference in preference response to total abundance. If the cryptoperidiniopsoids only detected a small subset of the prey, the damaged or ‘leaky’ cells could have been effectively limiting in both the high and low prey abundances tested. 5. Conclusions P. piscicida and a related cryptoperidiniopsoid dinoflagellate have the ability to graze on Rhodomonas sp. and fish blood cells and respond to increased prey abundance. Furthermore, the dinoflagellate strains tested here fed preferentially on fish blood cells over Rhodomonas. This preference was observed at all prey ratios tested, including when the preferred prey type was scarce. The strength of the preference apparently is influenced by the level of overall prey abundance. Possible differences in feeding behavior, including swarming, may also influence grazing rates and preferences. The preference for fish blood cells by both dinoflagellate taxa supports the need for further research to examine potentially harmful feeding behavior of other Pfiesteria-like-dinoflagellates. Acknowledgments Funding for this study was provided by the Virginia Department of Health and the Centers for Disease Control and Prevention and was a component of the M.S. degree program with the Department of Biological Sciences at Old Dominion University. We also thank three reviewers for their comments and insights.[SES] References Boenigk, J., Arndt, H., 2000. Comparative studies on the feeding behavior of two heterotrophic nanoflagellates: the filter-feeding choanoflagellate Monosiga ovata and the raptorial-feeding kinetoplastid Rhynchomonas nasuta. Aquat. Microb. Ecol. 22, 243–249. Bowers, H.A., Tengs, T., Glasgow, H.B., Burkholder, J.M., Rublee, P.A., Oldach, D.W., 2000. Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl. Environ. Microbiol. 66, 4641–4648. Brown, M.R., McCausland, M.A., Kowalski, K., 1998. The nutritional value of four Australian microalgal strains fed to Pacific oyster Crassostrea gigas spat. Aquaculture 165, 281–293. Burkholder, J.M., Glasgow, H.B., 1995. Interactions of a toxic estuarine dinoflagellate with microbial predators and prey. Arch. Protistenkd. 145, 177–188.
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Seaborn, D.W., Dunstan, W.M., Marshall, H.G., Gordon, A.S., Seaborn, A.M., 2001. Growth and toxicity studies of the dinoflagellates cryptoperidiniopsis sp., Gyrodinium galatheanum, and Pfiesteria piscicida. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal Blooms, Proceedings of the Ninth International Conference on Harmful Algal Blooms, IOC UNESCO, Paris, France, pp. 92–96. Seaborn, D.W., Tengs, T., Cerbin, S., Kokocinski, M., Marshall, H.G., 2006. A group of dinoflagellates, similar to Pfiesteria as defined by morphology and genetic analysis. Harmful Algae 5, 1–8. Sˇimek, K., Bobkova´, J., Macek, M., Nedoma, J., Psenner, R., 1995. Ciliate grazing on picoplankton in a eutrophic reservoir during the summer phytoplankton maximum: a study at the species and community level. Limnol. Oceanogr. 40, 1077–1090. Spero, H.J., More´e, M.D., 1981. Phagotrophic feeding and its importance to the life cycle of the holozoic dinoflagellate, Gymnodinium fungiforme. J. Phycol. 17, 43–51. Steidinger, K., Landsberg, J., Richardson, R.W., Truby, E., Blakesley, B., Scott, P., Tester, P., Tengs, T., Mason, P., Morton, S., Seaborn, D., Litaker, W., Reece, K., Oldach, D., Haas, L., Vasta, G., 2001. Classification and identification of Pfiesteria and Pfiesteria-like species. Environ. Health Perspect. 109, 661–665. Stoecker, D.K., Guillard, R.R.L., Kavee, R.M., 1981. Selective predation by Favella ehrenbergii (Tintinnia) on and among dinoflagellates. Biol. Bull. 160, 136–145. Strom, S.L., Buskey, E.J., 1993. Feeding, growth, and behavior of the thecate heterotrophic dinoflagellate Oblea rotunda. Limnol. Oceanogr. 38, 965–977. Throndsen, J., 1997. The planktonic marine flagellates. In: Tomas, C.R. (Ed.), Identifying Marine Phytoplankton. Academic Press, San Diego, CA, pp. 591–729. Vogelbein, W.K., Lovko, V.J., Shields, J.D., Reece, K.S., Mason, P., Haas, L.W., Walker, C.C., 2002. Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion. Nature 418, 967–970. Wegen, J.C., Segen, J.C., 1992. The Dictionary of Modern Medicine. Parthenon, Park Ridge, NJ.
Feeding preferences and grazing rates of Pfiesteria piscicida and a cryptoperidiniopsoid preying on fish blood cells and algal prey Todd A. Egerton *, Harold G. Marshall Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529-0266, USA Received 10 October 2005; received in revised form 29 March 2006; accepted 1 April 2006
Abstract The grazing rates and feeding preferences of the dinoflagellates Pfiesteria piscicida and a cryptoperidiniopsoid on the alga Rhodomonas sp. and fish blood cells were calculated at different ratios of the two food types and at different total food densities. Data from 6 h grazing periods within microcosms were used to calculate grazing rates. Grazing rates of both dinoflagellates increased linearly with an increased ratio of blood cells to Rhodomonas, and P. piscicida had a higher maximum grazing rate than the cryptoperidiniopsoid. The grazing rate of P. piscicida on Rhodomonas also increased with increased Rhodomonas densities relative to the blood cells, but increased densities of Rhodomonas did not increase the grazing rate of the cryptoperidiniopsoid, suggesting a lower feeding threshold for this species. Both dinoflagellates demonstrated a preference for fish blood cells over Rhodomonas cells, with no significant difference in the index of preference between the two species. Total food abundance affected the degree of preference differently for each dinoflagellate species. A higher index of feeding preference was attained by P. piscicida when resource levels were high, while the cryptoperidiniopsoid did not show this response. A preference for fish blood cells occurred at all food ratios for both dinoflagellates, including when blood cells were scarce relative to the alternate food type (15% of total available food). These results suggest that these strains of P. piscicida and the cryptoperidiniopsoid share similar feeding preferences for the prey types tested, although cryptoperidiniopsoids have not been associated with fish kills. # 2006 Elsevier B.V. All rights reserved. Keywords: Cryptoperidiniopsoid; Feeding preference; Heterotrophic dinoflagellates; Pfiesteria piscicida
1. Introduction Optimal foraging theory assumes that a predator will preferentially consume the resource which will maximize its fitness through maximum net energetic intake (MacArthur and Pianka, 1966; McNamara and Houston, 1985). Differences in resource availability will favor certain foraging behaviors (e.g. generalist, specialist and facultative). When a predator encounters changing levels of prey, a facultative strategy should provide maximal
* Corresponding author. Tel.: +1 757 683 3595; fax: +1 757 683 5283. E-mail address: [email protected] (T.A. Egerton). 1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2006.04.011
energy uptake (Glasser, 1984). Facultative predators would specialize on the most profitable food items when they are abundant, and expand their diet to less profitable prey when resources are scarce (Glasser, 1984). Feeding preference experiments of protists are relatively rare, although selective predation has been identified (Stoecker et al., 1981; Jacobson and Anderson, 1986; Sˇimek et al., 1995). Dinoflagellates have life histories with different stages that can respond to changing environmental situations (Rengefors and Anderson, 1998) and react to stimuli that include light, temperature, gravity, chemical and mechanical cues (Levandowsky and Kaneta, 1987; Cancellieri et al., 2001), making dinoflagellates a reasonable model to study a behavioral response to varying food resources.
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Due to toxin production and associated fish kills, Pfiesteria piscicida Steidinger et Burkholder and Pfiesteria shumwayae Glasgow et Burkholder (Marshall et al., 2006) have been studied extensively since their identification (Burkholder and Glasgow, 1997; Glasgow et al., 2001; Gordon et al., 2002; Vogelbein et al., 2002; Burkholder et al., 2005; Gordon and Dyer, 2005). Cryptoperidiniopsoids (Seaborn et al., 1999, 2001; Burkholder et al., 2001; Parrow and Burkholder, 2003) are closely related to Pfiesteria spp. based on morphological, ecological and genetic similarities (Marshall, 1999; Parrow and Burkholder, 2003; Seaborn et al., 2006). Both P. piscicida and cryptoperidiniopsoids feed by means of myzocytosis (Seaborn et al., 2001), a process where the predatory dinoflagellate inserts a feeding tube (peduncle) into the interior of the prey cell and ingests the cell contents (Schnepf and Deichgra¨ber, 1984). Pfiesteria spp. and cryptoperidiniopsoids can feed on a diverse assemblage of algae (Seaborn et al., 2001), bacteria (Burkholder and Glasgow, 1995), finfish (Burkholder and Glasgow, 1997; Gordon et al., 2002), shellfish (Burkholder and Glasgow, 1997) and mammalian red blood cells (Glasgow et al., 2001). In systems that include multiple omnivorous predators and multiple prey items, it is likely that competing predators have different feeding preferences both to maximize energy intake and avoid competitive exclusion. This may be the scenario with heterotrophic dinoflagellates, in particular P. piscicida and cryptoperidiniopsoid taxa due to the broad food selection available to these species. Previous studies to identify the optimal food source for these dinoflagellates relied on comparing growth curves (e.g. Seaborn et al., 1999) and did not measure food preference when given multiple food types. The objectives of this study were to determine whether a preference is shown by P. piscicida and a cryptoperidiniopsoid between fish blood cells and an algal food source, and if preference is influenced by the total and relative abundance of these resources. 2. Materials and methods 2.1. Culturing The clone of P. piscicida used in this study was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP1834), West Boothbay Harbor, ME. A cryptoperidiniopsoid dinoflagellate (00DEQ029; with identical SSU rRNA sequence as that submitted to GenBank as cryptoperidiniopsoid sp. brodyi AF080097 by Litaker et al.
(1999), and informally named as ‘Cryptoperidiniopsis brodyi’, e.g. Litaker et al., 2000) was established from samples collected from Nomini Creek, a tributary of the Potomac River, VA, USA. These dinoflagellates were identified with scanning electron microscope analysis following the suture swelling technique of Glasgow et al. (2001). Real-time PCR analysis was used to verify their species identification and test for cross-contamination (Bowers et al., 2000). The algal food source for the dinoflagellate cultures was the cryptophyte Rhodomonas sp. (CCMP 768). Cultures were grown in 200 ml Falcon tissue flasks using F/2-Si medium (Guillard, 1975) at 20 8C in a Precision incubator on a 12-h light:12-h dark cycle. The medium was made using filtered (0.2 mm pore size) Atlantic Ocean seawater diluted to 15 ppt. A 1 ml aliquot of Rhodomonas (103 cells ml1) was added to each flask at 2–3 day intervals, with media changes made monthly. 2.2. Prey preparation The Rhodomonas sp. in the study was the same strain used in maintaining the dinoflagellate cultures. Fish blood cells were obtained from freshly caught Atlantic croaker (Micropogonias undulatus), George’s Seafood, Norfolk, VA, USA. Fish blood cells were collected by dissecting the fish along the ventral section of the body and removing the internal organs. Blood was taken from the dorsal aorta with a syringe and filtered through a 100 mm aperture mesh. The blood was then mixed with F/2-Si medium at 9 ppt and diluted as necessary to obtain the desired cell density. 2.3. Grazing experiments The grazing experiments consisted of seven food treatments, each with a different ratio of fish blood cells to Rhodomonas cells. Total prey density in each treatment was 300 cells (Table 1). Additionally, one treatment consisted of equal densities of the two food types (1:1) with a total abundance of 100 cells. Dinoflagellate densities were determined by light microscopy using a Palmer–Maloney chamber and reduced as needed by dilution with F/2-Si medium to obtain equal initial cell densities (100 cells well1) in all treatments. Dinoflagellates and food treatments were added to a Nunclon# Microwell cell culture plate by micropipetting. The volume of each well was brought to 150 ml using F/2-Si medium. There were three well replicates per treatment for each species and three controls that contained no dinoflagellates. Three
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Table 1 Initial cell densities (ml1) of food treatments per 150 ml microwell (n = 3) Treatment
1 2 3 4 5 6 7 8
Total cell number
Fish blood cells
Rhodomonas cells
Mean
S.E.
Mean
S.E.
Mean
S.E.
325 334 364 347 326 302 308 90
26 28 16 9 4 16 19 15
325 305 307 210 121 46 0 51
26 30 15 5 1 6 0 7
0 29 57 138 206 255 308 39
0 3 4 5 5 18 19 8
Percentage of blood cells
100 91.3 84.3 60.4 37.0 15.4 0 56.5
Each well also contained 100 4 dinoflagellates (mean 1 standard error, S.E.) as either Pfiesteria piscicida or cryptoperidiniopsoids.
replicates for each treatment were added to a separate identical well plate and fixed immediately to represent the initial cell densities. After application of the food treatments, the covered well plates were kept in the dark at room temperature. Experiments were incubated for 6 h in darkness, after which 2 ml of 25% glutaraldehyde was added to each well. Each well was examined using an inverted light microscope at 200. All dinoflagellate and prey cells were identified and counted in each well. Numbers of cells grazed were calculated after 6 h by subtracting the abundance of prey cells per well from the cell numbers in the immediately fixed wells. Dividing this number by the number of dinoflagellates in each well gave the grazing rate over the 6 h period. Dinoflagellate feeding preference was measured as an index of preference (C) and was calculated using the following equation described by Murdoch (1969), where Nc1 and Nc2 are the numbers of prey types I and II consumed and N1 and N2 are the numbers of prey types I and II initially.
confounding factors such as algal growth or loss of cells to factors other than grazing. 3.2. Grazing rates P. piscicida and the cryptoperidiniopsoid fed on both Rhodomonas and fish blood cells. In all treatments, food levels of both types were significantly reduced by both dinoflagellates compared to the no-dinoflagellate controls. The maximum grazing rate of P. piscicida (3.49 cells dinoflagellate1 6 h1) on blood cells was significantly ( p < 0.001) higher than the cryptoperidiniopsoid (2.00 cells dinoflagellate1 6 h1). Overall, the grazing rates of the dinoflagellates on Rhodomonas cells were significantly lower ( p < 0.001) than grazing rates on fish blood cells. Grazing rates of P. piscicida (1.88 cells dinoflagellate1 6 h1) were significantly higher ( p < 0.001) than grazing rates of the cryptoperidiniopsoid (0.747 cells dinoflagellate1 6 h1).
Nc1 N1 ¼ C Nc2 N2 Analysis of variance (ANOVA) was used to compare the grazing rates and indices of preference. SPSS for Windows 10.0 was used for all statistical analyses. 3. Results 3.1. Controls Analysis of controls that were fixed immediately and those fixed after 6 h had no significant differences for blood cells ( p = 0.162), Rhodomonas ( p = 0.988) and dinoflagellates ( p = 0.326), indicating no differences in cell densities during the 6 h experiment due to potential
Fig. 1. Grazing rates of Pfiesteria piscicida (solid line, R2 0.980) and a cryptoperidiniopsoid (dashed line, R2 0.890) on fish blood cells at different prey abundances. Both taxa demonstrated a linear Type 1 functional response, with increased grazing activity at increased prey levels. The maximum grazing rate of P. piscicida was 3.49 blood cells dinoflagellate1 6 h1; the maximum grazing rate of the cryptoperidiniopsoids was 2.00 blood cells dinoflagellate1 6 h1. All treatments had a total of 300 food items.
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Fig. 2. Grazing rates of Pfiesteria piscicida (solid line, R2 = 0.974) and a cryptoperidiniopsoid (dashed line, R2 = 0.887) on Rhodomonas at different prey abundances. P. piscicida demonstrated a functional response to increasing Rhodomonas levels with a maximum rate of 1.88 Rhodomonas dinoflagellate1 6 h1. The cryptoperidiniopsoid demonstrated a Type 3 response, with grazing rates leveling off at a maximum of 0.747 Rhodomonas dinoflagellate1 6 h1. All treatments had a total of 300 food items.
Both dinoflagellates demonstrated a functional feeding response to different levels of fish blood cells (Fig. 1) and Rhodomonas cells (Fig. 2). P. piscicida and the cryptoperidiniopsoid had a linear Type 1 functional response with increases in grazing rate to available blood cells, with R2 values of 0.963 and 0.913, respectively. When feeding on Rhodomonas, the relationship between P. piscicida grazing rate and resource availability increased at all resource treatments (R2 = 0.974). Conversely, the grazing rates of the cryptoperidiniopsoid reached a maximum and did not increase when the food treatments exceeded 200 Rhodomonas cells well1, resulting in a sigmoid Type 3 functional response (R2 = 0.887). Total numbers of food cells eaten increased for both species at higher blood cell to Rhodomonas ratios as a response to the higher grazing rates on blood cells. 3.3. Feeding preferences Dinoflagellate feeding preference was calculated from their relative predation on Rhodomonas and fish blood cells compared to the initial abundance of the two food types. Feeding preference is shown in Fig. 3 as the percentage of blood cells grazed by the dinoflagellates in relation to the percentage of blood cells available. In all food treatments the percentage of blood cells grazed exceeded the percentage of blood cells available, indicating blood cells as the preferred food type by these strains of both dinoflagellates. The indices of preference were calculated and shown in Table 2, and compared using ANOVA. There was a significant treatment effect ( p = 0.006), with higher preference for blood cells when
Fig. 3. Grazing preference as shown by percentages of fish blood cells in the diets of Pfiesteria piscicida (white) and the cryptoperidiniopsoid (gray), compared to the percentage of blood cells available in each well (black). Both species had a significantly higher percentage of blood cells in their diet relative to their surroundings, indicating a preference for blood cells in all food treatments.
they made up a higher percentage of the diet. However, there was no significant difference in feeding preference between the two species ( p = 0.560) and no significant treatment–species interaction ( p = 0.476). Both species maintained a feeding preference for fish blood cells in all treatments even when blood cells represented only 15% of the total available prey. To test for an effect of total food abundance at equal prey ratios on the feeding preferences of the two dinoflagellates, data from treatments with equal relative levels of the two food resources (1:1), but different total abundances was compared (Table 2, treatments 4 and 8). Results of ANOVA indicated a significant ( p = 0.002) interaction effect on the feeding preferences between abundance and species. A significantly higher degree of preference was exhibited by P. piscicida at higher (3 prey:1 dinoflagellate) prey abundance than at lower prey abundance (1 prey:1 dinoflagellate). The cryptoperidiniopsoid showed the opposite relationship to prey concentrations. Table 2 Mean indices of preference (C) for fish blood cells over Rhodomonas by Pfiesteria piscicida and a cryptoperidiniopsoid for each food treatment Treatment
Pfiesteria piscicida
Cryptoperidiniopsoid
1 2 3 4 5 6 7 8
– 3.27 4.79 2.86 1.97 2.17 – 1.39
– 4.90 4.73 2.12 3.08 1.66 – 4.37
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4. Discussion 4.1. Preference for blood cells shown by both dinoflagellates The strain of P. piscicida and of the cryptoperidiniopsoid tested here preferred fish blood cells over Rhodomonas. This result was expected for P. piscicida and is consistent with previous studies showing fish predation and mortality caused by this species (Burkholder and Glasgow, 1997; Marshall et al., 2000; Gordon et al., 2002). While most heterotrophic dinoflagellates, including Pfiesteria-like species, feed on other algal taxa (Gaines and Elbra¨chter, 1987; Seaborn et al., 1999), predation on fish cells has not been widely observed in other species. Experimental studies with cryptoperidiniopsoids have not demonstrated toxin production leading to fish mortality (Marshall et al., 2000; Seaborn et al., 2001; Burkholder et al., 2005). Burkholder et al. (2001) observed larval fish attack by cryptoperidiniopsoids, but their attacks were only associated with low percentages of fish death, suggesting that fish are not as important in the diet of cryptoperidiniopsoids as in Pfiesteria spp. Although the apparent preference for fish blood cells by the cryptoperidiniopsoids in this study does not necessarily indicate evidence for these organisms as a cause of fish mortality. It does offer the possibility that fish may comprise some component of the cryptoperidiniopsoid diet. Several other as-yet-unnamed species of heterotrophic dinoflagellates are closely related to Pfiesteria and cryptoperidiniopsoids, based on their morphological and genetic similarities (Steidinger et al., 2001; Seaborn et al., 2006; Marshall et al., 2006). These results suggest that further studies are needed to investigate the behavioral traits of species similar to Pfiesteria and their potential predatory and/or toxic activity toward fish and other estuarine prey. 4.2. Intrinsic differences between fish blood cells and Rhodomonas cells There are several possible reasons for the observed differences in grazing rates between fish blood cells and Rhodomonas cells. Rhodomonas and other cryptophytes are common components of the plankton community within the Chesapeake Bay, often in subdominant densities (Marshall and Lacouture, 1986). Fish blood cells and Rhodomonas were chosen as food types because of their near uniform size and shape. However, the fish blood cells were slightly elliptical (10 mm 8 mm), with the Rhodomonas cells more
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elongated (12 mm 6 mm) in shape. While the size ranges of both food types were similar, small differences may have been a factor in the feeding preferences of the dinoflagellates. Dinoflagellates are rather unique among aquatic predators because often their preferred prey is equal in size or slightly larger than themselves (Hansen et al., 1994). If prey size had been an important factor influencing grazing by P. piscicida and the cryptoperidiniopsoids in this study, the slightly larger Rhodomonas cells might have been preferred over the smaller fish blood cells. Another difference between the two food types is that fish blood cells are non-motile and settle to the bottom of the well plates, while Rhodomonas cells are motile. Dinoflagellates may increase foraging efficiency by preferentially feeding on non-motile prey when it is available; thus, the blood cells may have been easier to capture than the Rhodomonas cells. However, it should be noted that the majority of the Rhodomonas cells in this study congregated on the well plate bottom in culture, and their swimming speed appeared to be much lower than that of the dinoflagellates. A third distinction between the two food types is the potential nutritional value of each. Cryptophyte cells have a varied composition of organelles that include a nucleus, chloroplasts, a pyrenoid, plus various storage products (Throndsen, 1997). A biochemical analysis of Rhodomonas salina indicated that the cells consisted of 48% proteins, 22% carbohydrates and 22% lipids (Brown et al., 1998). Fish blood cells have less complex structure, lacking the organelles present in Rhodomonas. Vertebrate blood cells also tend to have a lower percentage of carbohydrates, e.g. 52% proteins, 8% carbohydrates and 40% lipids composition (Wegen and Segen, 1992). Despite the apparent preference for fish blood cells shown by the dinoflagellates in this study, this food resource alone does not appear to be sufficient to sustain populations of P. piscicida or cryptoperidiniopsoids over long periods (e.g. Egerton, 2005; Parrow et al., 2005). While blood cells do not have a suitable mixture of nutrients to support the dinoflagellates independently, they may serve a supplemental role to an algal diet. Alternatively, the dinoflagellates may have been responding to a cue that is shared by fish blood cells and other fish tissues, including certain tissues that contain the nutrients needed to sustain the populations. Differences have been found between the two Pfiesteria species in this regard: P. shumwayae, but not P. piscicida, has been successfully maintained in culture on a diet of Chinook salmon epithelial cells. This distinction was observed even though both species were
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observed to feed on the fish cells (Parrow et al., 2005). It is also possible, as suggested by Parrow et al. (2005), that some species of fish may provide a certain nutritional requirement for the dinoflagellates that is lacking in other fish species. 4.3. Differences in feeding behavior between P. piscicida and cryptoperidiniopsoids Two similar predators that are found in the same habitat might be expected to have differing feeding behavior as a possible mode of coexistence (Boenigk and Arndt, 2000). In this study, two closely related heterotrophic dinoflagellates that co-occur in similar estuarine habitats were presented with different ratios and abundances of two prey types. Grazing data demonstrated both P. piscicida and the cryptoperidiniopsoid have the ability to feed preferentially. Multiple studies have been conducted on the diets of heterotrophic dinoflagellates (e.g. Jacobson and Anderson, 1986, 1996; Strom and Buskey, 1993), including those of Pfiesteria (e.g. Burkholder and Glasgow, 1995) and related species (Seaborn et al., 1999; Parrow and Burkholder, 2003). Heterotrophic dinoflagellates have been shown to be capable of feeding on various types of food, and some species may select prey based primarily on size (Hansen, 1992). The dinoflagellates in this study showed a feeding preference for fish blood cells over algal prey, even at low densities of the preferred food type relative to algal cells. Switching feeding preferences did not occur, and blood cells remained the preferred food type at all food ratios, indicating that both species actively searched for the preferred food type even when given a high abundance of alternative prey. Optimal foraging theory assumes that time spent on a specific aspect of an organism’s feeding behavior should be increased as long as the gain in energy exceeds the loss (MacArthur and Pianka, 1966). This concept is extended to assume that when there are plentiful prey items, a predator can ‘afford’ to specialize in its diet to include only the most profitable type of prey. In contrast, at low prey densities, a specialized feeding strategy might be expected to use more energy in searching for specific prey than is gained from its consumption. Thus, the predator must include a wider variety of prey in its diet to survive. P. piscicida exhibited classical optimal foraging behavior, with the degree of preference for fish blood cells higher at higher total prey availability than at lower prey levels (Table 2, treatments 4 and 8). This is consistent with what would be expected for an organism that can specialize on a
prey type while remaining capable of feeding on multiple prey items. The cryptoperidiniopsoids, however, did not display this behavior; instead, they showed less preference for fish blood cells when blood cells were more abundant. One hypothesis for the different response to prey availability between the two dinoflagellate taxa is a possible difference in their ability to discern between the experimental high and low treatments. The grazing rate of P. piscicida continued to increase as available Rhodomonas increased, while the grazing rate of the cryptoperidiniopsoids reached a maximum level that did not continue to increase when Rhodomonas densities exceeded 200 cells well1. The grazing of the cryptoperidiniopsoid both at the high and low treatments may have been limited by satiation, searching or digestion time. Another factor that may have influenced the outcome is how the two predators attacked the prey. Swarming behavior, in which multiple dinoflagellates feed on a single food item (e.g. Spero and More´e, 1981), was observed by both species on both prey types, but appeared to occur more in cryptoperidiniopsoids grazing on Rhodomonas. Often, dinoflagellates appeared to ignore the majority of the available Rhodomonas population and joined other dinoflagellates to actively feed on a single Rhodomonas cell. This aggregating behavior in P. piscicida and cryptoperidiniopsoids has previously been documented (e.g. Parrow and Burkholder, 2003). Often between 3 and 7 dinoflagellates were observed in contact with a single prey item, although occasionally there were in excess of 20 dinoflagellates. This multiple organism feeding strategy may explain why maximum grazing rates of these heterotrophic dinoflagellates can sometimes be low (e.g. for cryptoperidiniopsoids, less than 1 Rhodomonas dinoflagellate1 6 h). Peduncular feeding likely results in some release of the prey cell contents into the surrounding medium. Dinoflagellates are known to detect various chemical stimuli (e.g. Levandowsky and Kaneta, 1987). P. piscicida has been shown to detect and swim toward a gradient of fish tissue and excreta (Cancellieri et al., 2001). This behavior may indicate that P. piscicida prefers to feed on ‘leaky’ cells, as suggested by Parrow and Burkholder (2003), or that the dinoflagellates fail to detect intact cells as potential prey in the presence of leaky cells. If leaky cells are preferred, a higher level of undamaged/intact Rhodomonas cells would not necessarily promote increased feeding. The above scenario would suggest that intraspecific competition for food may be limiting the grazing rates of these species, especially cryptoperidiniopsoids, where swarming behavior appeared to occur more commonly than
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expected, considering that available prey were abundant. Thus, possible differences in chemosensory ability between the two dinoflagellate species may partly explain the difference in preference response to total abundance. If the cryptoperidiniopsoids only detected a small subset of the prey, the damaged or ‘leaky’ cells could have been effectively limiting in both the high and low prey abundances tested. 5. Conclusions P. piscicida and a related cryptoperidiniopsoid dinoflagellate have the ability to graze on Rhodomonas sp. and fish blood cells and respond to increased prey abundance. Furthermore, the dinoflagellate strains tested here fed preferentially on fish blood cells over Rhodomonas. This preference was observed at all prey ratios tested, including when the preferred prey type was scarce. The strength of the preference apparently is influenced by the level of overall prey abundance. Possible differences in feeding behavior, including swarming, may also influence grazing rates and preferences. The preference for fish blood cells by both dinoflagellate taxa supports the need for further research to examine potentially harmful feeding behavior of other Pfiesteria-like-dinoflagellates. Acknowledgments Funding for this study was provided by the Virginia Department of Health and the Centers for Disease Control and Prevention and was a component of the M.S. degree program with the Department of Biological Sciences at Old Dominion University. We also thank three reviewers for their comments and insights.[SES] References Boenigk, J., Arndt, H., 2000. Comparative studies on the feeding behavior of two heterotrophic nanoflagellates: the filter-feeding choanoflagellate Monosiga ovata and the raptorial-feeding kinetoplastid Rhynchomonas nasuta. Aquat. Microb. Ecol. 22, 243–249. Bowers, H.A., Tengs, T., Glasgow, H.B., Burkholder, J.M., Rublee, P.A., Oldach, D.W., 2000. Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl. Environ. Microbiol. 66, 4641–4648. Brown, M.R., McCausland, M.A., Kowalski, K., 1998. The nutritional value of four Australian microalgal strains fed to Pacific oyster Crassostrea gigas spat. Aquaculture 165, 281–293. Burkholder, J.M., Glasgow, H.B., 1995. Interactions of a toxic estuarine dinoflagellate with microbial predators and prey. Arch. Protistenkd. 145, 177–188.
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