1. Dinoflagellate Chemotaxis and Attraction to Fish Products…………………………………………………………

ABSTRACT

CANCELLIERI, PAUL JOSEPH. Chemosensory Attraction of Pfiesteria spp. to Fish Secreta. (Under the direction of Dr. JoAnn M. Burkholder).

Dinoflagellates represent a diverse group of both auxotrophic and heterotrophic protists. Most heterotrophic dinoflagellates are raptorial feeders that encounter prey using “temporal-gradient sensing” chemotaxis wherein cells move along a chemical gradient in a directed manner toward the highest concentration. Using short-term

“memory” to determine the orientation of the gradient, dinoflagellates swim in a “run- and-tumble” pattern, alternating directed swimming with rapid changes in orientation.

As the extracellular concentration of the attractant increases, a corresponding increase in the ratio of net-to-gross displacement results in overall movement toward the stimulus.

The dinoflagellates Pfiesteria piscicida and P. shumwayae are heterotrophic estuarine species with complex life cycles that include amoeboid, flagellated, and cyst stages, that have been implicated as causative agents in numerous major fish kills in the southeastern United States These organisms show documented “ambush-predator” behavior toward live fish in culture, including rapid transformations among stages and directed swimming toward fish prey in a manner that suggests the presence of a strong signalling relationship between live fish and cells of Pfiesteria spp.

Zoospores of the two species of Pfiesteria can be divided into three functional types:

TOX-A designates actively toxic isolates fed on fish prey; TOX-B refers to temporarily non-toxic cultures that have recently (1 week to 6 months) been removed from fish prey

(and fed alternative algal prey); and NON-IND refers to isolates without apparent ichthyotoxic ability (tested as unable to kill fish in the standardized fish bioassay process;

or without access to fish for ca. 1.5 years). Several Pfiesteria-like dinoflagellates have been isolated from samples in which P. piscicida and P. shumwayae are also present, including several cryptoperidiniopsoid species that have repeatedly been tested as lacking ichthyotoxic capability under ecologically relevant conditions (cell densities that occur in estuaries).

Microcapillary assay techniques were employed to determine the attraction of P. piscicida and P. shumwayae zoospores to sterile-filtered fish mucus and excreta.

Differences in attraction were measured among functional types, and between these two species and several isolates of cryptoperidiniopsoids, in ten-minute trials in which zoospores entering tubes filled with test substances were observed and counted. TOX-A zoospores of both P. piscicida and P. shumwayae were strongly and comparably attracted to fish secreta/excreta, relative to their behavior toward microcapillary tubes that were filled with filtered seawater. TOX-B zoospores of both Pfiesteria species showed intermediate attraction toward fish materials that appeared to be inversely related to time isolated from fish prey. NON-IND zoospores exhibited low attraction to fish materials.

Cryptoperidiniopsoid zoospores showed moderate attraction with no apparent influence of previous exposure to fish.

In an additional experiment that examined the signal activity in fish materials over time after collection from fresh fish, unfiltered fish materials ceased to attract P. piscicida zoospores after approximately 48 hours and ultrafiltered materials maintained attractive ability over the duration of the experiment (72 hours). These data show that filtration of fish materials may be used to extend the useful life of the chemical signal, possibly by removing bacteria that consume or degrade it.

In recent years, several researchers have identified fish kairomones (pheromones that benefit the recipient) present in fish mucus that induce life history and behavioral changes in a range of zooplankters. It is likely that one or more of these kairomones, or similar compounds, are responsible for the behavioral and developmental changes observed in Pfiesteria spp. in the presence of live fish. Data from these experiments support the current understanding that significant behavioral differences exist between functional types of Pfiesteria spp., and between these toxic dinoflagellate species and known lookalike dinoflagellates without ichthyotoxic activity under ecologically relevant conditions.

iii

BIOGRAPHY

Paul Cancellieri spent his childhood years in places like Massachusetts,

California, Puerto Rico, and Connecticut as his father’s U.S. Navy duties brought the family to a variety of coastal destinations. But the constant proximity of the family to the ocean and frequent excursions to the seashore stimulated his early interest in the oceans and their myriad of life. In high school, Paul took part in several marine science research programs, and decided to enroll at Southampton College of Long Island University after graduation. There he became interested in algae, particularly in toxic algal blooms and the ecology of the organisms responsible. After four years, and several research experiences, Paul graduated in 1998 with a B.S. (magna cum laude) honors degree in

Marine Science (Biology Concentration). His interests in toxic algae, and awareness of the intriguing story of Pfiesteria led him to apply to work toward a graduate degree in Dr.

Burkholder’s laboratory at NCSU. This thesis marks the completion of a Master of

Science degree at NC State University in Botany. Paul plans to begin a career in marine science education.

ACKNOWLEDGMENTS

I would like to thank the staff of the Center for Applied Aquatic Ecology at

NCSU for their research support and personal encouragement over the past several years, specifically Nora Deamer-Melia, Xandi Hamilton, Jenny James, Jerome Brewster, and

Hadley Cairns. I want to thank my advisory committee members, Dr. Nina Strömgren

Allen and Dr. Dan Kamykowski for their suggestions and assistance in preparing this thesis. I thank Dr. Sandra Shumway for her tireless efforts to aid me in advancing my career in science. I owe a debt of gratitude to Matt Parrow for acting as a sounding board for my ideas and theories, and to Howard Glasgow for insightful research suggestions. I wish to express my appreciation to my wife and family for their patience and encouragement. Most of all, I want to express my sincere gratitude to my major advisor for giving me so many opportunities and for providing the support and understanding that every graduate student deserves.

iii

TABLE OF CONTENTS

1. DINOFLAGELLATE CHEMOTAXIS AND ATTRACTION TO FISH PRODUCTS…………………………………………………………. 1

1.1. Abstract…………………………………………………………… 1

1.2. Dinoflagellate Chemosensory Responses………………………… 3

1.2.a. Chemotaxis by protists………………………………... 3 1.2.b. Dinoflagellate chemotactic behavior…………………. 7 1.2.c. Ecological considerations of chemotaxis in Pfiesteria spp…………………………………………. 11

1.3. Chemical stimuli in finfish excreta……………………………….. 13

1.4. Conclusions……………………………………………………….. 18

1.5. Literature Cited…………………………………………………… 20

2. CHEMOSENSORY ATTRACTION OF ZOOSPORES OF THE ESTUARINE DINOFLAGELLATES, PFIESTERIA PISCICIDA AND P. SHUMWAYAE SP. NOV., TO FINFISH MUCUS AND EXCRETA…… 27

1. DINOFLAGELLATE CHEMOTAXIS AND ATTRACTION TO FISH PRODUCTS

1.1. Abstract

This chapter presents a current understanding of chemotaxis in protists, particularly dinoflagellates, from an ecological perspective, and also reviews stimulatory effects of various bioactive components of finfish secreta. On a microscale, motile chemosensory responses by ciliates and flagellates impact the acquisition of prey and the availability of nutrient sources, and can be especially significant when harmful algae species are considered. Chemotaxis in dinoflagellates has particular ecological importance due to the roles of these organisms as parasites, symbionts, and toxin producers. The ability of dinoflagellates to detect targets (finfish, shellfish, ciliates, copepods, nutrient patches) directly affects the impact that they have on their environment, including fish kill events, parasitic fish disease outbreaks, and shellfish poisoning. As the extent and frequency of these negative environmental events has increased, researchers have begun to focus more attention toward understanding the dynamics of chemosensory attraction and its ecological role.

The toxic Pfiesteria complex (thus far, two species as P. piscicida and P. shumwayae sp. nov.) has repeatedly been implicated in major fish kills in estuaries of the southeastern United States, and has been described as displaying directed “ambush- predator” behavior toward fish prey. Experimental results indicate that Pfiesteria spp. respond to a specific signal in fresh fish secreta in a well-described manner that is linked to the onset of toxin production. This type of chemotaxis is not unique to this “ambush- predator,” but has been observed for several other species of protists in response to

threshold concentrations of specific sugars, amino acids and other nitrogenous compounds. Similarly, many planktivorous fish produce substances called kairomones that induce changes in the behavior and life history of several common zooplankton species. It is likely that the ambush-predator response of Pfiesteria zoospores to live fish is a result of chemosensory attraction to one or more of these fish kairomones, leading to initiation of toxicity.

1.2. Dinoflagellate Chemosensory Responses

1.2.a. Chemotaxis by protists

As the most readily observed behavior of motile protists (including ciliates, flagellates, amoebae, and some diatoms), ambulatory responses to chemical stimuli by these organisms have been known for more than a century (Jennings, 1906). Although early research, particularly ciliate experiments, focused on using these responses to screen for genetic mutants (Van Houten, 1978) and to test the effects of various drugs

(Doughty, 1979), more recent studies have sought to determine the ecological significance of chemotaxis1 (Antipa et al., 1983; Spero, 1985; Fenchel & Blackburn,

1999). Over the past century, researchers have identified numerous compounds that elicit attraction by various protozoans (Jennings, 1906; Fraenkel & Gunn, 1961; Dryl, 1973), including several specific chemical-taxon relationships (Verity, 1988, 1991; Berk et al.,

1990).

Protozoan chemotaxis serves several functions, including predator avoidance, gregarious responses (e.g., mating; Verity, 1988), and prey acquisition (Spero & Morée,

1981; Fenchel & Blackburn, 1999). Of these, the latter is the most critical to protist survival, owing to the relative scarcity of predators in the planktonic habitat and the use of asexual reproduction by most flagellates and ciliates (Antipa et al., 1983).

Biophysicists who have applied principles of fluid dynamics to studies of protozoan

1 Fenchel and Blackburn (1999) note that the formal definition of the term taxis is “an oriented response in the sense that organisms can at any point detect the direction of a stimulus.” These authors point out that protists are too small to detect differential concentrations across their cells, even with relatively steep gradients. Therefore, technically the term can be misused in describing protist behavior, and the term “kinesis,” meaning movement without directional bias, is more appropriate. However, since the term “chemotaxis” has more general use in the context of protozoology, it will be used here.

feeding have discriminated these organisms into three categories: filter-feeders, diffusion feeders, and raptorial feeders (Fenchel, 1980, 1986a, 1986b; Jørgensen, 1983). Although some level of particle selection has been observed in several filter-feeders (Nygaard et al., 1988), both filter feeders and diffusion feeders rely primarily on random prey acquisition and passive or “mechanical” processes (Fenchel, 1980, 1986b).

Chemosensory detection and location of food sources is necessary for raptorial feeders, including the majority of the flagellates, as these organisms encounter prey on an individual basis.

Raptorial feeders actively seek prey using one of two modes of chemotaxis: kinetic response or temporal-gradient sensing. The former is a procedure by which grazers/predators improve the statistical likelihood of encountering potential prey by increasing swimming velocity when prey are not present (Schnitzer et al., 1990). The result is more predator-prey interactions per unit of time, without directed movement toward a stimulus. In extreme cases, cells become immobile at patches of attractive material or stationary prey items, essentially becoming “trapped” by their own behavioral responses. This technique is often demonstrated by less sophisticated grazers such as bacteria and some mixotrophic phytoflagellates (Levandowsky et al., 1988), with chemoreceptors that apparently are only capable of detecting the ambient concentration of an attractant.

Temporal-gradient sensing is employed by protozoan grazers with more advanced detection capabilities, and it is far more energetically efficient than the kinetic response.

Prey acquisition by this method is based on motile responses to changes in concentration over time as the grazer/predator moves along a gradient, actively “seeking” higher

concentrations of attractant (Fenchel and Blackburn, 1999). Several studies have shown that cells which move within a concentration gradient integrate changes in signal intensity over <1 second intervals, utilizing a type of short-term “memory”, and a “run and tumble” swimming response (Berg, 1983). As the concentration of the signal external to the cell increases, random changes in swimming direction (tumbling) are repressed, and moderately directed swimming results. If the concentration outside the cell does not change or decreases, tumbling occurs more often and swimming direction fluctuates until re-orientation becomes beneficial (Köhidai, 1995). The result is a marked increase in the ratio of directed motion to random motion, often expressed as net-to-gross displacement in motion analysis studies, with increased proximity to prey items

(Kamykowski et al., 1992). On a larger scale, the effect of temporal-gradient sensitivity is observed as pseudo-random “swarming” at some distance from a stimulus and directed motion toward the same stimulus at close range. Dinoflagellates, rotifers, and most ciliated protozoans make use of this chemotactic technique (Schnitzer et al., 1990;

Köhidai, 1995; Buskey, 1997).

The type of chemosensory behavior (kinetic response or temporal-gradient detection) utilized by a motile protist is heavily influenced by the habitat of the organism, and its range of prey items. Phagotrophs that prey on motile organisms of similar size must engage in more active (and therefore more metabolically expensive) behavior to identify, track, and capture sufficient prey to maintain growth. It is thus advantageous, from an evolutionary standpoint, for these organisms to develop extensive chemosensory mechanisms for detecting and responding to specific stimuli over relatively long distances. Conversely, protists with a diet that is largely composed of nonmotile or slow-

moving prey, or relatively small (< 10% of predator size) and highly abundant prey, make more economical use of resources by employing the kinetic response to engage prey items.

An interesting example of these two modes of raptorial feeding is the planktonic ciliate Strombidium spiralis (18-20 µm), which feeds on Isochrysis galbana (Parke)

(flagellate, length ~7 µm) and the tiny (4-5 µm) heterotrophic nanoflagellate, Cafeteria roenbergensis (Fenchel and Patterson). When the weakly motile I. galbana cells are abundant, S. spiralis feeds on it using a kinetic response-type of feeding strategy that involves changes in swimming velocity in a seemingly random direction to encounter prey cells. However, when I. galbana is not present, and S. spiralis is instead preying on the faster swimming C. roenbergensis, it employs a gradient sensing technique to “hunt” individual cells (Van Houten et al., 1981).

At the population level, chemotactic responses have particular significance in terms of spatial and temporal patchiness (Mackas et al., 1985). Horizontal heterogeneity of marine plankton has been implicated as a reliable measure of many physical and chemical processes, including upwelling , mixing, and nutrient recycling. Chemosensory attraction adds an additional level of complexity to attempts at modeling oceanic patchiness, however, since all but the most sophisticated spatial pattern models treat microplankton as diffusive particles (Bennett & Denman, 1989; Edwards et al., 2000). In the same way, physical factors such as turbulence can distribute chemical signal to greater distances than diffusion alone, providing for large-scale chemotactic responses by motile protists.

The reproductive rate of many zooplankters is not sufficient to explain the speed at which some flagellate and ciliate blooms occur (Revelante & Gilmartin, 1987;

Montagnes & Lessard, 1999), suggesting that migration of predators to prey sources is a major factor in the initiation and prolongation of these events. Therefore, the ability of individual cells to respond to chemosensory stimuli, and the distance over which these stimuli can elicit a response, can play a critical role in bloom dynamics.

1.2.b. Dinoflagellate chemotactic behavior

Of the roughly 2,000 extant dinoflagellate species, approximately half are either mixotrophic or heterotrophic (Taylor, 1987). This group exhibits the widest range of raptorial feeding strategies of any protozoans, including phagocytosis (ingestion of entire prey cells), myzocytosis (transfer of body fluids by means of a pallium or peduncle to the dinoflagellate for digestion), and saprotrophy (external digestion of prey and subsequent ingestion of dissolved materials). The diversity of feeding appendage morphologies in the dinoflagellates is sufficient for use as a taxonomic character in many cases (Gaines and Elbrächter, 1987).

The variation in feeding extensions is likely a result of the wide range of prey items commonly utilized by dinoflagellates, ranging from bacteria to diatom chains and metazoan tissues several times larger than the dinoflagellate itself. Since there is often an inverse relationship between size and abundance of planktonic organisms, dinoflagellates that prey on cells larger than themselves must utilize some sort of active chemosensory attraction in order to locate individual food particles (Buskey, 1997). Observational feeding studies have repeatedly recognized feeding behavior that is indicative of

temporal-gradient sensing or “adaptive kinesis”, in that many heterotrophic dinoflagellate cells commonly swim in spiraling circles around a prey item (without physically contacting it) as they “home in” on its chemical signal (Jacobsen and Anderson, 1986).

Despite the implied utility of dinoflagellates for research on mechanisms for chemotaxis, very few researchers have investigated chemoreception and gradient sensing in this group of protozoans (Buskey, 1997). Buskey (1997) studied the feeding behavior of the pallium-feeding (i.e., uses a ‘veil’ to capture prey) dinoflagellate Protoperidinium pellucidum (Bergh) and found that this predator shows some difficulty in capturing motile prey but easily acquires diatoms. Spero and colleagues (Spero 1985; Spero and

Morée, 1981) have utilized a micro-capillary tube assay to examine chemosensory attraction of the heterotrophic dinoflagellate Katodinium [Gymnodinium] fungiforme

(Anissimova) to various amino acids and extracts from metazoan species. They found that this dinoflagellate was strongly attracted to several free amino acids that are known food sources for this species, including glycine and taurine, and that a chemotactic response was observed at threshold concentrations as low as 10-8 M.

Using agar plate methodology, in which motile cells are cultured on plates so that motility can be monitored, Hauser et al. (1975) were able to examine chemotaxis of

Cryptothecodinium cohnii (Seligo) cells in response to various chemical compounds.

This dinoflagellate feeds phagotrophically and osmotrophically on rotting seaweeds, and largely relies on water currents to transport cells to this habitat. These researchers measured the attraction of C. cohnii to a range of substances that included some that are found in high concentration near decomposing algae. Several of these, such as betaine, glucose, choline, and L-alanine that are found in this habitat, are known to be

metabolized by this dinoflagellate. Cells of C. cohnii showed active attraction to betaine and glucose, but were repelled by choline and L-alanine. Interestingly, cyclic AMP

(cAMP), which is known to play a role in signalling for aggregation and prey detection in many protozoa, did not have a chemotactic effect on C. cohnii in these experiments.

More recently, the protein moiety used as a recognition site for C. cohnii to choose attachment points on one red algal genus, Porphyridium, has been identified through the use of algal mutants lacking in the protein (Ucko et al., 1999). Several researchers suggest that the linkage between the utility of a nutritional source (prey, dissolved nutrient) and attraction of a grazer/predator to its chemical cue may require reconsideration in some cases (Hauser et al., 1975).

The autotrophic dinoflagellate, Dinophysis spp., is a known prey species of the ciliate, Tiarina fusus (Claperéde and Lachmann) in the pelagic waters of the North Sea

(Jacobsen & Anderson, 1986; Hansen, 1991). The ciliate and dinoflagellate are approximately the same size (60-100 µm), and T. fusus is rare among the ciliates in its ability to ingest same-size prey. The situation becomes strangely reversed when the heterotrophic Dinophysis rotundata (Claperéde and Lachmann) and D. hastata (Stein) encounter cells of T. fusus. These two raptorial feeders have been repeatedly observed attacking and ingesting the larger and faster-swimming ciliate in field samples and laboratory incubations, by attaching to it with extrusomes and ingesting internal fluids via peduncles (Hansen, 1991). Prior to direct physical contact between the prey ciliate and the dinoflagellate cells, D. rotundata display a pattern of “spiraling swimming” in tightening circles around the ciliate. Researchers have suggested that Tiarina fusus secretes a trailing gradient of the signal that Dinophysis rotundata uses to find its prey

(Hansen, 1991; Fenchel et al., 1995). The discrepancy in swimming speeds forces the dinoflagellate predator to detect its prey rapidly and respond in a very short time frame, implying a highly advanced chemosensory response system.

Another interesting case of specialized dinoflagellate chemotaxis is the symbiotic dinoflagellate, Symbiodinium microadriaticum (Freudenthal), which lives in association with a variety of marine invertebrates, but is often not passed to offspring. It is therefore necessary for the dinoflagellate to retain a motile stage in its life history that is capable of detecting and locating new hosts (Trench, 1979). In laboratory studies in which S. microadriaticum has been removed from a host, strong attractive responses are observed to dissolved nitrogenous compounds, especially ammonium (Fitt, 1985). Although Fitt

(1985) was the first to document chemosensory responses from a phototrophic dinoflagellate, his data showed that for chemotaxis to be observed, S. microadriaticum must be in the lag phase of growth and within 1 cm of the ammonium source. He concluded that chemosensory attraction alone could not account for the high rate of infection seen under field conditions, and that random encounters between host and symbionts play an important role (Fitt, 1985).

Interactions between motile dinoflagellates and their prey appear to depend almost exclusively on chemosensory signals, despite the remarkable diversity of prey items, signals, and methods of capture. Although it is evident that a diversity of receptors and biochemical pathways must be involved in dinoflagellate chemotaxis, only a limited number of studies have examined this aspect of dinoflagellate ecology and behavior.

Overall, it remains clear that further research in the area of chemical signalling between

predator and prey is needed to understand feeding dynamics in the plankton, and among dinoflagellates specifically.

1.2.c. Ecological considerations of chemotaxis in Pfiesteria spp.

The heterotrophic estuarine dinoflagellates Pfiesteria piscicida Steidinger &

Burkholder and P. shumwayae sp. nov. Glasgow & Burkholder have been implicated in fish disease events in the southeastern U.S., particularly in eutrophic waters of the

Albemarle-Pamlico and Chesapeake Bay (Burkholder et al., in press; Glasgow et al. in press). The life cycle of these two species includes swimming and amoeboid stages, as well as the capability to rapidly encyst and remain dormant in the benthos for long periods of time (Burkholder & Glasgow, 1997). Maintenance of actively toxic isolates

(TOX-A functional type; Burkholder et al., in press) has only been possible thus far in the presence of live fish or fresh fish tissues, suggesting that a component of the fish secreta acts as a biochemical trigger for toxin production (Burkholder & Glasgow 1997). After massive fish death at the site of a Pfiesteria-related fish kill, or in cultures from which live fish have recently been removed, rapid encystment and transformation into amoeboid forms is observed. This effect is believed to be a response to the absence of the toxicity trigger, or a related signal. The short time frame in which this response is observed would suggest that the signal is degraded, or otherwise made inaccessible to Pfiesteria zoospores, in a rapid manner consistent with bacterial consumption.

As discussed previously, bloom dynamics can be critically affected by chemotactic responses of bloom-forming species. This point is particularly significant in the case of Pfiesteria, which has demonstrated in vitro attraction to live fish (Burkholder

& Glasgow, 1997) at swimming velocities as high as ca. 760 µm sec-1 (Burkholder et al., in press), and for which the attractant (fish) also acts to induce toxicity.

Numerous observations of Pfiesteria spp. swimming behavior in proximity to prey cells and fish tissues suggest that these organisms, like most heterotrophic and mixotrophic dinoflagellates, utilize the ‘temporal-gradient sensing’ type of chemotaxis

(Burkholder & Glasgow, 1995, 1997; Burkholder et al, 1992; Glasgow et al., 1998).

Swimming movements are characterized as “run-and-tumble”, with net-to-gross displacement (NGD) increasing as the cell detects and subsequently approaches potential prey (Burkholder & Glasgow, 1997).

The variety of compounds that Pfiesteria utilizes as food sources (Burkholder &

Glasgow 1995; Glasgow et al., accepted), and the diversity of particles that it can detect, suggest that these cells have a complex array of chemoreceptors. Such an assortment of receptors would allow for a range of responses to various stimuli, and for discrimination between types of stimuli. In their natural estuarine habitat, Pfiesteria zoospores are presented with nutritional sources ranging from dissolved organic molecules to phytoplankton, to finfish and shellfish. The demonstrated success of this organism in estuarine habitats may be due to its ability to find and utilize these resources.

1.3. Chemical stimuli in finfish secreta

Fish secreta can act as extracellular signals for protozoans and other zooplankton, on the basis of correlative as well as experimental observations. Relatively extensive information is available about the interactions between parasitic dinoflagellates and finfish hosts, including the nature of the chemical signal used by parasites to identify specific hosts. These parasites have a motile dinospore stage, that is involved in host location and infection. The economical and ecological damage caused by these dinoflagellate parasites has stimulated an abundance of research studies into the nature of these interactions (Landsberg et al., 1994; Buckland-Nicks and Reimchen, 1994; Cobb et al., 1998).

The most commonly studied species of parasitic dinoflagellate, Hematodinium perezi (Chatton & Poisson), infects a wide range of shellfish including the commercially important Norway lobster (Nephrops norvegicus), snow crab (Chionoecetes opilio), and blue crab (Callinectes sapidus). Although the dinoflagellate stage that seeks out potential hosts is necessarily motile, all H. perezi cells found within crustacean tissues have lacked flagellae (Field et al., 1992) indicating that significant changes take place in the parasite cell after infection. The specificity of parasite-host relationships suggests that a type of signalling is used by the dinoflagellate parasite to find appropriate hosts, although evidence of this signalling is limited.

Immunity to infection by dinoflagellate parasite has also been observed in individual shellfish exposed to sublethal concentrations of the parasite for long periods of time (Cobb et al., 1998). Immunity is achieved through the development of specific

immunoglobulins (Ig), at potential points of infection, that are capable of inhibiting the biochemical pathways of attachment in the dinoflagellate (Cobb et al., 1998).

The components of fish secreta, including mucus and other compounds other than urine and feces (excreta), vary between fish species in both identity and quantity, although no broad-scale formal characterization of these constituents in any finfish has ever been published (Chivers & Smith, 1998). Research to date on finfish secretions has focused on (1) the functions of fish mucus (osmoregulatory, immune, and predator avoidance), (2) the nature of alarm substances produced by fish, and (3) bioactive compounds of commercial/pharmaceutical interest. This reason mostly has emphasized the identity of several specific compounds, the nature of the secretory cells themselves, and role of secreta in the ecology of finfish.

Fish secrete a variety of proteins, glycoproteins, amino acids, salts, and sugars into adjacent water by means of secretory cells in the gills, skin, and mouth. Chloride cells in the gills accomplish much of the necessary osmoregulation by secreting copious amounts of ions and salts and allowing the flow of water into the bloodstream (Hazon &

Balment, 1998). Mucus, in contrast, primarily performs immunological functions such as protecting the epidermis from ectoparasites. Although fish mucus also acts as a osmoregulatory barrier, its impact in this regard is dwarfed by the chloride cells (Hazon

& Balment, 1998).

The presence of alarm substances, pheromones produced by wounded fish, has been known for at least 30 years, through various studies that were reviewed recently by

Chivers and Smith (1998). Although the identities of these substances is not fully known, their mode of action is fairly well understood. In aquatic systems, alarm substances are

most often released by wounded prey species when secretory epidermal ‘club cells’ are damaged by a predatory attack. The signal dilutes quickly in water, and alerts conspecific individuals of the presence of a predator (Chivers and Smith, 1998). The response by conspecifics varies from ‘watchful behavior’ to ‘panicked escape’, and can be observed at distances of as much as 100 m. There is growing evidence that naïve juveniles learn to respond to predators that they have never encountered in a process mediated by the presence of alarm pheromones (Magurran, 1989), extending the impact of these chemical signals on vertical (generational) as well as horizontal (spatial) scales.

One class of fish pheromones, kairomones2, has stimulated many recent scientific findings (Berendonk, 1999; Forward & Rittschof, 1993, 1999, 2000; Loose et al., 1993;

McKelvey & Forward, 1995; Rose et al., 2000; Weber, 2001). In 1987, Steven Dodson and his colleagues discovered that the normal diel vertical migration (DVM) of zooplankton, which involves migration to the surface at night to feed and descend to depth during the day to avoid predators, is triggered by waterborne substances released by fish (Dodson et al., 1988). Over the past 20 years, researchers have shown that these kairomones are released by planktivorous fish, and can seriously affect cladocerans, rotifers, dipteran larvae, and other organisms. In Daphnia, the fish kairomone is a freely dissolved compound of molecular weight <500 Daltons, with hydroxyl groups but no amino acids. The Daphnia signal is also predator-specific, differing in several chemical groups among genera of planktivorous fish, and it initiates photoresponsive DVM (von

Elert & Loose, 1996; von Elert & Pohnert, 2000).

2 Pheromones, by definition, are subdivided into synonomes (in which both the producer and receiver benefit), allomones (where the recipient of another species is affected detrimentally), and kairomones (which benefit the recipient) [definitions from Morris, 1992]

Life history changes can also be induced by fish kairomones, in apparent response to the prey size range of the fish predators as controlled by feeding morphology. Gape- limited planktivores can consume prey up to a maximum size limited by mouth opening, whereas filter-feeders consume prey organisms down to a minimum size which can be trapped on their mouth parts and feeding appendages. The kairomone response is appropriate for each, i.e., gape-limited planktivorous fish produce a signal that triggers rapid growth and delayed maturation, while the chemical released by filter-feeding fish causes early maturation and smaller body size in organisms such as Ceriodaphnia dubia and Artemia salina (Havel & Dodson, 1987; Black & Hairston, 1988).

Some methylamines and methylaminoxides have been suggested as potential kairomones (Boriss et al., 1999), as they are present in substantial quantities in fish mucus of many species. Research thus far, however, indicates that these compounds stimulate only changes which would be expected from any mildly harmful substance

(Sakwińska, 2000). These researchers do not claim that methylamines do not elicit a response, only that the responses of kairomones are often linked to specific predator-prey relationships.

There is some evidence that intracellular signalling molecules such as calmodulins (CaMs) and immunoglobulins (Igs) may play a role in extracellular communication. Several types of CaM are known to stimulate stage transformations in marine flagellates that have amoeboid stages (Fulton et al., 1986; Shea & Walsh, 1987), and CaM-mediated calcium signalling is necessary for infection of the protozoan pathogen Toxoplasma gondii (Bonhomme et al., 1999). The extracellular effect of Ig is a result of the release of this immunological protein to combat antigens, localizing

macrophage activity at the site of invasion (Lin et al., 1996). There is even an example of a protozoan that has developed sensitivity to Ig in solution, and uses it as a signal for detecting and locating hosts (Laudan et al., 1986).

1.4. Conclusions

This chapter reviewed current understanding about the ecological importance of dinoflagellate chemotaxis and of chemical signals in finfish secreta, with secondary emphasis on the role of chemotaxis in Pfiesteria spp. toxic activity and bloom dynamics.

As a critical part of the trophic web in most aquatic systems, and the cause of fish kills, shellfish poisonings, and fish disease events, dinoflagellates demonstrate virtually every known type of heterotrophic feeding, including phagocytosis, saprotrophy, and myzocytosis; and they largely seek out their prey by means of temporal-gradient sensing, i.e., moving up a detectable gradient of a chemosensory stimulus. The attraction of motile dinoflagellates toward their prey can involve a specific predator-prey relationship, or these organisms can be prey generalists. Regardless, highly developed signalling pathways apparently are involved, which may not be linked to the prey components of highest nutritional value.

Many finfish and shellfish species are known to engage in extracellular signalling using molecules like calmodulin, immunoglobulins, and alarm pheromones, which have also been shown to stimulate several species of protozoans, including dinoflagellates.

The recent surge in research on fish kairomones has resulted in the identification of several compounds from fish mucus that are known to cause life history changes in various zooplankters. The toxic dinoflagellates Pfiesteria piscicida and P. shumwayae exhibit directed motion toward live fish tissue and freshly collected secreta, in a rapid manner that indicates a nominal propensity for detecting a chemical signal in these substances. They demonstrate high swimming velocities and tolerance over a wide range of environmental parameters, which enhance their ability to feed on a variety of prey

items including finfish, shellfish, and algae. They are capable of resting in a cyst stage until ambient conditions and prey availability are optimal, and can subsequently excyst in a rapid, synchronized manner.

There is ample evidence in support of a chemosensory relationship between estuarine fish and species of the toxic Pfiesteria complex species, both in a general sense and from specific experimental data. The nature of the relationship and the chemical signals involved are not well understood, although recent literature has provided some insights. Fish species affected by toxic Pfiesteria outbreaks, primarily Atlantic menhaden (Brevoortia tyrannus) which is an oily fish with particularly thin ventral epidermis tissue, likely release a kairomone that triggers a motile chemotactic response by zoospores in the sediments and the water column. This same stimulus, or a related pheromone, appears to induce toxin production by Pfiesteria spp. in close proximity to the fish which, in turn, can promote rapid lesion formation on the fish epidermis. This chain of events is supported by information to date on both behavior of Pfiesteria and chemistry of fish kairomones.

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2. CHEMOSENSORY ATTRACTION OF ZOOSPORES OF THE ESTUARINE DINOFLAGELLATES, PFIESTERIA PISCICIDA AND P. SHUMWAYAE SP. NOV., TO FINFISH MUCUS AND EXCRETA

Paul J. Cancellieri, JoAnn M. Burkholder, Nora J. Deamer-Melia, and Howard B. Glasgow

Journal of Experimental Marine Biology and Ecology (accepted)

Chemosensory attraction of zoospores of the estuarine dinoflagellates, Pfiesteria piscicida and P. shumwayae sp. nov., to finfish mucus and excreta

Paul J. Cancellieri, JoAnn M. Burkholder*, Nora J. Deamer-Melia,

and Howard B. Glasgow

* Corresponding author: Center for Applied Aquatic Ecology North Carolina State University 620 Hutton St., Suite 104 Raleigh, NC 27606

Telephone: (919) 515-2726. Fax: (919) 513-3194 Email: [email protected]

Abstract

Toxic strains of the estuarine dinoflagellates, Pfiesteria piscicida and P. shumwayae sp. nov., can cause fish death and disease, whereas other estuarine ‘lookalike’ species such as cryptoperidiniopsoids have not been ichthyotoxic under ecologically relevant conditions.

Chemosensory attraction of three functional types of these Pfiesteria spp. were separately evaluated for their attraction to fresh fish mucus. Clonal cultures of actively toxic (TOX-

A, engaged in killing fish) and temporarily nontoxic (TOX-B, tested as toxic but without access to live fish for > 1 week) functional types of P. piscicida and P. shumwayae sp. nov. were derived from the same clones whereas the non-inducible (NON-IND) cultures of necessity, were from different clonal isolates. NON-IND cultures were grown on algal prey, without fish for 1.5 years, and were incapable of re-initiating significant toxin production in the presence of fish when tested. Attraction to fish mucus was based on the number of zoospores that entered microcapillary tubes containing sterile-filtered 15-ppt water (controls), versus entry into tubes with sterile-filtered mucus (collected in 15-ppt water) that had been collected from live tilapia, bluegill, hybrid striped bass, and Atlantic menhaden (tested separately with mucus within 3 hr of removal from live fish). TOX-A zoospores of both Pfiesteria species exhibited the strongest attraction to the fish mucus/ excreta, with comparable response to the materials from all four test fish species. TOX-B zoospores showed an intermediate response that apparently depended on the duration of mucus separation from the live fish: the shorter the separation period, the stronger the zoospore attraction to the fish mucus. In contrast to TOX-A and TOX-B zoospores,

NON-IND zoospores generally showed little or no response to the fish mucus. Zoospores of the cryptoperidiniopsoid demonstrated a moderate attraction that did not appear to

2 depend on the time of isolation from fish. TOX-A zoospores were also tested for attraction to sterile-filtered versus non-filtered fish mucus (time separated from the live animal, 3-96 hr). These zoospores, which initially had been actively attracted, were no longer attracted to the unfiltered milieu after ca. 48 hr, whereas attraction to the sterile- filtered fish mucus persisted throughout the duration of the experiment. Thus, the attractant signal was degraded or effectively blocked by the bacterial community within hours of isolation from live fish. This study indicates the importance of functional type or toxicity status in the behavioral ecology of Pfiesteria spp. Attraction to fish materials strongly depended on the functional type and, thus, on the recent history of toxic activity.

Non-inducible strains of Pfiesteria spp., unlike toxic and potentially toxic strains, were virtually unresponsive to fish mucus.

Key words: Chemosensory, functional type, estuary, fish, mucus, non-inducible, toxic dinoflagellate, toxic Pfiesteria complex, cryptoperidiniopsoid

1. Introduction

Dinoflagellates within the genus Pfiesteria (two species known thus far, P. piscicida Steidinger & Burkholder and P. shumwayae sp. nov. Glasgow & Burkholder) form the toxic Pfiesteria complex (TPC; Glasgow et al., in press; Burkholder et al., in press). Their ichthyotoxic strains have been implicated as primary and secondary causative agents of numerous fish kills and fish epizootics in the largest and second largest estuaries on the U.S. mainland (Chesapeake Bay and the Albemarle-Pamlico

Estuarine System, respectively; Burkholder et al., 1992; Burkholder and Glasgow, 1997).

They are stimulated to produce toxin by live fish or their fresh secreta/excreta and tissues

(Burkholder and Glasgow 1997), to which they are strongly attracted (Pfiesteria

Interagency Coordination Working Group [PICWG], 2000; Burkholder et al., in press).

Toxic strains of Pfiesteria spp. have a complex life cycle including an array of flagellated, amoeboid, and cyst stages (Burkholder and Glasgow, 1995, Steidinger et al.,

1996). The presence of live fish stimulates amoebae and cysts to produce toxic zoospores (Burkholder and Glasgow, 1995, 1997; Burkholder et al., in press).

On the basis of repeated observations of Pfiesteria toxicity in the presence versus absence of fish (Burkholder et al., 1992, 1995; Burkholder and Glasgow, 1997), three functional types of Pfiesteria zoospores have been defined (Burkholder et al., in press).

TOX-A zoospores are actively toxic, and are found in the presence of live fish

(Burkholder and Glasgow, 1997; PICWG, 2000). Within hours of separation from fish prey, zoospore toxicity diminishes, then is re-induced after additional exposure to fish

(Burkholder and Glasgow, 1997). TOX-B zoospores are capable of toxicity, but cease toxin production in the absence of live fish and prey on other organisms such as bacteria

4 and algae. This functional type has been described as temporarily nontoxic (Burkholder and Glasgow 1997), although it may retain negligible or residual toxicity (Springer

2000). As in many other toxic algal species (here, including heterotrophic dinoflagellates; e.g., Anderson, 1990; Gentien and Arzul, 1991; Bates et al., 1998; Edvardsen and

Paasche, 1998), non-inducible strains (NON-IND functional type) of both Pfiesteria spp. have been isolated. These strains are apparently incapable of toxin production based on present knowledge about toxicity, although molecular controls that can re-initiate toxin production in this functional type may eventually be found (Burkholder et al., in press).

As a separate phenomenon and an artifact of culture conditions, toxic strains become non-inducible, or lose toxin-producing ability, after extended culture with fish (typically,

≥ 6 months), accelerated when Pfiesteria is given other prey in the absence of fish

(toxicity loss in most clonal cultures within ≥ 6 weeks).

Other researchers have described Pfiesteria lookalike species (Steidinger et al.,

1996; Litaker et al, 1999; Seaborn et al., 1999; Marshall et al., 2000) that have been informally named as ‘shepherd’s crook,’ ‘Lucie,’ and Cryptoperidiniopsis gen. ined.

(e.g., C. brodyii gen. et sp. ined.; Dr. K. Steidinger, Department of Environmental

Protection – Florida Marine Research Institute, St. Petersburg, Florida, U.S.A., pers. comm.). These organisms have been present in estuaries where Pfiesteria spp. have been documented. Unlike toxic strains of Pfiesteria spp., thus far none of the lookalike species have been ichthyotoxic under ecologically relevant conditions (live or sonicated cells and fish in the standardized fish bioassay process, at similar densities as found under field conditions; Marshall et al., 2000; Burkholder et al., in press). Our research team has sought to compare the two known toxic Pfiesteria spp. and lookalike species by

5 investigating their cellular and behavioral differences in response to various environmental stimuli.

Toxic Pfiesteria outbreaks require that benthic and suspended stages of TPC species (amoebae and cysts, and TOX-B zoospores, respectively) produce/transform to actively toxic zoospores (TOX-A functional type) when sufficient live fish are detected

(Burkholder and Glasgow, 1997; Burkholder et al., 1999). TPC zoospores have shown positive chemotaxis toward fresh fish tissues in laboratory trials; therefore, it is expected that toxic strains of TPC zoospores exhibit positive chemotaxis toward fish and their fresh secreta in estuarine waters (Burkholder and Glasgow, 1997). The stimulatory substance in fish materials is believed to be a water-soluble chemical released by live fish, including potential candidates within methylamines, methylaminoxides, mucus proteins, or mucopolysaccharides (Burkholder et al., 1995; Burkholder and Glasgow,

1997; Boriss et al., 1999). Fresh fish secreta has been documented to affect aquatic microfauna other than dinoflagellates. For example, zooplankton species respond to glycosaminoglycans in fish mucus, which induce life cycle changes and migratory activity as defenses against the fish predators (Forward and Rittschof, 1999, 2000). In

Pfiesteria, cessation of toxin production soon after fish death (e.g., Burkholder and

Glasgow, 1997) suggests that the fish signal is rapidly removed or ‘lost’ from the water.

Aside from tests for toxicity by TOX-A zoospores in the presence/absence of fish materials, little is known about comparative responses of the two known Pfiesteria species to fresh fish secreta/excreta, or behavioral differences in zoospore response depending on the fish species. The objectives of this research were to compare the behavior of TOX-A, TOX-B, and NON-IND P. piscicida and P. shumwayae vs. a

6 cryptoperidiniopsoid species in response to a mixture of fish mucus/excreta; to test whether/how the behavioral responses differ depending on the fish species; to test whether/how the behavioral responses of TOX-B and NON-IND Pfiesteria spp. functional types differ depending on the duration away from live fish or re-exposure to live fish; and to assess the duration of the response by Pfiesteria zoospores when bacteria are present versus absent in the fish-materials substrate. We hypothesized that TOX-A,

TOX-B, and NON-IND zoospores would show progressively less attraction to fresh fish mucus/excreta, with TOX-A zoospores most strongly attracted and NON-IND zoospores least attracted. Moreover, considering information from other research indicating that

NON-IND Pfiesteria becomes increasingly ‘plant-like’ or mixotrophic, with higher apparent reliance on kleptochloroplasts from algal prey (Burkholder et al., in press;

Parrow et al., accepted), we predicted that NON-IND isolates would show little or no attraction to fresh fish mucus/excreta. Based on experience with cryptoperidiniopsoids, we anticipated that these isolates would show moderate to low attraction, comparable to the behavior of TOX-B or NON-IND Pfiesteria zoospores. We expected that comparison of the stability of the attractant signal in sterile-filtered versus unfiltered fish materials over time would enable insights as to whether the substance(s) in fish secreta/excreta that attracts TOX-A Pfiesteria is chemically degraded, or lost via microbial consumption.

2. Methods and Materials

2.1 Pfiesteria and cryptoperidiniopsoid isolates

A toxic culture of Pfiesteria piscicida (#271A-1) was isolated from the mesohaline Neuse Estuary, North Carolina (Glasgow and Burkholder, 2000) and cloned in uni-dinoflagellate culture with algal prey (cryptomonads) as a food source at NCSU.

Pfiesteria spp. have not been cultured successfully without a prey source (Burkholder et al., 1992; Burkholder and Glasgow, 1995; Lewitus et al., 1995; Glasgow et al., 1998), and it has not been possible to induce toxin production unless live fish are added

(Burkholder and Glasgow, 1997). Clonal Pfiesteria thus is defined as an isolate of (uni- dinoflagellate) P. piscicida or P. shumwayae sp. nov., with associated endosymbiont bacteria that may be present (Steidinger et al., 1995; Lewitus et al., 1999) and its prey

(Burkholder et al., in press). The clone was isolated from water samples taken from fish- killing bioassays (fish bioassay process of Burkholder et al. 1992, 1995; Burkholder and

Glasgow, 1997; Burkholder et al., in press) using a flow cytometer (Coulter® EPICS®

AltraTM with HyPerSortTM system; Coulter Corporation, Miami, Florida, U.S.A.) equipped with a water-cooled INNOVATM Enterprise IITM Ion Laser (Coherent, Inc.,

Santa Clara, California, U.S.A.), and excitation provided by a 150 mW/488 nm argon laser line.

This isolate was used to supply TOX-A and TOX-B functional types of P. piscicida zoospores. TOX-A P. piscicida was maintained on fish prey (tilapia,

Oreochromis mossambicus Peters, length 3-5 cm) for ca. 2 months after being isolated from natural phytoplankton samples, and prior to the experiments. Toxicity of the clone was cross-confirmed by Dr. H. Marshall, Old Dominion University, Norfolk, Virginia,

U.S.A.; Marshall et al. 2000). Assessment of toxicity by fish death in the presence of clonal Pfiesteria spp. has been used routinely in such research because the toxins of these dinoflagellates have been, at present, only partially characterized. Thus, purified standards are not yet available to verify the reliability of assays that are being developed to detect Pfiesteria toxins in fish cultures and estuarine samples (e.g., Fairey et al., 1999).

The clone was cross-confirmed as uni-dinoflagellate using a Heteroduplex mobility assay

(Dr. D. Oldach, University of Maryland, Baltimore, MD; Oldach et al., 2000).

The species identification from scanning electron microscopy of suture-swollen cells (Glasgow, 2000), was cross-checked using polymerase chain reaction (PCR) species-specific molecular probes of our laboratory (18S rDNA), and a fluorescent in situ hybridization (FISH) probe of Dr. P. Rublee, UNC-Greensboro (Rublee et al., 1999).

TOX-B zoospores were cultured as a sub-culture of TOX-A P. piscicida that was separated from fish after 1 month of fish-killing activity, cleaned and re-cloned, and grown for up to 6 months prior to experiments using cryptomonad algal prey (cloned from CCMP757 [Rhodomonas sp., Culture Collection for Marine Phytoplankton,

Bigelow Laboratory, Bigelow, Maine, U.S.A.]). Three other isolates from the mesohaline Neuse (#B93BN3, #101296, and #416T) were used for additional strains of

TOX-A and TOX-B P. piscicida, prepared as above. Each P. piscicida TOX-B isolate was tested at several time intervals of isolation from live fish, including 0.2, 0.5, 2, and 5 months for #271A1; 1 and 1.25 months for #416T; 0.25 and 0.75 months for #101296; and 0.5, 1, 1.5, and 3 months for #B93BN3.

Four other Neuse clones (#114160, #89B2, #140B, and #271A), isolated from the same location using algal assays (Burkholder and Glasgow, 1995; PICWG, 2000), were

9 necessary for NON-IND P. piscicida (species identification cross-confirmed as above; non-inducibility confirmed for isolates grown on algal prey > 28 months). The NON-

IND isolates were grown with Rhodomonas prey for ca. 3 months after being isolated from the Neuse. All four clones were tested as unable to kill fish in repeated, standardized fish bioassays two weeks prior to the experiments.

Four cultures each of toxic (TOX-A, TOX-B) functional type, and three cultures designated as NON-IND P. shumwayae sp. nov. zoospores were isolated from the mesohaline Neuse Estuary using similar procedures as those given for P. piscicida isolates. Cultures #104TPA, #101127, #121B2, and #101272 were used for TOX-A and

TOX-B P. shumwayae sp. nov.; and cultures #270A2, #130T1 and #598A were used for

NON-IND P. shumwayae sp. nov. TOX-B P. shumwayae cultures were assayed at several periods of isolation from fish prey, including 0.5, 1.0, and 1.25 months for

#104TPA; 0.75 and 3.0 months for #101127 and #121B2; and 1.5 months for #101272.

Of the three cultures used as NON-IND P. shumwayae, two cultures had been grown on cryptomonad prey for 1.5 years prior to the experiments. The third culture had been grown on algal prey for nine months, and was tested as incapable of re- initiating toxin production and killing fish in repeated, standardized fish bioassays four weeks prior to the experiments. Eight clones of cryptoperidiniopsoids (#508A, #543A, #547A, #548A,

#599A, #1040C, #1039C, and #101240) were isolated from field samples and fish bioassays, and were cultured on cryptomonad prey (cloned from Rhodomonas CCMP

#757) for 1 week to 15 months. All eight isolates were verified as cryptoperidiniopsoids using PCR primers specific to that group (Rublee et al., 1999), although not specific to

10 one species; therefore it is possible, and likely, that more than one species of cryptoperidiniopsoid dinoflagellate is represented by these isolates.

2.1. Fish mucus/excreta collection

Fish mucus/excreta was collected from live tilapia, Atlantic menhaden

(Brevoortia tyrannus Latrobe; from the mesohaline Neuse Estuary, North Carolina,

U.S.A.), hybrid striped bass (Morone saxatilis x Morone chrysops Rafinesque; from the

Pamlico Aquaculture Field Laboratory, NCSU, Aurora, North Carolina, U.S.A.), and bluegill (Lepomis macrochiris L.; from Mr. T. Shedd, U.S. Department of Defense, Fort

Detrick, MD). All fish were juveniles with an average mass of 15 g, and all (except for

Atlantic menhaden) had been fed Tetra Marin® fish food once daily prior to collecting excreta and mucus. The fish were rinsed in 15-ppt water (made with deionized water and

Instant Ocean salts, then filtered using 0.22 µm-porosity, Pall-Gelman Acrodisc® filters).

Fish were placed into a Ziploc® plastic bag (1 individual per bag) with 50 mL of sterile- filtered 15 ppt water, and the bag was sealed. Mucus/ excreta production was stimulated by repeated gentle squeezing of the bagged fish; after 4 min the fish were removed, and

30 mL of material from the bag were collected. Since the fish were completely submerged, the water contained both mucus and excreta. The medium was sterile-filtered immediately upon collection (0.22 µm-porosity, Pall-Gelman Acrodisc® filters, including filtration of the menhaden materials in the field, followed by transport to the laboratory on ice) or unfiltered depending on the experiment. The excreta/secreta samples were held at 4oC in darkness until use (within 3 hr of collection from the live animals).

2.2. Assay for chemosensory attraction

Microcapillary tubes were prepared using a Flaming-Brown micropipette puller

(Model P-97, Sutter Instruments, Novato, CA) to an aperture size of 30 ± 2 µm (mean ± 1 standard error [SE], n = 30; modified from Spero, 1985). The tubes were filled by capillary action with the sterile-filtered fish mucus/excreta solution, and one end was capped with inert Teflon® stopcock grease. We also tested Pfiesteria general response to organic molecules, using microcapillary tubes filled with an amino acid standard solution

(mixture of 12 amino acids in 15-ppt seawater at 2.5 nM each, #NCI0180; Pierce

Chemical Company, Rockford, Illinois, U.S.A.) or with a bovine serum albumin [BSA] solution (#A3350; Sigma Chemical Company, St. Louis, Missouri, U.S.A.) in 15-ppt water. These two solutions were tested for response of Pfiesteria spp. functional types and the algal-fed cryptoperidiniopsoid dinoflagellates at several concentrations each, spanning the range of amino acid levels measured in the Neuse Estuary (total dissolved amino acids 25 µM to 2.5 nM; BSA 3-300 mg/mL; Center for Applied Aquatic Ecology,

NCSU, unpubl. data). Control microcapillary tubes were filled with sterile-filtered 15- ppt water.

Each trial consisted of one microcapillary tube immersed into a small dish containing 4 mL of culture water that had been filtered with a 30-µm mesh to remove large contaminants. Activity at the tube aperture was observed using light microscopy

(Olympus AX-70 research light microscope, 600×, water immersion objective; Olympus,

Inc., Melville, New York, U.S.A.) and videotaped for 10 min (Figure 1). The videotaping equipment included a cooled-chip digital video camera (Optronics

Corporation, Goleta, California, U.S.A.), S-VHS video recorder (model SVO-9500MD,

Sony Electronics, Inc., Park Ridge, New Jersey, U.S.A.), and 53-cm video monitor (Sony

Electronics, Inc.). Total cells entering and leaving the microcapillary tube over the course of the experiment (to determine net cells entering) were recorded. The average rate of entry per minute was also calculated over the duration of each trial. Results were adjusted to the appropriate scale by normalizing total cell density to the median value for all experiments (104 zoospores mL-1), and then adjusting the value of net cells entering accordingly. Trials (control and experimental) were performed in triplicate (that is, 3 trials per isolate; also 4 isolates per functional type for each species [3 isolates of P. shumwayae NON-IND due to seasonal lack of availability], and 8 periods of TOX-B zoospore separation from live fish for each Pfiesteria species). Assays on cryptoperidiniopsoid isolates were carried out with similar replication (3 trials per isolate,

5 periods of separation from live fish), and used similar controls.

To test the influence of re-exposure to live fish on the response of TOX-B and

NON-IND Pfiesteria species, 4 isolates each of TOX-B P. piscicida (#101301, #101302,

#101295, #101296) and of NON-IND P. piscicida (#101298, #101299, #101300,

#101593) that had been grown on algal prey (CCMP757) for 1-5 months and 10-30 months, respectively, were re-inoculated into fish bioassays. After 48 hours of exposure, attraction to fish secreta/excreta was measured as above (3 trials per isolate, control and experimental). Introduction of fish prey to the cryptoperidiniopsoid isolates was repeatedly attempted (n = 3 trials per isolate for the 8 isolates), with and without high abundance of available algal prey (3000 Rhodomonas cells/mL), but poor growth resulted in insufficient cell densities for the microcapillary assays (≤ 800 cells mL-1; similar data reported by Marshall et al., 2000).

2.4. Time series experiment

A time series experiment was completed to assess the duration of viability of the chemical signal attractant for TOX-A Pfiesteria spp. in fish materials. Tilapia mucus/excreta was collected as above. Sub-samples were not filtered, for comparison with fish material that was sterile-filtered to remove the microbial community. We inspected the filtered fish material with light microscopy, and did not observe bacteria in either fresh or stained samples (stained with acidic Lugol’s solution [Vollenweider,

1974], held at 4oC, and observed within 4 hr at 400x), after examining 10-20 fields of view. In contrast, unfiltered material contained abundant bacteria (> 200 cells per field of view, 400x). Attraction of TOX-A zoospores (cultures #271A and #104TPA, P. piscicida and P. shumwayae sp. nov., respectively) was assayed (in triplicate) using the microcapillary tube method at 12-hr intervals over a 72-hr period, based on preliminary trials which indicated a degradation time for unfiltered fish materials of 30-50 hr. The duration over which the mucus/excreta attracted the zoospores was compared, to determine the potential influence of the microbial population in degradation of the chemical signal.

2.5. Statistical analyses

It was hypothesized that cell density affects net entry of zoospores into microcapillary tubes based on a non-biological, linear relationship. In preliminary experiments net entry of zoospores was measured in a dilution series, in which all factors other than density were held constant. The data showed that for both Pfiesteria spp., with or without fish mucus/secreta, density and net entry increased in a linear fashion (Figure

2; n = 3 trials dilution-1 species-1; r2 = 0.986-0.992). To correct for density range due to isolate variability, all values for ‘net zoospores entering tube’ were normalized to the median cell density (104 zoospores mL-1), as follows:

net cells entering tube ⋅1×104 = normalized net zoospores entering tube cell density ⋅ culture volume

Multi-factor ANOVA (MANOVA; α = 0.05; dependent variable = normalized tube entry values) was performed on the dataset to identify variables of interest (i.e., variables that accounted for variation in attraction; SAS Institute, Inc., 1997). Net entry values from triplicate trials were averaged, and mean zoospore entry values were used for statistical comparisons. Comparisons were made between control and experimental values, and between treatments in experimental trials using a Student’s t test. Differences were considered significant at p < 0.05, and behavior was regarded as positive attraction only when a significant difference was found between controls and experimental trials.

3. Results

Statistical analysis (using General Linear Model testing, SAS Institute, Inc., 1997) indicated that variations between isolates, dinoflagellate species, and fish species were not significant (p > 0.05). TOX-A and TOX-B Pfiesteria spp. zoospores exhibited a strong attraction to the microcapillary tubes filled with fish mucus/excreta, relative to zoospore response to filtered-water controls (Figures 1, 3). The attraction progressively diminished as isolates were separated from fish prey for increasing duration, with the data generally following a second-order decay function. The response of the algal-fed cryptoperidiniopsoids was intermediate, and comparable to that of a TOX-B Pfiesteria piscicida isolate that had been separated from live fish for 10 months. Time isolated from fish prey did not affect the attraction of the cryptoperidiniopsoids to fish excreta/secreta (Figure 4). Least attraction was observed when NON-IND zoospores were exposed to microcapillary tubes filled with tilapia mucus/excreta, although even at this low level the attraction was statistically significant when compared with Pfiesteria behavior toward controls. There was no significant difference in attraction toward fish species. None of the concentrations of the amino acids or the BSA solution elicited a chemosensory response from any functional type of TPC zoospores or the cryptoperidiniopsoid, even at concentrations ten-fold higher than normal in situ levels in the Neuse.

TOX-A Pfiesteria spp. zoospores entered the mucus-filled microcapillary tubes at a significantly higher rate than did other functional types (Figure 5). These actively toxic zoospores appeared to respond more rapidly to the chemical signal, and reached maximum rate of entry within 6 min. In contrast, NON-IND zoospores reached a

16 maximal rate of entry into the microcapillary tubes within 8 min, and this rate of entry was only ca. one-fourth the rate of the TOX-A zoospores. The cryptoperidiniopsoid zoospores exhibited an intermediate in rate of entry that was approximately half that of

TOX-A Pfiesteria spp. zoospores (Figure 5).

TOX-B and NON-IND Pfiesteria spp. isolates that had been re-introduced to fish prey 48 hr before the experimental trials showed marginally increased attraction to fish secreta/excreta. For the four TOX-B isolates, the average normalized net entry value increased from 65 ± 5 (mean ± 1 SE) previously fed algal prey, to 81 ± 7 when tested following re-exposure to live fish for 48 hr.

The attractant signal in tilapia mucus/excreta continued to significantly stimulate the zoospores of P. piscicida and P. shumwayae sp. nov. for at least 72 hr, if bacteria were removed from the fish materials immediately after collection (Figure 6). In contrast, unfiltered fish materials rapidly lost attractant capability within 48 hr after removal from live fish, indicating that loss of the signal is related to the presence of the microbial community.

4. Discussion

In this study, Pfiesteria piscicida and P. shumwayae sp. nov. actively toxic (TOX-

A) zoospores showed strong attraction to the mucus/excreta from all four test fish species, indicating that these dinoflagellates are positively chemotactic toward a substance(s) commonly produced by fish. The lack of an observed response (by any functional type) to the amino acid or BSA solutions suggests that the positive chemotaxis of TOX-A Pfiesteria toward fish materials is not simply a nutritional response to organic molecules but, rather, a more specific stimulation by a substance in fish mucus/excreta.

The attractant response to materials from Atlantic menhaden was comparable to, but not notably greater than, the attraction to mucus/excreta from the other species tested, of interest since 90% of the fish killed in estuarine toxic Pfiesteria outbreaks have been menhaden (Burkholder et al., 1995; Burkholder and Glasgow, 1997; Burkholder et al., in press). Other characteristics of menhaden -- such as high production of oily materials, schooling behavior, and tendency to feed in poorly flushed shallows where there are abundant phytoplankton and, often, Pfiesteria (Manooch, 1988; Noga, 1993; Burkholder et al., 1995; Burkholder and Glasgow, 1997) – probably enhance detection by these dinoflagellates. In addition, the unusually thin epidermal covering on the ventral surface of this species (Dr. J.M. Law, College of Veterinary Medicine, NCSU) could facilitate exposure to Pfiesteria toxins and toxin impacts on the underlying viscera.

The observed decrease in attraction to fish mucus/excreta by TOX-B zoospores indicates that cultured Pfiesteria spp. lose the ability to rapidly detect and respond to live fish after feeding for extended periods on non-fish prey. Subsequent re-exposure to fish prey appears to stimulate some degree of increased response to fish materials. Whether

18 this phenomenon occurs in the natural environment is unknown, but if certain biochemical pathways interact with molecular controls to elicit the positive chemotactic response toward fresh fish substances, a lag period may be required for the pathways to be activated. These findings support earlier studies which indicated that the history of

Pfiesteria exposure to fish is an important factor affecting toxicity and other aspects of the nutritional ecology of toxic strains (Burkholder et al., 1998; Burkholder et al., in press). Moreover, strains in the natural environment that inherently lack toxin-producing capability may not prey on juvenile fish, as suggested by the behavior of NON-IND

Pfiesteria spp. in this study. Other research has demonstrated that NON-IND Pfiesteria responses to nutrient enrichment and algal prey are distinct from responses of toxic strains (Burkholder et al., in press; Parrow et al., accepted).

The consistent response of the cryptoperidiniopsoids to the fish excreta/secreta, and the low cell production of cryptoperidiniopsoids in the presence of live fish, indicates that their attraction is not linked to the recent availability of fish prey. This information is consistent with current understanding of these dinoflagellates and their role in fish kills. Our difficulty in culturing this organism with fish in the absence of abundant algal prey would suggest that these cryptoperidiniopsoid isolates have limited capability to switch from algal to live fish prey. Cryptoperidiniopsoids have, however, been shown to attack and consume larval fish (Burkholder et al, in press). Thus, the significant chemosensory response that we observed may reflect the presence of a general attractant in fish excreta/secreta for some dinoflagellates.

The data from the time series experiment with sterile-filtered versus unfiltered fish mucus/excreta point to an important role of the co-occurring bacterial consortium in

19 the rapid breakdown of the attractive signal. The labile nature of the signal apparently results from its degradation by primary consumers, suggesting that bacteria may factor significantly in the dynamics of toxic Pfiesteria outbreaks. As instructive information for laboratory studies, sterile filtration of fish substances can extend their utility in research to understand the mechanisms of Pfiesteria spp. stimulation by fish. This technique also proved to be of value in reducing variability among secreta/excreta sources because it enabled use of one collected sample of fish substances over experiments lasting 1-3 days.

Pfiesteria spp. are dinoflagellates with complex life cycles (Burkholder and

Glasgow, 1995; Steidinger et al., 1996; Burkholder et al., in press; Glasgow et al., in press) and complex behavior toward a wide array of prey and other environmental stimuli

(Burkholder and Glasgow, 1997; Burkholder et al., 1998; Glasgow et al., 1998;

Burkholder et al., in press; Parrow et al., accepted). This study supports the current understanding of functional types of TPC species (Burkholder and Glasgow, 1997;

PICWG, 2000; Burkholder et al., in press; Parrow et al., accepted), and the importance of working with toxic rather than NON-IND Pfiesteria spp. strains in research to understand the behavior, physiology, and ecology of these [toxic] dinoflagellates. Although other research has demonstrated that many other so-called ‘toxic algae’ have strains with undetectable toxin production (e.g., Anderson, 1990; Gentien and Azam, 1991; Skulberg et al., 1993; Bates et al., 1998, 1999; Edvardsen and Paasche, 1998), the potential for significant differences in the comparative ecology of toxic versus non-inducible strains within a given ‘toxic algal species’ generally has been ignored. Such comparative information takes on greater significance, given that most commercially available cultures are not screened for toxicity but are used, nonetheless, to make broad inferences

20 about the behavior and ecology of toxic species. We hypothesize that, as for Pfiesteria, use of verified toxic strains will eventually be recognized to be of critical importance in research to understand the ecology and physiology of many harmful algae. We also note that commercial algal culture collections are not properly equipped (with specially designed biohazard III facilities) to maintain toxic strains, and generally list “pfiesteria- like” species. We caution against applying data from use of such cultures toward understanding the cell biology, behavior, and ecology of TPC species, on the basis of this and other studies (Burkholder et al., in press).

6. Acknowledgments

Funding support for this research was provided by the U.S. Environmental

Protection Agency, an anonymous foundation, the North Carolina General Assembly, and the Department of Botany at NCSU. We thank A. Hamilton and N. Leaver of the Center for Applied Aquatic Ecology, NCSU, for technical support, and M. Parrow and J.

Springer for their counsel. N. Allen, D. Kamykowski, M. Mallin, S. Shumway, and two anonymous colleagues kindly reviewed the manuscript.

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A B

Figure 1. Frames from videotapes of microcapillary tube assays, illustrating zoospore attraction to tubes filled with an ultra-filtered (0.22 µm-porosity) solution of fish mucus/excreta. Frame A shows moderate to strong attractant (positive chemosensory) behavior (TOX-A Pfiesteria zoospores; scale bar = 10 µm), and frame B represents non-attraction (NON-IND Pfiesteria functional type; scale bar = 10 µm).

) 200 -1 P. piscicida P. shumwayae 160 P. piscicida (controls)c

P. shumwayae c(controls)

120

(Number · 10 min Zoospores Attracted 0 0 3000 6000 9000 12000 15000 18000

Density ( Zoospores · mL-1 )

Figure 2. Dilution series experiments to determine the relationship between zoospore density and net entry into microcapillary tubes filled with ultra-filtered (0.22 µm-porosity) tilapia mucus and secreta, or filled with ultra-filtered 15-ppt medium (controls), including data for all of the dinoflagellate species tested. The strong linear trend indicates a non-biological relationship, justifying use of normalization to allow comparison of trials with isolates of varying densities.

) -1 200 bluegill menhaden striped bass 160 tilapia control

120

0 (Number · 10 min Zoospores Attracted 0 5 10 15 20 25 30

Time Away From Fish (months)

Figure 3. Attraction of zoospores of Pfiesteria spp. to fish secreta/excreta from several species of estuarine fish. TOX-A zoospores (taken from cultures that had been in actively toxic, fish-killing mode for ≥ 1 month) and TOX-B (temporarily nontoxic) zoospores (fed algal prey for 1 week to 6 months without live fish) consistently showed significant attraction to fish mucus/excreta ( = bluegill, ● = Atlantic menhaden, = striped bass, ▲ = tilapia) relative to zoospore activity in controls ({, with microcapillary tubes containing ultra-filtered 15-ppt water). In contrast, NON-IND cultures (non-inducible status; verified for cultures isolated from live fish for > 28 months) showed very limited attraction to the fish materials. Overall, the TOX-A isolates of both Pfiesteria spp. were significantly more strongly attracted to the fish materials than cultures previously fed algal prey (TOX-B, NON-IND functional types). The data indicate a negative second-order trend for attraction of TOX-A and TOX-B zoospores to fish materials with increasing duration of separation from live fish; and the data also show no differential response of zoospores among the 4 species of fish tested (for each species of Pfiesteria, n = 3 replicates per isolate and 4 isolates per functional type; note that each isolate was tested for its response to the mucus/excreta from each fish species).

)

-1 200

P. piscicida P. shumwayae 160 cryptoperidiniopsoids controls

120

Zoospores Attracted (Number · 10 min Zoospores Attracted 0

0 5 10 15 20 25 30

Time Away From Fish (months)

Figure 4. Attraction of zoospores of the two species of Pfiesteria and a cryptoperidiniopsoid to a mixture of fish mucus and excreta. All zoospores were exposed to secreta/excreta of all four fish species, with the exception of the cryptoperidiniopsoid which was tested with only tilapia mucus. For all functional types, Pfiesteria piscicida () and P. shumwayae (z) attraction to fish secreta/excreta showed no difference between species. The cryptoperidiniopsoid () showed an intermediate response, relative to seawater controls ({), at all time intervals isolated from fish, here referring to initial sample collection since re-introduction of cryptoperidiniopsoid cultures to fish was unsuccessful. It is apparent that the history of recent exposure to fish does not affect the attraction of the cryptoperidiniopsoid dinoflagellate to fish materials.

)

-1 35 TOX-A TOX-B 28 NON-IND cryptoperidiniopsoids

(number · min Rate of Zoospore Entry 0 0246810

Duration of Experiments (minutes)

Figure 5. Rate of entry per minute of TOX-A, TOX-B, and NON-IND functional types of Pfiesteria spp. zoospores into microcapillary tubes filled with fish mucus/excreta over 10-minute experimental trials (means ± 1 SE; n = 3 for each of 4 isolates of each species and functional type). The asymptotic shape of the curves for TOX-A and TOX-B Pfiesteria spp. zoospores indicates that the 10-minute period was of sufficient length to observe the greatest differences among the functional types. The data indicate that TOX-B zoospores, separated from fish for an extended period (1 week to 6 months), were less strongly attracted to the fish materials than TOX-A zoospores which had consistently been exposed to live fish. That is, isolation from fish prey (and subsistence on algal prey) apparently promotes gradual loss of chemosensory attraction to fresh fish mucus and excreta by toxic strains of Pfiesteria spp.

150 Filtered fish secreta / excreta )

-1 Unfiltered fish secreta / excreta Controls

100

(number · 10 min Zoospores Attracted 0 0 1530456075

Duration Away From Fish (hours)

Figure 6. The extended time series experiment, showing the change in net number of Pfiesteria spp. TOX-A zoospores in attraction to sterile-filtered versus unfiltered fish mucus and excreta, in comparison to TOX-A zoospore response to control tubes filled with ultra- filtered 15 ppt water, over a 72-hour period (means ± 1 SE, n = 3). Attraction to unfiltered fish materials was lost by 48 hr, whereas the filtered fish materials attracted zoospores throughout the 72-hr duration. The data indicate that the microbial consortium consume or degrade the attractant substance(s) in the fish mucus/excreta.