Zhou Yang

Hae Jin Jeong

David J S Montagnes

The role of Oxyrrhis marina as a model prey: current work and future directions

Keywords: model organism, dinoflagellate, protozoa, Oxyrrhis

The role of Oxyrrhis marina as a model prey: current work and future directions

INTRODUCTION

There is now, and has been for some time, compelling evidence that protozoa hold a key position and are an essential link in pelagic food webs [START_REF] Conover | Interrelations between microzooplankton and other planktonic organisms[END_REF]. A high portion of primary and bacterial production is consumed by these "protozooplankton", and their role as consumers has been well established through field and laboratory studies (e.g. [START_REF] Calbet | The trophic roles of microzooplankton in marine systems[END_REF].

Possibly not as fully appreciated or understood is their fate, by predation from upper trophic levels and through viral lyses (Fig. 1), although there has been, and continues to be, a growing emphasis on this link (e.g. Hansen, 1991a;[START_REF] Sanders | Planktonic protists and metazoa: predation, food quality and population control[END_REF][START_REF] Pesan | Significance of the virus-rich 2-200 nm size fraction of seawater for heterotrophic flagellates. I. Impact on growth[END_REF][START_REF] Jakobsen | Escape of protists in predator-generated feeding currents[END_REF][START_REF] Irigoien | Feeding of Calanus finmarchicus nauplii in the Irminger Sea[END_REF][START_REF] Fileman | Grazing by the copepods Calanus helgolandicus and Acartia clausi on the protozooplankton community at station L4 in the Western English Channel[END_REF][START_REF] Montagnes | The protozooplanktonichthyoplankton trophic link: an overlooked aspect of aquatic food webs[END_REF].

One typical approach to examine the loss of protozooplankton via predation is to employ model species in lab-based experiments, rather than attempting to follow populations in natural samples. Inevitably, such model species are rarely ideal representatives of natural systems, but various attributes (e.g. easy of culture and manipulation) facilitate experimentation and thus, in many cases, provide more precise, if not more accurate, data.

For instance, the ciliate Strombidium sulcatum has been extensively used as model for the planktonic ciliates, which are often the dominant protozooplankton. Studies on this one species include consumption by zooplankton (e.g. [START_REF] Wiadnyana | Selective feeding of Acartia clausi and Centropages typicus on microzooplankton[END_REF][START_REF] Bamstedt | Trophodynamics of the scyphomedusae Aurelia aurita. Predation rate in relation to abundance, size and type of prey organism[END_REF][START_REF] Broglio | Effect of heterotrophic versus autotrophic food on feeding and reproduction of the calanoid copepod Acartia tonsa: relationship with prey fatty acid composition[END_REF], swimming and trophic behaviour (e.g. [START_REF] Fenchel | The functional biology of Strombidium sulcatum, a marine oligotrich ciliate (Cilophora, Oligotrichina)[END_REF], and nutrient recycling and processing (e.g. [START_REF] Allali | Culture characteristics and orthophosphate excretion of a marine oligotrich ciliate, Strombidium sulcatum, fed heat-killed bacteria[END_REF][START_REF] Dolan | Processing of ingested matter in Strombidium sulcatum a marine ciliate (Oligotrichida)[END_REF]. Strombidium sulcatum, however, is more epibethic than planktonic and is not generally found in the open water column; rather it has been isolated from shallow waters and tide pools (e.g. [START_REF] Fenchel | The functional biology of Strombidium sulcatum, a marine oligotrich ciliate (Cilophora, Oligotrichina)[END_REF][START_REF] Montagnes | Strombidium inclinatum n. sp. and a reassessment of Strombidium sulcatum Claparède and Lachmann (Ciliophora)[END_REF][START_REF] Modeo | A multidisciplinary approach to describe protists: redescriptions of Novistrombidium testaceum Anigstein 1914 and Strombidium inclinatum Montagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia)[END_REF].

Nonetheless, because of its morphological and behavioural similarities to other plankters and its ease of maintenance, S. sulcatum has provided invaluable data. Such data (e.g. growth and grazing responses) may then be used to stimulate and direct field-based studies, encourage researchers to attempt to culture more representative taxa, and in many cases, may be used directly in ecosystem models, as proxies for more common taxa that are impossible or difficult to study. Here we examine the use of another, easily maintained model species, a heterotrophic dinoflagellate (the other dominant group of protozooplankton; [START_REF] Jeong | The ecological roles of heterotrophic dinoflagellates in marine planktonic community[END_REF] and suggest it can, and should be, used as suitable model in predation studies.

The heterotrophic dinoflagellate Oxyrrhis marina is planktonic, sensu stricto, in that it remains in the water column, is moved by currents, is the same size as many planktonic species (15-40 µm), and generally behaves like many planktonic protozoa. Furthermore, similarly sized heterotrophic gymnodinoid dinoflagellates and a host of 10-30 µm mixotrophic dinoflagellates occur in planktonic samples (e.g. [START_REF] Kim | Feeding by the heterotrophic dinoflagellates Gyrodinium dominans and G. spirale on the red-tide dinoflagellate Prorocentrum minimum[END_REF]; thus, O. marina might act as a useful proxy for these planktonic taxa that are less tractable to culture.

However O. marina is rarely found in coastal waters and virtually never occurs in the open ocean. Rather, O. marina thrives in shallow waters and littoral and supralittoral pools [START_REF] Scott | Further nutritional studies on the marine rotifer Encentrum linnhei[END_REF][START_REF] Johnson | Physical control of plankton population abundance and dynamics in intertidal rock pools[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF], although "red-tide" blooms occur in large bays, reaching up to 10 5 cells ml -1 , and it can regularly be found in some estuaries at abundances of 10 -100 ml -1 [START_REF] Fenchel | Microbial diversity and activity in a Danish fjord with anoxic deep water[END_REF][START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF][START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF][START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF]. Thus, occasionally O. marina may be trophically important as a grazer and prey for other organisms, but in terms of large-scale plankton dynamics, assessing the specific importance of O. marina in natural systems is probably a moot point.

In contrast, there are significant benefits associated with examining O. marina as a representative of other planktonic protozoa. It is easy to obtain from natural samples, identify, maintain in culture, and manipulate in experiments. Furthermore, there is a long history of research on this species, including extensive work that has examined its growth and feeding (e.g. [START_REF] Droop | Phagotrophy in Oxyrrhis marina Dujardin[END_REF]Montagnes et al., this issue).

At present, however, many of the studies on the fate of O. marina, are incidental, using it as a convenient prey and not focusing on it as a model to assess general questions regarding predation on protozooplankton. Consequently, as an aid to continued research on this species, we consider it timely to evaluate the dispersed works that uses O. marina as prey and synthesise their findings to assess its general utility. Our premise, throughout the review, is that O. marina is a versatile and extremely useful model prey that should be adopted in future planktonic studies, ranging from those that examine nutrition to feeding behaviour. In conducting this review, however, we recognised a further point: O. marina appears to be a valuable food for maintaining many laboratory species. We, therefore, have augmented our study with a brief assessment of the pragmatic nature of using this species to help maintain its predators.

CONSUMPTION WITHIN THE MICROBIAL FOOD WEB

There is a growing recognition that predation occurs within the microbial food web, at a variety of levels (Fig. 1). Potential impacts of predation are a reduction in the trophic transfer efficiency towards upper levels, such as copepods and ultimately fish; recycling of nutrients within the smaller sized fraction of the food web; and increased pelagic food web respiration (CO 2 generation) due to added heterotrophic activity. One such link is "groupcannibalism", a term applied to describe when protozoa eat other protozoa [START_REF] Weisse | Trophic interactions among heterotrophic microplankton, nanoplankton, and bacteria in Lake Constance[END_REF]; O. marina may be used to help assess this process. For instance, O. marina is be preyed upon by the planktonic ciliate Strombidinopsis jeokjo [START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF]Fig. 2). To our knowledge, however, there have been no other empirical studies on the feeding by heterotrophic protists on O. marina, although [START_REF] Montagnes | Responsibility of microzooplankton and parasite pressure be responsible for the demise of toxic dinoflagellate blooms[END_REF] have incorporated an O. marina-ciliate link into a model of toxic dinoflagellate bloom control. There is, without doubt, scope here to use O. marina in further experiments to assess group-cannibalism within the microbial food web.

In contrast to group-cannibalism, true cannibalism also occur within protozoan, and specifically dinoflagellate, populations, generally as a survival strategy to cope with food limitation [START_REF] Jeong | Growth and grazing rates of the heterotrophic dinoflagellates Protopendinum spp. on red tide dinoflagellates[END_REF][START_REF] Latz | Effect of red tide dinoflagellate diet and cannibalism on the bioluminescence of the heterotrophic dinoflagellates Protoperidinium spp[END_REF][START_REF] Hausmann | Protistology. E. Schweizerbart'sche Buchhandlung[END_REF]. Again, O. marina may act as a model, as it has long been known to be cannibalistic [START_REF] Öpik | The digestive process of the dinoflagellate, Oxyrrhis marina Dujardin, feeding on the chlorophyte, Dunaliella primolecta Butcher: a combined study of ultrastructure and free amino acids[END_REF][START_REF] Davidson | Predator-prey interactions between Isochrysis galbana and Oxyrrhis marina III. Mathematical modelling of predation and nutrient regeneration[END_REF][START_REF] Flynn | Prey selection and rejection by a microflagellate implications for the study and operation of microbial food webs[END_REF]. Recently, [START_REF] Martel | Morphological controls on cannibalism in a planktonic marine phagotroph[END_REF] reviewed cannibalism for O. marina, obviating the need for us to do so in detail. In short, a small proportion of a population (~ 2% or fewer) are cannibals, and this number seems to depend on prey availability and the nutritional state of O. marina. However, unlike some other protozoa (e.g. the freshwater ciliate Blepharisma, where some cells become cannibalistic "monsters" when food is depleted, [START_REF] Giese | Blepharisma, the Biology of a Light-Sensitive Protozoan[END_REF], when O. marina is starved all cells decrease in size and there appears to be a decrease in cannibalism. These findings suggest that multiple cannibalstrategies occur within the protozoa (and even within the dinoflagellates), which should stimulate further research, possibly using O. marina as a useful representative for comparison.

Furthermore, the indication that cannibalism is relatively rare in cultures should provide solace to those researchers who use O. marina to examine population dynamics, either in the laboratory or in models. Likely, in laboratory work cannibalism can be ignored as a confounding factor, and as [START_REF] Martel | Morphological controls on cannibalism in a planktonic marine phagotroph[END_REF] suggest loss can be considered (if at all) to be a constant in population models, rather than having to apply more complex population dependent relationships.

Possibly the most unexpected microbial-predator of O. marina can be, paradoxically, its prey. Although in most cases, O. marina engulfs prey, there is one case where the roles become reversed, and the prey ingest O. marina. Specifically, the mixotrophic flagellate Prymnesium parvum consumes O. marina under some conditions [START_REF] Tillmann | Phagotrophy by a plastidic haptophyte, Prymnesium patelliferum[END_REF][START_REF] Tillmann | Kill and eat your predator: a winning strategy of the planktonic flagellate Prymnesium parvum[END_REF]: when P. parvum is not nutrient-stressed it is a suitable prey for O. marina, eliciting growth and grazing responses similar to those produced by other suitable prey. However, when exposed to toxin producing P. parvum cultures (induced through nutrient stress), O. marina loses its motility, becomes rounded, hyaline, and finally lyses. Under these conditions, individual O. marina are consumed, sometimes by several P. parvum at a time.

A wide range of protozoa are detrimentally influenced by this toxin producer (Tillmann, 2003, and references within), and it is well accepted that protozoan grazing (similar to that exhibited by O. marina) may prevent algal blooms (e.g. [START_REF] Tillmann | Interactions between planktonic microalgae and protozoan grazers[END_REF]. Consequently, this reversal in roles, by reducing grazing pressure on P. parvum, may allow the latter to bloom. In this case, O. marina has acted as a model to assess trophic role-reversal, a process that has been recognised for some time (e.g. Hansen, 1991b) and may be more prevalent than expected. We suggest that further studies on other mixotrophic prey be conducted on O. marina, to assess the extent of role-reversal.

Finally, no recent review of "microbial predation" on protozooplankton would be complete without including viruses, as although they are parasitic, they result in the demise of their host, and thus from the hosts perspective and in terms of nutrient recycling the consequence remains the same. Some of the earliest work linking viruses into the microbial food web used O. marina as a convenient model to indicate that viral-like particles reduce growth rate of protozoa [START_REF] Pesan | Significance of the virus-rich 2-200 nm size fraction of seawater for heterotrophic flagellates. I. Impact on growth[END_REF]. Since the early 1990s there has been a substantial amount of research on how viruses will alter planktonic populations and trophodynamics (the reader is directed to the review by [START_REF] Suttle | Marine viruses -major players in the global ecosystem[END_REF], as a good starting place on this topic). Given the interest in viruses, it is surprising that there appears to have been little further research conducted on O. marina and viruses, or more importantly on viruses and any other planktonic protozoa. We, therefore, propose that, given the ease of conducting experiments with O. marina, it be reemployed to investigate viral infection on protozoa, using new methodologies developed over the last 15 years (see [START_REF] Suttle | Marine viruses -major players in the global ecosystem[END_REF].

CONSUMPTION BY METAZOOPLANKTON

A range of planktonic invertebrates consume and grow on O. marina, including rotifers and copepods. Furthermore, especially in the context of mesozooplankton, but also for other invertebrates, O. marina has been used as both a dietary-staple and as a prey in a wide range of experiments (Table 1). By assembling these studies, we hope to illuminate the versatility of this species as a prey for invertebrates, and in the next two sections we suggest how such versatility might be exploited.

However, our review of the literature has also recognized that some planktonic vertebrates (i.e. fish larvae) may also consume O. marina. Such a "protozooplanktonichthyoplankton trophic link" could be important for larval survival, and there is a long history of protozoa being used as food for larval fish, in aquaculture and the tropical fish trade (for a recent review on this subject see [START_REF] Montagnes | The protozooplanktonichthyoplankton trophic link: an overlooked aspect of aquatic food webs[END_REF]. Although O. marina has been successfully used to rear young stages of larval black porgy, Mylio macrocephalus, supplemented the diet of larval lemonpeel angelfish, Centropyge flavissimus, and, anecdotally, used to raise grey mullet larvae [START_REF] Kasahara | A study on the growth and rearing methods of black porgy, Mylio macrocephalus (Basilwesky)[END_REF][START_REF] Nash | Hypotheses for problems impeding the mass propagation of grey mullet and other finfish[END_REF][START_REF] Olivotto | Spawning, early development, and first feeding in the lemonpeel angelfish Centropyge flavissimus[END_REF], there is limited research into its use, in this respect. Given the ease of growing O. marina to high abundances, its potential nutritional quality (see below), and its appropriate size for small fish larvae [START_REF] May | Feeding larval marine fishes in the laboratory: a review[END_REF], O. marina may prove to be useful. We propose that feeding it to fish larvae is a direction for aquaculture-based research and laboratoryexploration of the role of protozoa as food for larval fish in natural waters.

ASPECTS OF OXYRRHIS MARINA ACTING AS A NUTRITIONAL-MODEL

Evidence suggests that protozoa, in general, are nutritious for a range of organisms (Sanders and Wickham, 1993). Specifically, Table 1 indicates that O. marina is a suitable food source for a broad taxonomic range of organisms, and it is an appropriate prey size for many protists and invertebrates such as large (~100 µm) ciliates, rotifers, and many copepods [START_REF] Hansen | The size ratio between planktonic predators and their prey[END_REF].

Like other protozoa, the composition of O. marina, and hence its potential nutritional quality, is in part determined by its diet (typically microalgae but also bacteria), but O. marina synthesises a range of compounds that do not occur in its prey. Thus, feed-cultures containing O. marina and microalgae may enhance the growth of higher order predators. For instance, O. marina grown on the autotrophic flagellate Dunaliella sp., produces long-chain fatty acids and sterols that are not abundant in Dunaliella sp.; the combined diet of O. marina and Dunaliella sp. then allows the copepods Temora longicornis and Pseudocalanus elongatus, to grow successfully, whereas they do not survive well on Dunaliella sp. alone [START_REF] Klein Breteler | Trophic upgrading of food quality by protozoans enhancing copepod growth: role of essential lipids[END_REF]. [START_REF] Lund | Mechanism(s) of long chain n-3 essential fatty acid production in two species of heterotrophic protists: Oxyrrhis marina and Gyrodinium dominans[END_REF] have also indicated that O. marina does not simply elongate and desaturate fatty acid precursors from its prey to produce long chain fatty acids but produces these de novo. In a similar study on fatty acids and amino acids [START_REF] Kleppel | Nutrition and the regulation of egg production in the calanoid copepod Acartia tonsa[END_REF] Heterotrophic dinoflagellates are argued to play a unique role within the microzooplankton, being able to produce key nutritional substances [START_REF] Klein Breteler | Trophic upgrading of food quality by protozoans enhancing copepod growth: role of essential lipids[END_REF]. If this is so, then it seems O. marina may act as a useful model to pursue such research. However, of equal interest, in some cases predators consume O. marina but are unable to reproduce on this diet (e.g. [START_REF] Scott | Further nutritional studies on the marine rotifer Encentrum linnhei[END_REF], indicating that it can fail to provide all the essential dietary nutrients to predators. One promising direction for future study is to establish which nutrients occur and which are missing in O. marina and then assess if those that are missing can be provided by trophic upgrading; i.e. providing O. marina with a defined, complete diet. In this sense, we may be able to use O. marina to better understand fundamental processes of survival of heterotrophic dinoflagellates in natural systems. Such work will be facilitated by research that now indicates O. marina can easily be grown axenically and on heat-killed bacteria [START_REF] Droop | Phagotrophy in Oxyrrhis marina Dujardin[END_REF]C. D. Lowe, pers. comm.).

FUTURE DIRECTIONS

Given that O. marina is a suitably sized, nutritious prey for a wide range of organisms, it is not surprising that, over the years, numerous experiment have used it (Table 1). Below, we briefly expand on these types of experiments, suggesting how continued use of the O. marina might be appropriate.

Much of the experimental work on O. marina has been directed towards assessing why it is such a good supplement or sole diet for grazers and how this influences the growth and reproduction of the grazers (Table 1). This work has focused on several calanoid copepods commonly used to elucidate plankton dynamics. Furthermore, the work has focused on how O. marina provides essential fatty acids and sterols to its predator and how these improve their development and egg production. It is, therefore, surprising that comparable studies have not been conducted on the nutritional benefits obtained through group-cannibalism (i.e. between protozoa eating protozoa). Given the evidence that O. marina is nutritious and may act as a trophic-upgrader (see the previous section), using this species to investigate the potential importance of group-cannibalism seems a logical future direction of study.

There are also surprisingly few studies of grazing and growth rates of predators fed on O. marina; those that do so describe a classic Holling type-II response (Fig. 2). Grazing experiments using O. marina and Gymnodinium dominans (a similarly sized heterotrophic dinoflagellate) as prey for the large ciliate Strombidinopsis jeokjo suggest that predator growth rate is similar when fed these two species, but grazing rates differ (Fig. 2a,b). Noting the difference in these responses, [START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF] postulated that O. marina may yield a higher gross growth efficiency than G. dominans. Two other data sets that examine predation rates on O. marina both use the calanoid copepod genus Acartia (Fig. 2c); these data suggest similar rates of ingestion, although there are study-and predator-differences. Finally, there are only two studies that assess the bioenergetics of copepods (in both cases using the Acartia tonsa) when provided with O. marina: one examines faecal pellet production [START_REF] Feinberg | Effects of diet on dimensions, density and sinking rates of fecal pellets of the copepod Acartia tonsa[END_REF], while the other is more extensive and examines how food concentration alters feeding, egestion, and ultimately assimilation efficiency [START_REF] Besiktepe | Coupling of ingestion and defecation as a function of diet in the calanoid copepod Acartia tonsa[END_REF]. Clearly, if O. marina is to be included in food web models (e.g. [START_REF] Lee | Parameterising a microplankton model[END_REF][START_REF] Montagnes | Responsibility of microzooplankton and parasite pressure be responsible for the demise of toxic dinoflagellate blooms[END_REF], then there is a need to better parameterise these responses in the future, using other predators.

There is a growing recognition that mesozooplankton, and specifically copepod swimming behaviour may play an important role in pelagic ecosystems (e.g. [START_REF] Kiørboe | A Mechanistic Approach to Plankton Ecology[END_REF].

To this end, O. marina has been used in a model that assess mesozooplankton swimming and feeding (e.g. [START_REF] Mariani | A numerical investigation of the impact of turbulence on the feeding rates of Oithona davisae[END_REF]. Unfortunately, though, there has been little work on the behavioural changes of zooplankton, when feeding on O. marina. [START_REF] Schmitt | Multifractal random walk in copepod behavior[END_REF] examined the three-dimensional swimming of Temora longicornis and noted changes in behaviour when it was fed O. marina and the smaller flagellate Nannochloropsis occulata.

Furthermore, unpublished data (M. Steinke, pers. comm.) indicate that the feeding history of O. marina may affect dimethyl sulphide (DMS) production, and this, in turn, influences copepod swimming behaviour (Centropages hamatus, Acartia tonsa); thus, indirectly, the prey may alter the predator. These few, and disparate, studies suggest there is substantial scope for future work using O. marina as a prey, directed at paramaterising models such as the one developed by [START_REF] Mariani | A numerical investigation of the impact of turbulence on the feeding rates of Oithona davisae[END_REF]. Finally, O. marina has played a role in multi-trophic level laboratory microcosm studies and computer models (e.g. [START_REF] Hansen | Phaeocystis blooming enhanced by copepod predation on protozoa: evidence from incubation experiments[END_REF][START_REF] Montagnes | Responsibility of microzooplankton and parasite pressure be responsible for the demise of toxic dinoflagellate blooms[END_REF][START_REF] Mariani | A numerical investigation of the impact of turbulence on the feeding rates of Oithona davisae[END_REF]Malzahn et al., 2010) and may continue to do so given its extensive use as a model (Montagnes et al., this issue). It thus would seem appropriate that further work focuses on a range of behaviours associated with grazers on O. marina, to better understand and model predator-prey dynamics.

A BRIEF NOTE ON MAINTAINING PREDATORS

Although O. marina may act to help model predator-prey dynamics and behaviours, possibly its most pragmatic use has been simply as food, for maintaining stock and experimental cultures of laboratory organisms. For most applications, O. marina is mixed with other smaller organisms, to provide a composite diet for an upper level grazer. For instance many studies on copepods indicate that a combined diet of O. marina plus autotrophic flagellates provides a better diet than either prey alone, and similar findings have been obtained for feeding O. marina to rotifers (Table 1). In addition, benthic bryozoa have been successfully grown on a prey mixture of O. marina and yeast [START_REF] Kitamura | Suitable food plankton for growth of a Bryozoan Bugula neritina under laboratory conditions[END_REF]. All of these cases illustrate that there is a general improvement in predator growth when they are supplemented with O. marina.

From purely empirical observations, we might then suggest that this species should be a recommended food for planktivorous proto-and metazoa that have proven difficult to rear in the lab, and researchers conducting studies on such organisms should maintain stocks of O. marina. Given that it is simple to establish O. marina cultures (e.g. [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF], and strains are available from commercial culture collections (the CCMP, CCAP) or can be obtained from colleagues, it would seem sensible for researchers to consider this species as a routine stock prey item.

CONCLUSIONS

What then makes O. marina so special? It is a robust experimental protozoa that acts as a proxy for many of the more common, but difficult to culture, planktonic species. Furthermore, this review indicates that O. marina has the capability to be used to examine many aspects of predation associated with the microbial food web (Fig. 1), from viral infection through to group-and real cannibalism, trophic role-reversal, and ultimately to predation by mesozooplankton and possibly larval fish. Finally, O. marina provides either all the dietary needs or supplements the diet of many predators and can be used to reveal processes associated with trophic-upgrading. Thus, O. marina acts as a useful, if not ideal, model in this sense, and we recommend its use in future experiments.

We do provide the caveats that: 1) O. marina is not found in open waters and therefore does not necessarily act as ideal representative of planktonic systems; 2) almost by definition, as O. marina is a weed (i.e. easy to grow), it is robust and, therefore, not representative of many planktonic protozoa; and finally, 3) recent work indicates that there are many strains of O. marina (e.g. [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] that can have different ecophysiological responses to salinity [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF] and to temperature and prey types (Montagnes, unpublished data). Therefore, there is a need for researchers to work on defined, well characterised strains of O. marina (note this concern is, undoubtedly applies to many model species). Thus, after reviewing the literature, we recommend the following directions: 1) establish more functional and numerical responses of predators (viral, proto-, and metazoan) feeding on O. marina; 2) further examine predator behavior associated with the presence of O. marina (e.g. swimming, prey rejection); and 3) compare 1 and 2 (above) to other non-toxic, similar sized dinoflagellate prey, to establish O. marina's appropriateness as a model organism. Finally, there is a growing potential for the use of dinoflagellates to produce nutraceuticals for human and agricultural consumption (e.g. [START_REF] Mansour | The effect of growth phase on the lipid class, fatty acid and sterol composition in the marine dinoflagellate, Gymnodinium sp. in batch culture[END_REF]; possibly O. marina, with its ability to trophically upgrade the quality of fatty acids could be a focus for investigating commercial products, extending the range of predators to ourselves.

FIGURE LEGENDS

Fig. 1 An illustration of the potential links by predation within and extending from the microbial food web: c, cannibalism; f, larval fish; g, "group-cannibalism"; mz, micrometozooplankton; v, viral, z mesozooplankton. The illustration is liberally modified from a food web presented by [START_REF] Conover | Interrelations between microzooplankton and other planktonic organisms[END_REF] using information from a range of sources [START_REF] Stoecker | Predation on protozoa: its importance to zooplankton[END_REF][START_REF] Gifford | The protozoan-metazoan trophic link in pelagic ecosystems[END_REF][START_REF] Sanders | Planktonic protists and metazoa: predation, food quality and population control[END_REF][START_REF] Pesan | Significance of the virus-rich 2-200 nm size fraction of seawater for heterotrophic flagellates. I. Impact on growth[END_REF][START_REF] Irigoien | Feeding of Calanus finmarchicus nauplii in the Irminger Sea[END_REF][START_REF] Tillmann | Kill and eat your predator: a winning strategy of the planktonic flagellate Prymnesium parvum[END_REF][START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF][START_REF] Figueiredo | Do protozoa contribute significantly to the diet of larval fish in the Irish sea?[END_REF][START_REF] Martel | Morphological controls on cannibalism in a planktonic marine phagotroph[END_REF] [START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF]; data on Acartia spp. are from [START_REF] Jeong | Interactions among the toxic dinoflagellate Amphidinium carterae, the heterotrophic dinoflagellate Oxyrrhis marina, and the calanoid copepods Acartia spp[END_REF]; data on A. tonsa are from [START_REF] Besiktepe | Coupling of ingestion and defecation as a function of diet in the calanoid copepod Acartia tonsa[END_REF]. Where necessary O. marina volume was converted to carbon following the equation for heterotrophic dinoflagellates presented in [START_REF] Menden-Deuer | Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton[END_REF] and volume estimates presented in [START_REF] Jeong | Feeding by the marine planktonic ciliate Strombidinopsis jeokjo on common heterotrophic dinoflagellates[END_REF]. et al., 1982;1994;[START_REF] Tillmann | Interactions between planktonic microalgae and protozoan grazers[END_REF][START_REF] Klein Breteler | Influence of cultivation and food concentration on body length of calanoid copepods[END_REF][START_REF] Klein Breteler | Culture and development of Temora longicornis (Copepoda, Calanoida) at different conditions of temperature and food[END_REF][START_REF] Fryd | Development, growth and egg production of the two copepod species Centropages hamatus and Centropages typicus in the laboratory[END_REF][START_REF] Klein Breteler | Development of Acartia clausi (Copepoda, Calanoida) cultured at different conditions of temperature and food[END_REF][START_REF] Kirchner | Influence of the heterotrophic dinoflagellate Oxyrrhis marina on the population dynamics of Tisbe holothuriae[END_REF][START_REF] Klein Breteler | Development and grazing of Temora longicornis (Copepoda, Calanoida) nauplii during nutrient limited Phaeocystis globosa blooms in mesocosms[END_REF][START_REF] Van Duren | Copepod feeding currents: flow patterns, filtration rates and energetics[END_REF][START_REF] Besiktepe | Coupling of ingestion and defecation as a function of diet in the calanoid copepod Acartia tonsa[END_REF][START_REF] Koski | Post-bloom feeding of Calanus finmarchicus copepodites: Selection for autotrophic versus heterotrophic prey[END_REF]Koski et al., 2006 Fish larvae: Centropyge flavissimus, Mylio macrocephalzis, Mugil cephalus, Mugilidae sp.

Diet of first feeding [START_REF] Morris | Some aspects of the problem of rearing marine fishes[END_REF][START_REF] Kasahara | A study on the growth and rearing methods of black porgy, Mylio macrocephalus (Basilwesky)[END_REF][START_REF] May | Feeding larval marine fishes in the laboratory: a review[END_REF][START_REF] Nash | Hypotheses for problems impeding the mass propagation of grey mullet and other finfish[END_REF][START_REF] Olivotto | Spawning, early development, and first feeding in the lemonpeel angelfish Centropyge flavissimus[END_REF]) Nutrition, food quality, and trophic upgrade Acartia longiremis (copepod)

Fatty acid content effect on egg production and hatching success [START_REF] Augustin | Food and climate effects on copepod reproduction in the North and Baltic Seas[END_REF] Acartia tonsa (copepod) Amino and fatty acids regulation of egg production

  indicated that the copepod Acartia tonsa grew better when fed mixed cultures containing O. marina, again suggesting a trophic upgrade by O. marina. Furthermore, Scott (1987) indicated that O. marina provides a needed source of tryptophan to rotifers that was lacking in the algal prey. Finally, in a recent work, Malzahn et al. (2010) indicated that whenAcartia tonsa was fed O. marina that had been maintained on low-quality autotrophic flagellates (Rhodomonas salina) the copepod was not affected, but when the copepod fed low-quality primary producers, their respiration rates were higher and development rate was lower; thus, O. marina was used to examine the general principle that an intermediate step in the food chain may ultimately improve upper level productivity when primary producers are of low nutritional quality. These studies above not only use O. marina but also include other model plankters (e.g. Dunaliella, Rhodomonas, Acartia, Temora, Pseudocalanus) illustrating the utility of model systems and the versatility of O. marina.

Fig. 2 .

 2 Fig. 2. A comparison of grazing and growth rates on Oxyrrhis marina. a. grazing by the

  Fig. 2 2 3

Table 1 .

 1 Predators on Oxyrrhis marina, indicating the type of grazer and type of study(ies), a brief indication of the scope of the study(ies), and the authors of the studies.

	Type of study/grazer	Direction of study	Source
	Animal husbandry
	Bryozoa: Bugula avicularia, B. neritina, B.	Supplement to diet of alga and yeast Jebram, 1970; Jebram and
	stolonifera, Bowerbankia gracilis, Conopeum		Everitt, 1982; Kitamura and
	reticulurn, Conopeurn seurati, Electra		Hirayama, 1984 and references
	monostachys, Electra pilosa, Farella repens,		therein
	Farrella repens, Tanganella appendiculata,
	Tanganella mulleri, Victorella pseudoarachnidia
	Copepod: Acartia clausi, Acartia tonsa, Calanus	Supplement to diet of alga	Klein Breteler
	finmarchicus, Centropages hamatus, Centropages
	typicus, Pseudocalanus elongatus, Pseudocalanus
	sp., Temora longicornis, Tisbe holothuriae

  Yang et al. 11. Is Oxyrrhis marina a model prey?

		(copepod)			larvae to maturity
	1	Pseudocalanus elongatus, Temora longicornis Fig. 1	Body size in the adult stage and rate	Klein Breteler et al., 1990
		(copepod)			of development
		Pseudocalanus elongatus (copepod)		Rate of development from egg to	Klein Breteler et al., 1995
					adult
		Pseudocalanus elongatus, Temora longicornis	Prosome length and biomass	Klein Breteler and Gonzalez,
		(copepod)			1988
		Temora stylifera (copepod)			Postembryonic development	Carotenuto et al., 2002
		Tisbe holothuriae (copepod)			Population dynamics	Guérin et al., 2001
		Behaviour
		Oithona davisae (copepod)			Impact of turbulence on feeding	Mariani et al., 2008
		Oxyrrhis marina			Cannibalism in protozoa	Martel and Flynn, 2008
		Temora longicornis (copepod)		Swimming behaviour	Schmitt and Seuront, 2001
		Bioenergetics
		Acartia tonsa (copepod)			Diet effect on faecal pellet	Besiktepe and Dam, 2002
					production, grazing rate, and
					assimilation efficiency
		Acartia tonsa (copepod) Ecotoxicology Oithona davisae (copepod) Grazing and ingestion	F o r F o r F o r	Diet effect on faecal pellet characteristics Subleathal/lethal bioassay and toxin transfer in food chain	Feinberg and Dam, 1998 Saiz et al., 2009
		Acartia omorii, Acartia hongi (copepod) Acartia tonsa (copepod)	P Ingestion rates O. marina used as non fluorescing particle to assess gut clearance P P	Jeong et al., 2001 Kiørboe and Tiselius, 1987
		e e r Establishing functional response Pseudocalanus eiongatus, Temora longkornis Acartia tonsa (copepod) (copepod) Weight-specific ingestion e e Strombidinopsis jeokjo (ciliate) Grazing impact on populations Food web dynamics e r e r	Besiktepe and Dam, 2002 Klein Breteler et al., 1990 Jeong et al., 2004
		Acartia spp. (copepod)			Toxic dinoflagellates bloom	Jeong et al., 2001
		Acartia tonsa (copepod) Acartia tonsa (copepod) Acartia tonsa (copepod) Calanus glacialis (copepod) Pseudocalanus elongatus, Temora longicornis (copepod) Acartia tonsa (copepod) Calanoid copepods (various life stages) Temora longicornis (copepod) Virtual (i.e. models) Oithona davisae (copepod) Strombidinopsis multiauris (ciliate) Interaction Encentrum linnhei (rotifer) No specific predator Prymnesium parvum (alga)	Kleppel et al., 1998 Establishing a trophic upgrade index Tang and Taal, 2005 Trophic modification of essential fatty acids Veloza et al., 2006 3 and 4 trophic-level interactions Malzahn et al., 2010 Production of polyunsaturated fatty acids Stevens et al., 2004 Production of unsaturated fatty acids and sterols R e v i e dynamics Food chain stoichiometry, review Boersma et al., 2008 Microcosm manipulation R R Ptacnik et al., 2004 Laboratory study of trophic cascade Hansen et al., 1993 e e Impact of turbulence on feeding Mariani et al., 2008 Parasite control of toxic algal blooms Montagnes et al., 2008 v i e v i e Klein Breteler et al., 1999 Trophic transfer of tryptophan Scott, 1987 Production of long-chain n-3 Chu et al., 2008a w Mixotrophic prey eat O. marina Tillmann, 1998; 2003 w w
					essential fatty acids and sterols
		No specific predator			The kinetics and efficiency of sterol	Chu et al., 2008b
					production and bioconversion of
					phytosterols
		Reproduction, development, and growth
		Acartia clause, Centropages hamatus, Temora	Breeding success and population	Klein Breteler, 1980
		longicornis (copepod)			characteristics
		Acartia longiremis (copepod)			Effect of fatty acid content on egg	Augustin, 2006
					production rate and egg hatching
					success
		Acartia tonsa (copepod)			Egg production	Kleppel et al., 1998
		Acartia tonsa (copepod)			Egg production and hatching success Kleppel and Burkart, 1995
		Acartia tonsa (copepod)			Egg production rate and egg	Tang and Taal, 2005
					hatching success
		Pseudocalanus elongatus, Temora longicornis	Development from young nauplius	Klein Breteler et al., 1999
					1

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

ACKNOWLEDGEMENTS

We would like to thank Mark Breckels, Keith Davidson, Chris Lowe, Emily Roberts, Michael Steinke, and three anonymous reviewers for their constructive comments on this manuscript.

FUNDING

This work was, in part, supported by a Project of International Cooperation and Exchanges from the National Natural Science Foundation of China (30970500) awarded to YZ and a UK NERC grant (NE/F005237/1) awarded to P. C. Watts, C. D. Lowe, and DJSM.


David J S Montagnes

Christopher D Lowe

Phill Laura Martin

C Watts

Naomi ; Downes-Tettmar

Zhou Yang

Emily Clare Roberts

Keith Davidson

Marine Plymouth

Zhou ; Laboratory, - Yang

Phillilp C Watts

Yang Zhou

la diffusion de documents scientifiques

Keywords: model organism, dinoflagellate, protozoa, life history, Oxyrrhis model organism, dinoflagellate, protozoa, life history, Oxyrrhis

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

There is now, and has been for some time, compelling evidence that protozoa hold a key position and are an essential link in pelagic food webs (e.g. [START_REF] Calbet | The trophic roles of microzooplankton in marine systems[END_REF]. Furthermore, there is a growing awareness that heterotrophic flagellates and specifically dinoflagellates, are a significant component of the protozoa in planktonic ecosystems [START_REF] Laybourn-Parry | Flagellates and the microbial loop[END_REF][START_REF] Sherr | Heterotrophic dinoflagellates: a significant component of microzooplankton biomass and major grazers of diatoms in the sea[END_REF]. It is, therefore, important for researchers interested in plankton dynamics to have at least a basic knowledge, and at times a detailed understanding, of the life and cell cycles of these organisms.

Protozoa differ from most metazoa in that reproduction (and hence population growth) and sex are typically decoupled (Fig. 1). The protozoan cell cycle sensu stricto refers to mitotic cell division, and hence binary, asexual, reproduction (of either diploid or haploid cells). In contrast, the protozoan life cycle sensu stricto may include a number of cell cycles and ends-begins with sex; i.e. the recombination of genetic material, through syngamy or conjugation, where two cells exchange, or combine, gametic nuclei, either by fusing into a single cell, as is the case for dinoflagellates, or temporarily fusing and then separating, as is the case for ciliates. Of course, growth also occurs by cells increasing in size during the cell cycle, where maximum, pre-division cell size is determined by biotic and abiotic factors, such as food abundance and ambient temperature (e.g. [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF].

Beyond their importance in pelagic food webs, protozoa are also useful tools to understand fundamental biological processes. Because of their tractability, protozoa (including planktonic taxa) have been employed as models to examine cell cycles (e.g. [START_REF] Berger | Riding the ciliate cell cycle -a thirty-five-year prospective[END_REF], life cycles (e.g. [START_REF] Bell | Sex and Death in Protozoa[END_REF], and the constraints of external factors on optimal size (e.g. [START_REF] Atkinson | Protists decrease in size linear with temperature: ca. 2.5 °C-1[END_REF]. Furthermore, repeated cell cycles result in population growth, and protozoa have historically been, and continue to be, used as models to examining concepts associated with population dynamics (e.g. [START_REF] Gause | Further studies of interaction between predators and prey[END_REF][START_REF] Fenton | Parameterising variable assimilation efficiency in predator-prey[END_REF]. Thus, uncovering the mechanisms underlying the cell and life cycles of planktonic protozoa is useful to understand both ecological and fundamental biological processes.

Why then should we focus on the heterotrophic dinoflagellate Oxyrrhis marina?

First, this species is planktonic: it remains in the water column, is moved by currents, and behaves (swimming, growth, feeding) like many planktonic protozoa including other heterotrophic flagellates. Although O. marina blooms occur in large bays, producing "red tides" (10 5 cells ml -1 ) and it can regularly be found in estuaries at abundances of 10-100 ml -1 [START_REF] Fenchel | Microbial diversity and activity in a Danish fjord with anoxic deep water[END_REF][START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF][START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF], O. marina rarely occurs in the open ocean. Rather, O. marina is typical of shallow waters and littoral and supralittoral pools [START_REF] Johnson | Physical control of plankton population abundance and dynamics in intertidal rock pools[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. Thus, at times O. marina may be trophically important as a grazer and prey for other organisms, but in terms of open-ocean plankton dynamics assessing the specific importance of O. marina is of a lesser concern. There are, nonetheless, significant benefits to using O. marina as a model for other open-ocean species, as, unlike many planktonic protozoa, it is easy to obtain from natural samples, identify, maintain in culture, and manipulate in experiments.

Directly pertinent to this review, over the last 100 years, O. marina has been extensively studied, and much of this research has been associated with the cell cycle and population growth; in contrast, far less work has been conducted on its life cycle (Montagnes et al., this issue). Here we examine the literature on O. marina cell and life cycles, population growth, and production (growth rate × cell size).

LIFE CYCLE

The generalised life cycle of dinoflagellates includes several stages (Pfeister and Anderson, 1987;Fig. 1). Under favoured conditions (e.g. sufficient nutrients) cells (which are typically but not always haploid) divide vegetatively by binary fission, allowing population growth.

Due to some trigger (e.g. lack of nutrients, shift in temperature), haploid cells act as gametes and fuse to form a diploid zygote that may continue to be motile but in some cases forms a resting cyst. Diploid cells then undergo a single meiotic division to produce haploid cellsnote that some dinoflagellates, such as Noctaluca, are diploid, and thus do not follow this generalised dinoflagellate model (see Fig. 1). Most dinoflagellates are homogamous (i.e. gametes do not differ from vegetative cells), but some are isogamous (i.e. gametes differ from vegetative cells in size, pigmentation, or presence of a theca but are similar to each other), and others may be anisogamous (gametes differ from each other). Furthermore, although most dinoflagellates are monoecious (homothallic), some may be dioecious (heterothallic), not being able to conjugate within a clone.

As indicated above, we know little about the life cycle of O. marina. It is even unknown if cells are haploid, diploid, or polyploid [START_REF] Sano | Localization and copy number of the protein-coding genes actin, α-tubulin, and HSP90 in the nucleus of a primative dinoflagellate, Oxyrrhis marina[END_REF]. To our knowledge only one researcher has reported on sex in O. marina (von Stosch, 1972a,b): O. marina is isogamous, with gametic cells being "considerably" smaller than vegetative cells, and gametes were suggested to be formed by two typical meiotic divisions (although data were lacking to support this); the zygote enlarges after fusion (von Stosch, 1972a). Zygotes are motile during meiosis, and both flagella are retained in "skiing-track fashion" and are "distributed singly to the daughter cell of the first meiotic division" (von Stosch, 1972b).

Below, we quote directly from von Stosch (1972a), as we are unsure of his meaning (no illustrations accompany his text), but "as chance favours the prepared mind" (L. Pasteur), we provide his description in the hope that a keen observer will make sense of it: "In Oxyrrhis, however, the chromosomes could not be seen in the living meiocyte and only circular motion of the nucleoli, which are driven around with the chromosomes, could be observed. The cytology of fertilization and meiosis in this organism is rather well, if not completely, known to us, and older zygotes or meiocytes respectively can easily be recognized by large size attained by voracious feeding.... Even in Oxyrrhis, where the chromosomes appear short in vegetative interphase, they are considerably stretched in zygotene.". Sex may be induced by changing food type, from Pyraminonas to Dunaliella (von Stosch pers. comm. in [START_REF] Pfiester | Dinoflagellate reproduction[END_REF].

To the observations above, we can add one new insight: we routinely observe conspicuously small O. marina cells (~8 µm) in many of our cultures, occurring across all of the currently recognised Oxyrrhis phylotypes (see [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] for a description of the major genetic lineages). The small cells may be gametes (see Mini-cells, below; Fig. 2); however, we have not discovered the factors that stimulate their production, observed fusion of these mini-cells, nor ascertained their ploidy. Clearly, there is great scope for study of the life cycle of O. marina. The implications of sex in cultures are worth considering, as a clonal culture unable to have sex may reduce in fitness over time [START_REF] Bell | Sex and Death in Protozoa[END_REF]; possibly undetected sex is the reason for O. marina cultures surviving for years. Likewise, if it becomes possible to perform mating experiments with O. marina, a new field of studies, associated with classical genetic recombination and survival strategies (e.g. [START_REF] Bell | Sex and Death in Protozoa[END_REF] will be opened to plankton researchers using this species. Given the existing, extensive, application of O. marina as a model for planktonic species (Montagnes et al., this issue), the added ability to examine life cycle traits would broaden its utility to assess planktonic processes.

CELL CYCLE

Here we do not provide a detailed description of the cell cycle but indicate some major features and offer a guide to the extensive literature on the subject. For a review of the dinoflagellate cell cycle see Pfeister and Anderson (1987).

Cell division

Over the last 100 years (e.g. [START_REF] Senn | Oxyrrhis, Nephroselmis und einige Euflagellaten, nebst Bemerkungen über deren System[END_REF][START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF][START_REF] Triemer | A unique mitotic variation in the marine dinoflagellate Oxyrrhis marina (Pyrrophyta)[END_REF][START_REF] Gao | Nuclear division in the marine dinoflagellate Oxyrrhis marina[END_REF][START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF][START_REF] Kato | Isolation of the major basic nuclear protein and its localization on chromosomes of the dinoflagellate, Oxyrrhis marina[END_REF][START_REF] Kato | Dynamic changes in microtubule organizations during division of the primitive dinoflagellate Oxyrrhis marina[END_REF][START_REF] Sano | Localization and copy number of the protein-coding genes actin, α-tubulin, and HSP90 in the nucleus of a primative dinoflagellate, Oxyrrhis marina[END_REF], there have been a number of detailed descriptions of the intracellular processes and stages involved in the cell cycle of O. marina, and we direct the interested reader to these works. Cell division in O. marina is transverse, and during the cell cycle the chromosomes are always condensed, as is typical for dinoflagellates, but there are differences from other dinoflagellates in the structural organisation of division that make O. marina phylogentically unique (see the above references).

As is typical of eukaryotes, in exponentially growing cultures of O. marina there are three phases to the cell cycle: G 1 (between the end of mitosis and the beginning of DNA replication), S (DNA replication), and G 2 (between the end of DNA replication and the beginning mitosis) + M (meiosis) [START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF][START_REF] Sano | Localization and copy number of the protein-coding genes actin, α-tubulin, and HSP90 in the nucleus of a primative dinoflagellate, Oxyrrhis marina[END_REF]. Most of the cell cycle, of exponentially growing cells, is spent in G 2 +M, and G 1 is reduced [START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF][START_REF] Sano | Localization and copy number of the protein-coding genes actin, α-tubulin, and HSP90 in the nucleus of a primative dinoflagellate, Oxyrrhis marina[END_REF]. However, [START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF] indicate stationary-phase cells accumulate in G 1 and G 2 , and nutrient-limited cells accumulate at G 2 , rather than G 1 ; they conclude that nutrient-dependent control-points exist at both G 1 and G 2 , with the main control point in G 2 . Their data led [START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF] to suggest that this is a mechanism adapted to feast-or-famine conditions, often experienced by protozoa; they proposed a dual limitation of the cell cycle that would allow cells to slowly progress through G 1 to G 2 when F o r P e e r R e v i e w key nutrients are limited but not absent, allowing cells to accumulate in G 2 in anticipation of feast conditions, when they could rapidly divide and exploit the resource.

Intra and Inter population size-distributions

Cell size (i.e. volume) changes during the cell cycle; this is expected and clearly depicted in early observations of dividing O. marina (e.g. [START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF] and in cell-size distributions revealed by more modern techniques, such as flow cytometry (e.g. [START_REF] Sano | Localization and copy number of the protein-coding genes actin, α-tubulin, and HSP90 in the nucleus of a primative dinoflagellate, Oxyrrhis marina[END_REF].

However, it should be noted that cell size of O. marina also changes with nutritional state (see Biotic factors and Abiotic factors, below), temperature [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF], and it appears, from our initial evaluation of >400 isolated strains of O. marina, that strains may vary in maximum size (Lowe, unpublished data).

Cannibalism

Large cannibals in O. marina populations can occur [START_REF] Öpik | The digestive process of the dinoflagellate Oxyrrhis marina Dujardin, feeding on the chlorophyte, Dunaliella primolecta Butcher: a combined study of ultrastructure and free amino acids[END_REF]Flynn and Davidson, 1993a;[START_REF] Martel | Morphological controls on cannibalism in a planktonic marine phagotroph[END_REF]: a few cells (≤2% in a population; [START_REF] Martel | Morphological controls on cannibalism in a planktonic marine phagotroph[END_REF] are cannibalistic, and this may depend on available prey and the nutritional state of O. marina. Clearly, the occurrence of cannibals will alter the distribution of cell sizes in a population and potentially alter population growth (see below). However, it is unknown if cannibals initially grow larger and then ingest other O. marina or if the increase in size is a consequence of prey consumption.

Mini-cells

Here we provide, to our knowledge, the first report of mini-cells occurring within O. marina cultures. Within our routine observations of the >400 isolates of O. marina that we are presently maintaining (at the University of Liverpool, School of Biological Sciences), small F o r P e e r R e v i e w cells (~8 µm) occur, compared to the typical 20-30 µm cells (Fig. 2). These have been confirmed to be O. marina, by PCR sequencing of 5.8S ITS rDNA fragments (following methods outlined in [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF], and are observed in all four clades of Oxyrrhis, including the lineage we have suggested to be a different species (Lowe et al., submitted).

As indicated above, we do not know the origin of these mini-cells and can only speculate that they may be gametic (see Life Cycle, above).

Cysts

Post conjugation resting cysts form in many dinoflagellates (Pfeister and Anderson, 1987; Fig. 1). Although [START_REF] Goodman | Dinoflagellate cysts in ancient and modern sediments[END_REF] suggests Oxyrrhis does not produce cysts, there are reports in the literature of cysts. However, neither of the two descriptions of cyst formation in Oxyrrhis are related to sex. Although we are not convinced that we have ever seen cysts (but see Fig. 2), and to our knowledge neither have other recent studies, O. marina may form thin-membrane covered cysts (Fig. 3) induced by both excess and lack of food [START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF].

A more recent study indicated that Oxyrrhis sp. (possibly not O. marina; see [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF], found in intertidal rock pools, formed robust adherent cysts [START_REF] Jonsson | Tidal rhythm of cyst formation in the rock pool ciliate Strombidium oculatum Gruber (Cilophora, Oligotrichida): a description of the functional biology and analysis of the tidal synchronization of encystment[END_REF] that were speculated to allow the dinoflagellate to maintain its place in the pools when they were flushed by the incoming tide. We suggest that, as for observations of the life cycle, researchers also should be vigilant for the occurrence of cysts when they examine cultures; indications of conditions that encourage cyst formation would direct future research into the biological relevance, if at all, of this stage in the life cycle.

GROWTH RATE, CELL SIZE, PRODUCTION, AND ASSIMILATION

Below, we examine factors that alter the specific growth rate, cell size, production, and assimilation of O. marina under various biotic and abiotic conditions. Specific growth rate, or per capita growth rate, is typically determined as the slope of the relationship of the natural log of abundance vs. time, in exponentially growing cultures; it is often examined in relation to prey abundance (the numerical response) but is also examined in response to abiotic factors (e.g. temperature). Cell size (i.e. volume), estimated from linear measurements made with a microscope, or by automated means such as electronic particle counters or flow cytometers is often used as a proxy for biomass and may be converted to carbon (see [START_REF] Menden-Deuer | Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton[END_REF]. Given that both specific growth rate and volume may be influenced by biotic and abiotic factors, some authors (e.g. [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF] have examined their product (growth rate volume) to determine production; i.e. the amount of biomass elaborated per time. Finally, the amount of material assimilated by an organism is a key component of most population models (Turchin, 2005;[START_REF] Fenton | Parameterising variable assimilation efficiency in predator-prey[END_REF], and consequently there is a need to determine how biotic and abiotic factors alter assimilation.

Biotic factors

Food type and abundance

Oxyrrhis marina will grow on a wide range of taxa (from bacteria to cells its own size) and prey abundances; clearly though, some prey elicit a higher growth rate than others (Fig. 4, Table 1; [START_REF] Tarran | Aspects of the grazing behaviour of the marine dinoflagellate Oxyrrhis marina[END_REF]. Furthermore, the quality of the prey, in terms of nutrient stoichiometry and chemical cues, may alter uptake and presumably then growth (this topic is beyond the scope of the present review, and the interested reader is directed to the reviews on this topic: Davidson et al., this issue; Roberts et al., this issue).

Cell size and production also vary with prey concentration, both following a similarly shaped, rectangular hyperbolic response, akin to the functional response [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. Surprisingly, O. marina cell size rarely reaches an asymptote over the prey abundance range where growth rate has become asymptotic (e.g. [START_REF] Jeong | Interactions among the toxic dinoflagellate Amphidinium carterae, the heterotrophic dinoflagellate Oxyrrhis marina, and the calanoid copepods Acartia spp[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]Fig. 5, Table 2), which results in biomass-production continuing to increase with prey abundance, even when growth rate is constant. Thus, in this case, O. marina has provided a model to illustrate that both growth rate and cell size must be measured when studies are concerned with the biomass-production of planktonic protozoa.

Assimilation and gross growth efficiency

The proportion of ingested material assimilated by protozoa is exceedingly difficult to determine, as measurements of this parameter typically require estimates of egestion, which is not easily measured in protozoa. Rather, gross growth efficiency (or yield), the proportion of ingested material converted to consumer biomass, is typically determined for protozoa, and this has been done for O. marina [START_REF] Fuller | The grazing and growth rates of some marine protozoa measured in batch and continuous culture with particular reference to the heterotrophic dinoflagellate Oxyrrhis marina[END_REF][START_REF] Tarran | Aspects of the grazing behaviour of the marine dinoflagellate Oxyrrhis marina[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF], with values ranging between 0 and ~70%, depending on prey concentration and temperature (see Abioitc factors, below). However, recently, using data on O. marina (from [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF], [START_REF] Fenton | Parameterising variable assimilation efficiency in predator-prey[END_REF] have provided a means to calculate assimilation efficiency when data are available for both functional (i.e. ingestion vs. prey abundance) and numerical responses. For O. marina, this analysis indicated a significant decrease in assimilation efficiency with increasing prey abundance (from ~1 to 0.5 over a range of 0 to 10 5 Isochrysis galbana ml -1 ). Thus, using O. marina, [START_REF] Fenton | Parameterising variable assimilation efficiency in predator-prey[END_REF] have paved the way for exploration of this phenomenon in other protozoa.

Prey stoichiometry

The "quality" of phytoplankton (prey for O. marina and other protozoa), in terms of nutrient and metabolic composition, changes within the growth cycle and as a response to various biotic and abiotic factors [START_REF] Davidson | Non-steady state ammonium-limited growth of the marine phytoflagellate, Isochrysis galbana Parke[END_REF]Flynn et al., 1993). This variability can be a major factor influencing the growth of protozoa [START_REF] Goldman | Dynamics of prey selection of an omnivorous flagellate[END_REF][START_REF] John | Prey selectivity and the influence of prey carbon:nitrogen ration on microflagellate grazing[END_REF][START_REF] Montagnes | Selective feeding behaviour of free-living protists: views on, and avenues for, continued study[END_REF]. While the composition of a range of metabolites may be important in this process, the stoichiometric balance of bulk cell carbon and nitrogen are often chosen as indices of prey quality, being relatively easily quantified and composing a large fraction of the biomass of the cell.

A number of studies based on O. marina have investigated this topic. Flynn and Davidson (1993b) noted that a proportion of ingested C is lost through respiration associated with the energy demands of growth and motility. Carbon is also lost through the formation of C-rich "micro-particles" that void excess C when prey are N deplete. [START_REF] Davidson | Predator-Prey interactions between Isochrysis galbana and Oxyrrhis marina III. Mathematical modelling of predation and nutrient regeneration[END_REF], using a combined experimental-modelling approach, suggested that O. marina optimises growth by maintaining a balanced C:N ratio, and Hammer et al. (2001) have used O. marina grazing and growth as an experimental model system to study artificial carbohydrate-protein food particles that mimic natural C:N stoichiometry of prey. Further studies [START_REF] Goldman | Dynamics of hervivorous grazing by the heterotrophic dinoflagellate Oxyrrhis marina[END_REF][START_REF] Davidson | The role of prey nutritional status in governing protozoan nitrogen regeneration efficiency[END_REF] have highlighted the differential regeneration of N by O. marina as a function of prey stoichiometry as a means of optimising growth. Finally, a range of mathematical modelling studies (e.g. [START_REF] Mitra | The influence of changes in predation rates on marine microbial predator/prey interaction: a modelling study[END_REF][START_REF] Mitra | Predator-prey interactions: is "ecological stoichiometry" sufficient when good food goes bad?[END_REF] based on O. marina experimental data have investigated the role of prey stoichiometry in governing protozoan growth. Thus, in this sense, O. marina has acted as a useful model to examine the effects of prey stoichiometry on growth.

Strain differences

Primarily through our work, there is a growing awareness that there are genetically distinct strains of O. marina (see [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF], and many of these exhibit distinct growth responses [START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF]Yang et al., in prep). Our main methodological conclusion from this is that researchers examining biological parameters, including those associated with growth, must define the strain of O. marina that they are working on (see [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF].

Population growth

To our knowledge, there are no data that illustrate a typical sigmoidal logistic-growth response (i.e. abundance vs. time) for O. marina (but see Fig. 6); such responses occur when resources (e.g. prey for O. marina) are replete, and it is the population abundance of the target organism that influences its own growth rate. However, from the literature, we can estimate maximum growth rate (r) may range around 1.0 d -1 (e.g. Fig. 5); this figure agrees with general estimates for protozoa (and specifically dinoflagellates and other heterotrophic flagellates) of a similar size [START_REF] Hansen | Zooplankton grazing and growth: scaling within the 2-2,000-µm body size range[END_REF], supporting the suitability of O. marina as a model, in this respect. The carrying capacity (K) may be up to 10 5 O. marina ml -1 [START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF]Fig. 6;Lowe;Davidson;Roberts, unpublished data). Combined, these estimates allow us to make some predictions of logistic growth, assuming that growth rate decreases linearly with increasing O. marina abundance (but see [START_REF] Sibly | Population growth rate and its determinants: an overview[END_REF], for modifications on the general logistic growth curve that allow growth rate to decrease in a nonlinear manner with increasing prey abundance). However, it is unlikely that under most conditions (natural or batch cultures in the laboratory) resources will remain replete, and thus logistic growth is an unlikely scenario.

Most of our laboratory data on population dynamics of O. marina and its prey indicate either predators and prey exhibit population cycles or populations become roughly stable (Montagnes, unpublished data). Similarly, models of O. marina based on functional and numerical response data suggest when O. marina is incubated with a single prey, population cycling occurs [START_REF] Davidson | Predator-Prey interactions between Isochrysis galbana and Oxyrrhis marina III. Mathematical modelling of predation and nutrient regeneration[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]Yang et al., in prep). As is discussed in detail by Davidson et al. ( studies (e.g. [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF] and may act as a model to assess protozoan, and possibly more general population phenomena.

Starvation and nutritional shifts

Little is known regarding the response of O. marina to low prey concentrations, where it starves. Studies such as that by [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF] provide a threshold level, where food is just sufficient to sustain the cells (Table 1), and a mortality response at sub-threshold concentrations (see [START_REF] Montagnes | Growth responses of planktonic ciliates in the genera Strobilidium and Strombidium[END_REF]. However, to our knowledge there are no data on how O. marina will respond to changes in prey levels (either nutritional shifts up or down). Some protozoa will respond in a non-intuitive way to these shifts: e.g. ciliates may initially increase in numbers (but get smaller) when starved, and they may increase in cell size and not divide when they experience an increase in food (e.g. [START_REF] Lynn | Divider size and the cell cycle after prolonged starvation of Tetrahymena corlissi[END_REF].

Our own perception is that O. marina has a long lag-phase when transferred to increased prey levels or a different prey type (Lowe, Roberts, unpublished data). This is in agreement with the above argument made for ciliates but seems to contradict the speculations of [START_REF] Whiteley | Rapid method for cell cycle analysis in a predatory marine dinoflagellate[END_REF] that the population progresses to G 2 -phase, to allow rapid exploitation of new resources (see Cell Division, above). Clearly, to understand its population dynamics, it will be beneficial to develop studies to examine the response of O. marina to transitional and rapidly shifting prey levels; these can then be compared to other planktonic protozoa to assess the utility of O. marina as a model. Researchers interested in pursuing this area are directed to the classic works of Fenchel (e.g. [START_REF] Fenchel | Ecology of heterotrophic microflagellates. III. Adaptations to heterogeneous environments[END_REF][START_REF] Fenchel | Adaptive significance of polymorphic life cycles in Protozoa: responses to starvation and refeeding in two species of marine ciliates[END_REF].

Abiotic factors

Temperature

Classically, biotic rate processes are expected to increase exponentially with temperature, following a Q 10 or Arrhenius response, but [START_REF] Montagnes | Using Q 10 : can growth rates increase linearly with temperature?[END_REF] have argued that the response of protozoan growth rate to temperature is better modelled by a linear response. Data on O. marina seem to support the linear model [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. [START_REF] Atkinson | Protists decrease in size linear with temperature: ca. 2.5 °C-1[END_REF] have indicated another general temperature rule: protist cell size decreases linearly with increasing temperature, and again data on O. marina follow this trend [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. Thus, O. marina has been useful in helping to further assess these general trends.

Oxyrrhis marina is able to grow between 8-30 ºC [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF][START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF], with notable differences between clones (Lowe, Montagnes, Yang, et al., in prep), so this range may not be as great for any one isolate. The estuarine and intertidal environments in which O. marina occurs (e.g. [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF][START_REF] Jonsson | Tidal rhythm of cyst formation in the rock pool ciliate Strombidium oculatum Gruber (Cilophora, Oligotrichida): a description of the functional biology and analysis of the tidal synchronization of encystment[END_REF][START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF][START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF] will vary, rapidly and extensively ,in temperature (e.g. as air temperatures change), compared with typical water column conditions, possibly explaining why this species is eurythermal.

Salinity

In general O. marina grows over a wide range of salinities, between 4 and 130 PSU, with maximum growth at ~20, although there are strain-specific responses [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF][START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF]. Furthermore, O. marina is better at surviving transfers to lower salinities, rather than to higher salinities. Given its apparent global distribution and prevalence in intertidal pools and estuaries [START_REF] Fenchel | Microbial diversity and activity in a Danish fjord with anoxic deep water[END_REF][START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF][START_REF] Lowe | There are high levels of functional and genetic diversity on Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] unpublished data on >400 isolates from around the globe), it is not surprising that O. marina is euryhaline and adaptable. The two studies that have investigated the effect of pH on O. marina growth indicate that it can tolerate a wide range, growing between 7.8 and 9.8, but it dies below 7.5 [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF][START_REF] Pedersen | Effects of high pH on the growth and survival of six marine heterotrophic protists[END_REF]. Like the data on salinity, these should not be surprising, as rock pools often exhibit a wide range of pH, due to high levels of photosynthetic activity, altering CO 2 production-consumption, which in turn alters pH. Given the interest in pH shifts in the ocean and its effect on microbes [START_REF] Joint | Will ocean acidification affect marine microbes[END_REF], O. marina may be useful as a study organism to assess short and long-term phenotypic and genotypic changes on protists. [START_REF] Strom | Light-aided digestion, grazing and growth in herbivorous protists[END_REF] indicated that light may increase population growth of heterotrophic protists, and [START_REF] Klein | Digestion of chlorophylls and carotenoids by the marine protozoan Oxyrrhis marina studied by h.p.l.c. analysis of algal pigments[END_REF] found that light enhanced alloxanthin and chlorophyll c degradation by O. marina, suggesting that light may have an effect on its growth rate. Furthermore, Jakobsen and Strom (2004) indicated that O. marina exhibits a light-induced deil cycle in its feeding and growth rates. As O. marina contains rhodopsin, it may be that this protozoan can use light to facilitate cellular processes that increase growth rate (C. Slamovits et al., unpublished data). Clearly, this is an area for future study, not just for O. marina but to generally assess aspects of light-mediated metabolism in planktonic heterotrophs.

Light

Oxygen

There is one report of O. marina being associated with the microaerophilic zone of a Danish fjord [START_REF] Fenchel | Microbial diversity and activity in a Danish fjord with anoxic deep water[END_REF]. Although O. marina occurred in abundance at this interface, there was no indication that it was growing, and given this flagellate's ability to migrate and accumulate at interfaces [START_REF] Menden-Deuer | Individual foraging behaviors and population distributions of a planktonic predator aggregating to phytoplankton thin layers[END_REF], it may be as Fenchel et al. (1995) suggest: O. marina was abundant at this site because there were few predators. The ability of plankton to relocate to thin-layer conditions where growth is optimal, due to lack of predation or an increase in growth rate, is undoubtedly another area of research where O. marina could be employed as a model.

Mixing and turbulence

There is an expected negative effect of high turbulence on protozoan growth, although increased encounter with prey due to low-level turbulence may increase ingestion, and hence growth (see [START_REF] Peters | Effects of turbulence on plankton: an overview of experimental evidence and some theoretical considerations[END_REF]. [START_REF] Havskum | Effects of small-scale turbulence n the interactions between the heterotrophic dinoflagellate Oxyrrhis marina and its prey Isochrysis sp[END_REF] found that O. marina growth significantly decreased, by ~20%, at high but not low turbulence (dissipation, ε, was 1 and 5 -2 cm 2 s 3 , respectively). We also found that turbulence, measured as dissipation, had no apparent effect on the growth of O. marina, except at very high levels (Fig. 6). Note also, that our previous work (Downes-Tettmar and Montagnes, 2008) indicated no effect of turbulence (i.e. mixing) on Isochrysis galbana, one of the prey routinely used to grow O. marina (e.g. [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF].

It might be expected, given that one of the natural habitats of O. marina is intertidal pools (e.g. [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF][START_REF] Jonsson | Tidal rhythm of cyst formation in the rock pool ciliate Strombidium oculatum Gruber (Cilophora, Oligotrichida): a description of the functional biology and analysis of the tidal synchronization of encystment[END_REF], that it would be robust to mixing (turbulence), as a consequence of wave action and wind mixing. This ability to grow when mixed vigorously means that O. marina is probably a poor model organism to study the effect of turbulence on "planktonic" species. However, our data (Fig. 6) and those of [START_REF] Havskum | Effects of small-scale turbulence n the interactions between the heterotrophic dinoflagellate Oxyrrhis marina and its prey Isochrysis sp[END_REF] indicate that researchers need not concern themselves with detrimental influences of routine mixing when examining the effects of other parameters (e.g. temperature, prey levels), making O. marina a useful model, in this respect. It is also important to note that continuous mixing on a plankton wheel produced different population dynamics to those in containers that were only mixed once per day (Fig. 6); this may be due to increased encounter by predators and prey in the continually mixed cultures, but it could equally be due to aggregation of predators and prey on the bottom of cultures that were not mixed. These data, on O. marina, illustrate the benefits of continually mixed cultures, a fact well-recognised by most planktologists.

Interactions between biotic and abiotic factors

Most population and ecosystem models that include both abiotic and biotic factors do so in a additive fashion, as there are rarely measurements that allow parameterisation of interactive effects. However, interactions may produce surprising results. For instance, the intensive work on O. marina by [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF] is a good example of how abiotic (temperature) and biotic factors (prey concentration) influence growth rate, cell size, and ultimately production and population dynamics of organisms, in non-intuitive ways: briefly, the responses were not additive, and there were non-linear responses to a number of variables, including gross growth efficiency. It might, thus, be expected that combinations of other biotic and abiotic factors (see above) will also yield non-intuitive information about how protozoa respond to their environment. In this sense, O. marina may prove useful to explore such interactions.

Population growth in the environment

There are few data that examine population growth of O. marina in the environment. [START_REF] Kimmance | The interactive effects of temperature and food concentration on growth responses of aquatic protists, with particular reference to the heterotrophic dinoflagellate Oxyrrhis marina[END_REF] recorded seasonal variation in O. marina abundance in intertidal pools on the Isle of Man and indicated peaks in abundance during late June and between late July and September, when prey would have been abundant. [START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF] followed bloom dynamics of O. marina for two months in an enclosed bay in the Sea of Japan; they suggested that reduced salinity, caused by freshwater inflow may have stimulated blooms that reached as high as 4 10 5 O. marina ml -1 , although their data are also in keeping with the notion that freshwaters increased nutrient levels and thus increased the abundance of autotrophic (or bacterial) prey that stimulated blooms. Finally, [START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF], examining summer plankton in Chesapeake Bay, USA noted that O. marina was at times abundant (0-100 ml -1 ) and was positively correlated with its prey, but it also was most abundant at lower salinities (10)(11)(12)(13)(14). From these studies we might conclude that there are interactive effects of salinity and prey concentration on the abundance (a proxy for growth in the natural environment), as well as the known interactions between prey abundance and temperature on growth [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. These field data emphasise the need for further long-term collections of O. marina, to improve our understanding of this species and appreciate it as a potential model.

OXYRRHIS AS A MODEL

Cell and life cycles

Oxyrrhis marina is an unusual dinoflagellate, having a basal position within this group [START_REF] Saldarriaga | Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage[END_REF] and, therefore, is not necessarily an ideal candidate to act as a model of either the cell or life cycle of dinoflagellates. Furthermore, the lack of data available on O. marina, reduce its immediate use to elaborate cell or life cycle patterns, in general. Thus, at present, in this respect, O. marina is a poor model. However, the basal position of O. marina in dinoflagellate phylogeny makes it a target for further evolutionary study, and specific cell cycle processes have illustrated its unique position; possibly if life cycle attributes were better understood, they too would be illuminating. Finally, if O. marina is to be used as a model for other processes (see Ecology, below), there is a need to understand both its life and cell cycles; for instance, if, as for most dinoflagellates, O. marina is homothallic, and the minicells that we have observed are, indeed, gametes, then sex within clonal cultures may bias long-term studies. We, therefore, strongly recommend that future research is conducted on

Ecology

Oxyrrhis marina is used as a model organism to examine ecological processes (Montagnes et al., this issue). Reasons for this are: its ability to be recognised and isolated from the environment, its ease of culturing, and its ability to be maintained for many years in culture (for details on culturing beyond the scope of this review, see Lowe et al., this issue). The last of these factors might be attributed to sex within clones, as protozoa often exhibit clonal decline when sex is prevented [START_REF] Bell | Sex and Death in Protozoa[END_REF]. Furthermore, O. marina responds to many biotic and abiotic factors that are considered important drivers of marine productivity in pelagic environments (see above) and is able to survive over a wide range of these, possibly because in nature it experiences extremes. Thus, although O. marina is not routinely found in openocean waters, it is potentially useful as a proxy to model the range of environmental factors experienced by planktonic protozoa, including the affects of environmental perturbations.

Given that many truly planktonic protozoa are notoriously difficult to maintain for extended periods, O. marina seems well suited as a model organism to examine both aut-and synecological processes associated with their growth. likely less than half the size of the dividing cell, suggesting that they are not simply young daughter cells. Cells were fixed with 2% Lugol's iodine, which may result in cells being ~90% of their linear dimensions [START_REF] Montagnes | Estimating carbon, nitrogen, protein, and chlorophyll a from cell volume in marine phytoplankton[END_REF]. Scale bar = 10 µm. [START_REF] Jeong | Interactions among the toxic dinoflagellate Amphidinium carterae, the heterotrophic dinoflagellate Oxyrrhis marina, and the calanoid copepods Acartia spp[END_REF]; Heterosigma akashiwo [START_REF] Jeong | Feeding by the heterotrophic dinoflagellate Oxyrrhis marina on the red-tide raphidophyte Heterosigma akashiwo: a potential biological method to control red tides using mass-cultured grazers[END_REF]; Pfiesteria piscicida, Stoeckeria algicida (Jeong et al., 2007a); Cafeteria sp. (Jeong et al., 2007b); mixed natural bacteria [START_REF] Jeong | Feeding and grazing impact by small marine heterotrophic dinoflagellates on heterotrophic bacteria[END_REF]; Dunaliella primolecta, Chlamydomonas spreta, Brachiomonas submarina, Nanochloropsis oculata [START_REF] Fuller | The grazing and growth rates of some marine protozoa measured in batch and continuous culture with particular reference to the heterotrophic dinoflagellate Oxyrrhis marina[END_REF]; Isochrysis galbana [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]; Dunaliella primolecta*, Dunaliella primolecta** (unpublished data, Yang and Montagnes, using two strains of O. marina as predators,*351_FAR01, **45_BOG01, Univ. Liverpool cultures of O. marina). All responses were fit to the equation r = r max ×(P-P')/(k+P-P'), where r is growth rate and r max is the asymptotic growth maximum (both with units of d -1 ), P is prey concentration, P' is the prey concentration where growth rate is zero, and k is a constant; P, P', and k have units of (ng carbon ml -1 ).

Fig. 5.

A comparison of volume responses of Oxyrrhis marina feeding on a range of prey, over a range of prey biomass, all conducted at 20 ºC. Responses are for Amphidinium carterae [START_REF] Jeong | Interactions among the toxic dinoflagellate Amphidinium carterae, the heterotrophic dinoflagellate Oxyrrhis marina, and the calanoid copepods Acartia spp[END_REF]; Isochrysis galbana [START_REF] Kimmance | Do temperature -food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]; Dunaliella primolecta*, Dunaliella primolecta** (unpublished data, Yang and Montagnes, using two strains of O. marina as predators,* 45_BOG01, **351_FAR01, Univ. Liverpool cultures of O. marina). All responses were fit to the equation V = (V max ×P/k+P)+V', where V is growth rate, V max is the asymptotic maximum volume, and V' is cell volume at zero prey (all three with units of µm 3 ), P is prey concentration and k is a constant (both with units of ng carbon ml -1 ). 16 °C and 30-35 µmol photons m -2 s -1 of 24 h light. Flasks contained ~200 ml of 32 PSU artificial seawater, enriched with f/2 media (Sigma). Daily, 3-ml sample was collected, fixed with Lugol's Iodine (2 % final concentration), and cells were counted. Treatments, quantified as dissipation (ε) m 3 s -1 were: "No mixing", gently mixed once per day (ε = 0); "Low mixing", rotated on a plankton wheel (ε = 5.3 × 10 -5 ), and shaken on agitating tables (ε = "Medium mixing", 8.2 × 10 -3 ; "High mixing", 2.32 × 10 -2 ). Calculations of dissipation are described in Downes-Tettmar and Montagnes (2008).

  support the ongoing efforts of those researchers who are pursuing this work.

Fig. 1 .

 1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. An indication of Oxyrrhis marina cell sizes and shapes. a. a large cell; b. mini-cells; c.

Fig. 3 .

 3 Fig. 3. Temporary cysts of Oxyrrhis marina, from Hall (1925); note the lack of flagella and

Fig. 4 .

 4 Fig. 4. A comparison of numerical responses of Oxyrrhis marina feeding on a range of prey,

Fig. 6 .

 6 Fig. 6. Mixing and turbulence effects on Oxyrrhis marina and the prey flagellate Isochrysis

  Fig. 4 4

  Our aim is to provide an overview of what is known regarding aspects of O. marina growth, indicate where information is needed, and suggest ways in which this species can and cannot be used as a general model, in this respect.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

  this issue), O. marina lends itself to such population

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 .

 1 Parameters for the numerical responses depicted in Fig. 4. See Fig. 4 legend for an explanation of the parameters and sources of the data. Montagnes et al. Oxyrrhis marina growth, sex, and reproduction 34

	r max (d -1 )	p' (ng C ml -1 )	k (ng C ml -1 )	Prey
	1.43	8.0	104 Heterosigma akashiwo
	1.17	1.3	12.5 Amphidinium carterae
	0.19	0.06	0.48 Cafeteria sp.
	0.66	0.4	21.0 Pfiesteria piscicida
	0.22	5.0	21.0 Stoeckeria algicida
	1.47	-22	458 Dunaliella primolecta
	0.53	146	387 Chlamydomonas spreta
	0.73	40.8	57.6 Brachiomonas submarina
	1.31	-114	626 Nanochloropsis oculata
	0.94	44.5	98.7 Isochrysis galbana
	0.50	46.2	91.8 Dunaliella primolecta*
	0.77	21.3	49.4 Dunaliella primolecta **

Table 2 .

 2 Parameters for the volume responses depicted in Fig. 5. See Fig. 5 legend for and explanation of the parameters and sources of the data. Montagnes et al. 7. Oxyrrhis marina growth, sex, and reproduction 4

	V max (µm -3 )	V' (µm -3 ) k (ng C ml -1 )
	4770	908	378 Amphidinium carterae
	5910	1800	2510 Isochrysis galbana
	6710	5890	3970 Dunaliella primolecta*
	8750	4160	1280 Dunaliella primolecta**

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Journal of Plankton Research

Fig.3

ACKNOWLEDGEMENTS

The Authors would like to thank Mark Breckels, Claudio Slamovits, Michael Steinke, and Ewan Minter for their constructive comments on this manuscript. We would also like to thank the anonymous reviewers for their suggestions.

FUNDING

This work was, in part, supported by: a UK NERC grant (NE/F005237/1) awarded to PCW, CDL, and DJSM; a Project of International Cooperation and Exchanges from the National Natural Science Foundation of China (30970500) awarded to YZ; SAMS/NERC Oceans 2025 programme awarded to KD; and NERC grants NE/C519438/1 and NE/G010374/1 awarded to ER and Ed Dudley. Page 21 of 40 http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research