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The effect of harmful algae on the summer mortality of juvenile pacific oysters (Crassostrea gigas) Cassis, David 2005

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THE EFFECT OF H A R M F U L A L G A E ON THE S U M M E R M O R T A L I T Y OF JUVENILE PACIFIC OYSTERS (Crassostrea gigas) by DAVID CASSIS Licenciado in Marine Biology, Universidad de Valparaiso 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Oceanography) THE UNIVERSITY OF BRITISH C O L U M B I A April, 2005 © D. Cassis, 2005 ABSTRACT During the summer of 2001, a mass mortality of early juvenile Pacific oysters, Crassostrea gigas Gmelin, was observed at a farm site in Jervis Inlet, British Columbia. During this episode, several toxin producing and potentially harmful algae were detected within the phytoplankton community, with a bloom of Protoceratium reticulatum (Claparede et Lachmann) Buetschli preceding and including the mortalities. Searching for a cause we examined experimentally the rapid response behaviour of juvenile oysters to various species of microalgae. The behavioural response was a strong rejection, complete closure and feeding cessation when exposed to cultures of P. reticulatum and Alexandrium tamarense (Lebour) Balech. While exclusion in pseudo-feces (Amphidinium carterae Hulburt) and mixed reactions were observed with other species (Heterosigma akashiwo (Hada) Hada ex Sournia, Karenia mikimotoi (Miyake et Kominami ex Oda) Hansen and Moestrup, Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle, and Gonyaulax spinifera Diesing). The juvenile oysters feed well on the controls provided (Dunaliella tertiolecta Butcher, Isochrysis galbana Parke, Phaeodactylum tricornutum Bohlin, and Chaetoceros calcitrans Paulsen). Our study suggests that several of these phytoplanktonic species could have contributed to the oyster mortality in 2001 by causing starvation. The bloom of P. reticulatum was identified as the main probable cause for the die-off. In order to avoid juvenile losses, the phytoplankton composition and the size of the oysters should be considered at the time of introduction to farm sites. This study is one of the first to focus on the qualitative and quantitative responses of early juvenile oysters (+/- 5mm shell length) to various potentially harmful phytoplanktonic species. ii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES , vi ACKNOWLEDGEMENTS ix 1. INTRODUCTION 1 1.1. A Q U A C U L T U R E OF T H E PACIFIC OYSTER (CRASSOSTREA GIGAS) 2 1.2. O Y S T E R S A N D P H Y T O P L A N K T O N 3 1.3. H A R M F U L A L G A L B L O O M S (HABs) A N D THEIR POTENTIALIMPACT O N SHELLFISH 5 1.4. M A S S I V E M O R T A L I T Y OF JUVENILE OYSTERS D U R I N G T H E S U M M E R OF 2001 A T S Y K E S I S L A N D 8 1.5. O B J E C T I V E S 10 2. MATERIALS AND METHODS 11 2.1 . O Y S T E R M O R T A L I T Y 11 2.2. E N V I R O N M E N T A L PARAMETERS 11 2.3. P H Y T O P L A N K T O N 12 2.4. ISOLATION A N D CULTURE OF POTENTIALLY H A R M F U L SPECIES 14 2.5. R A P I D RESPONSE OF JUVENILE OYSTERS TO P O T E N T I A L L Y H A R M F U L A L G A E 15 2.6. QUANTITATIVE RESPONSE OF JUVENILE OYSTERS TO POTENTIALLY H A R M F U L A L G A E 17 2.7. Y E S S O T O X I N 18 iii 3. RESULTS 19 3.1. E N V I R O N M E N T A L PARAMETERS 19 3.2. P L A N K T O N COMPOSITION 21 3.2.1. Diatoms 22 3.2.2 Dinojlagellates 23 3.2.3 Silicoflagellates and other organisms 26 3.2.4. Biomass 26 3.3. O Y S T E R M O R T A L I T Y A N D E N V I R O N M E N T A L PARAMETERS 28 3.4. R A P I D RESPONSE OF JUVENILE OYSTERS W H E N EXPOSED TO A L G A L CULTURES 31 3.5. REJECTION LOCUS 3 4 3.6. SINGLE-SPECIES C L E A R A N C E R A T E 35 3.7. TWO-SPECIES C L E A R A N C E R A T E 40 3.8. Y E S S O T O X I N PRODUCTION 42 4. DISCUSSION 44 5. CONCLUSIONS 48 6. LITERATURE CITED 50 7. TABLES 63 iv LIST OF TABLES Table 1: Carbon per cell (pgC cell-1) estimations modified from Haigh (1992), used for calculations of total biomass for the species identified during 2001-2002 63 Table 2: Algal species used for rapid response experiments and their source 65 Table 3: Planktonic species and groups identified during 2001 and 2002 66 Table 4: Harmful planktonic species identified during the sampling period, and their possible effect on other organisms (modified from Landsberg, 2002, Taylor et al. 1994, Moestrup, 2004, Fryxell and Hasle, 2003) 69 Table 5: Rapid feeding responses observed in 5mm shell length oysters to selected algal species: pseudo-feces production, feces production, feeding and digested state in incrementing qualitative scale 70 Table 6: Rapid responses observed in 5mm shell length oysters to selected algal species: clapping frequency shown in an incrementing qualitative scale 71 Table 7: Algal species and their grouping according to their trophic acceptance (or palatability) by 5mm shell length oysters, during the rapid response experiments 72 Table 8: Average clearance rate (mL hr"1) measured during the first six hours after introduction into medium containing high abundance of the selected species 73 Table 9: Average clearance rate (mL hr"1) measured during the first six hours after introduction into medium containing different proportions of Dunaliella tertiolecta and Protoceratium reticulatum 73 LIST OF FIGURES Figure 1.1: Pacific cupped oyster (Crassostrea gigas) a) grown in a tray system and b) beach grown (modified from DeJager, 2003) 1 Figure 1.2: Location of the areas of the west coast of Canada h which Pacific oysters were introduced around 1903 (red arrows), and the main current culture areas in the Strait of Georgia (green arrows) 3 Figure 1.3: (a) Whole cells of live Amphidinium carterae in pseudo- feces and (b) digested and semi-digested cells in the feces produced by juvenile oysters with an abundant food supply 4 Figure 1.4: Oyster mortalities (individuals) by size class detected during the summer of 2001 9 Figure 2.1: Jervis Inlet system and location of sampled stations 11 Figure 3.2: (a) Temperature (?C), (b) salinity (PSU) and (c) transparency (m) registered during summer, fall and winter of 2001 20 Figure 3.3: Depth profile of abundance (cells L" 1) of the main phytoplanktonic groups: diatoms, dinoflagellates, and other groups present during the summer and fall of 2001 22 Figure 3.4: Scanning electron micrograph of Protoceratium reticulatum 24 Figure 3.5: Scanning electron micrograph of a Protoceratium reticulatum (Pr) bloom, with a cell of Ceratium fusus (Cf) and skeletons of Dictyocha speculum (Ds) 25 Figure 3.6: Estimated carbon (pgC L" 1) concentration in the seawater from diatoms (a), dinoflagellates (b), other planktonic groups (c), and total biomass (d) during the summer and fall of 2001 27 Figure 3.7: Total abundance (cells L" 1) of selected dinoflagellate species and total oyster mortality during the summer of 2001 29 v i Figure 3.8: (A) Depth distribution of P. reticulatum cells (cells L"1) in the water column. (B) Size composition of dead juvenile oysters 30 Figure 3.9: Oyster mortalities during the summer of 2001 and environmental parameters: (a) Oyster mortality (individuals), Protoceratium reticulatum total abundance (cells L" 1), transparency (m) and (b) temperature (C) 30 Figure 3.10: Rejection sites of some algal species observed within the oysters during the rapid response experiments 35 Figure 3.11: Abundance (cells L" 1) of (a) Alexandrium tamarense and (b) Protoceratium reticulatum during clearance rate experiments. Solid line represents the control, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates 36 Figure 3.12: Abundance (cells L" 1) of (a) Karenia mikimoti and (b) Amphidinium carterae during clearance rate experiments. The solid line represents the control, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates 37 Figure 3.13: Abundance (cells L" 1) of Heterosigma akashiwo during clearance rate experiments. The solid line represents the control, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates 38 1 Figure 3.14: Abundance (cells L" 1) of (a) Isochrysis galbana and (b) Dunaliella teriolecta during clearance rate experiments. The solid line represents the control, while the bars represent the abundance of the "food" species when the oyster was present. Error bars indicate the standard deviation between the triplicates 39 » Figure 3.15: Abundance (cells L"1) of Dunaliella tertiolecta during two-species clearance rate experiments, when D. tertiolecta and P. reticulatum were introduced in a proportion of 10:1. vn The solid line represents the control, while the bars represent the abundance of the algae when the oyster was present. Error bars indicate the standard deviation between the triplicates 41 • Figure 3.16: Abundance (cells L"1) of Dunaliella tertiolecta during two-species clearance rate experiments, when D. tertiolecta and P. reticulatum were introduced in a proportion of 3:1. The solid line represents the control, while the bars represent the abundance of the algae when the oyster was present. Error bars indicate the standard deviation between the triplicates 42 • Figure 3.17: Theoretical concentration of jessotoxin (mg L 1 ) at different depths, during the summer of 2001 43 • Figure 4.1: Percentage of the total planktonic abundance represented by P. reticulatum, and levels of deleterious effects on the feeding response of juvenile oysters, due to potentially harmful microalgae 45 viii ACKNOWLEDGEMENTS I would like to thank Dr. F.J.R "Max" Taylor for his knowledgeable advice and support throughout the course of this study. I would also like to thank my supervisory committee, Dr. Maria Teresa Maldonado and Dr. Alan Lewis for their valuable input during the past three years. This research project was supported by grant AP23 from AquaNet. It was carried out through collaboration with Sam Bowman and James Manders of Pearl Seaproducts. The following people were generous with their time, equipment, algal cultures, and advice: Elaine Humphrey of the E M lab, Donna Dinh of the C C C M , Tawnya Peterson, Nadene Ebell, and Bi l l Hardstaff and Mike Quillam for the toxin analysis. Special thanks go to Joel Gray, Lauren Moccia, Matthias Behr, and F.J.R. "Max" Taylor for reading countless pages in the quest for the final version. I would also like to thank Adrian Marchetti, Eric Galbraith, Helen Drost, Kevin Lin, Shannon Harris, Jeff Babuin, Juan Saldariaga, and Tonny Wagey for all their helpful comments. Thanks to my parents for supporting me all these years, and for trying to ask when would it be done as few times as possible. Finally, I extend my deepest gratitude for all the support and encouragement to Mariana, my wife. ix 1. INTRODUCTION This thesis is the result of an investigation into a severe mortality of juvenile Pacific or Japanese cupped oyster (Crassostrea gigas Thunberg) (Fig. 1.1) at an aquaculture farm site at Sykes Island, near the mouth of Jervis Inlet, B.C., in the summer of 2001. The juveniles were held in baskets on a floating upwelling system (Ralonde, 1998), exposed to natural phytoplankton. The hypothesis tested was that the deaths were the result of exposure to a Harmful Algal Bloom (HAB) species. The study consisted of three main components: an analysis of the composition of the phytoplankton concurrent with the oyster juvenile mortality, culture of the main suspect species, and exposure experiments in the laboratory. The evidence strongly implicated a common summer dinoflagellate, Protoceratium reticulatum. Since the smallest juveniles (l-5mm shell length) were most vulnerable, the practical implication is that the seasonal abundance of this species should be considered in the timing of the introduction of post-spat oyster juveniles into farm sites. Figure 1.1: Pacific cupped oyster {Crassostrea gigas) a) grown in a tray system and b) grown intertidally (modified from DeJager, 2003). 1 1.1. Aquaculture of the Pacific oyster (Crassostrea gigas) The Pacific oyster is the most aquacultured mollusc throughout the world, reaching more than 2.92 million metric tons annually since 1996 (Rana and Immink, 1997). In British Columbia the Pacific oyster has been cultured since 1903. The main farming areas are now located in Baynes Sound, around Cortes Island, and in Okeover Inlet (DeJager, 2003) (Fig. 1.2).This mollusc has been a preferred species to culture because it grows rapidly, can be cultured in variety of estuarine environments, and has high market value (Pauley et al. 1988). This oyster grows up to 25cm in shell length, normally averaging 10 to 15cm. It develops rapidly from a microscopic larva, reaching up to 4cm during the first year, and grows to commercial size in approximately 3 years (Pauley et al., 1988), depending on the seawater temperature and food availability (Ray, 2002, Brown and Hartwick, 1988, Brown et al, 1998). The aquaculture of these molluscs starts when the farmers buy juveniles, called "spat" or "seed", from hatcheries. When introduced into the farms, the oysters are only a few millimetres long (Andrews, 1987, DeJager, 2003). From the highly controlled environment of the hatcheries, the oysters are introduced to sea-based nursery systems, in intensive floating upweller rafts, where they grow up to 2cm in length These intensive nursery rafts basically follow the FLUPSY approach (Ralonde, 1998), where water is forced to flow through baskets h which the seed is kept. These rafts draw water from near the thermocline during summer, where most of the phytoplankton can be found during strong stratification conditions, thus increasing the number of oyster juveniles to be kept in a limited space. Oyster seed of this small size are then introduced into the desired grow-out system until harvest, which can be intertidal or deepwater, depending on the available space and type of 2 product desired (DeJager, 2003). The suspended deepwater culturing methods have higher growth rates and better physiological condition, with no detectable losses due to predation or siltation observed in intertidal cultures (Quayle, 1969 in Pauley et al, 1988). -127" 30' -126' 15' -125" 00' -123" 45' -122" 30' Figure 1.2: Location of the areas of the west coast of Canada in which Pacific oysters were introduced after 1903 (red arrows), and the main current culture areas in the Strait of Georgia (green arrows). 1.2. Oysters and phytoplankton Unlike other sessile filter feeders, such as mussels, oysters are especially selective with the phytoplankton that constitutes the main part of their diet. Most of particles that constitute their food are between 3 and 10um in diameter, being comprised of small diatoms and flagellates (Andrews, 1987, Coil-Morales, 1991, Thompson etal, 1993, Baldwin and Newell , 1995, Brown et al, 1998). According to Kusuki they can also capture smaller and larger particles, although it 3 is less efficient (Pauley et ai, 1988, Lees, 2000, Levinton et ai, 2002). Like other bivalves, digestion in oysters is quite inefficient, resulting in the production of feces that contain numerous whole and partially digested cells (Fig. 1.3b). Unwanted particles are ejected in bundles as pseudo-feces (Fig. 1.3a) in which they are surrounded by a sheath of mucus (Pauley et al, 1988, Coil-Morales, 1991, Jorgensen, 1996). Oysters also capture a wide variety of suspended particles including organic and inorganic detritus, bacteria, and small zooplankton. Figure 1.3: (a) Whole cells of live Amphidinium carterae in pseudo-feces and (b) digested (d) and semi-digested (sd) cells in the feces produced by juvenile oysters with an abundant food supply. The spring bloom of diatoms is normally regarded as the "fattening season" for this species of bivalve. This season is usually used by farmers for the introduction of the juveniles into the sea (Quayle, 1969 in Pauley et al., 1988). The summer is considered as a generally low nutrition period. This low availability of nutritive particles is alleviated in the autumn, when a second bloom of diatoms normally occurs. During the winter season, the suspended material decreases markedly, most of the particles consisting of nano and picoplankton, mainly small flagellates, re-suspended detritus and a few hardy diatoms. 4 1.3. Harmful Algal Blooms (HABs) and their potential impact on shellfish While most species of phytoplankton are beneficial to oysters, some can be harmful, or temporarily contaminate them with potent toxins, rendering them unfit for human consumption. Sometimes phytoplankton blooms are formed by toxigenic species of the dinoflagellate genus Alexandrium, or the diatom Pseudo-nitszchia. These species produce saxitoxins, and domoic acid, the causative agents of Paralytic Shellfish Poisoning (PSP) and Amnesic Shellfish Poisoning (ASP) (Fryxell and Hasle, 2003), also known as Domoic Acid Poisoning (DAP) (Trainer et al, 2000), respectively. They are accumulated by filter-feeding bivalves and can be lethal to humans and animals that are exposed to the toxins (Taylor et al., 2003). These H A B species cause severe losses in the aquaculture operations, by imposing closures and delaying the sale of their products. The most common H A B species responsible for outbreaks in the Strait of Georgia is the raphidophyte Heterosigma akashiwo. It has caused severe damage to the salmon farming industry since the 1980s. This was particularly a problem during this industry's early development in British Columbia (Taylor and Haigh, 1992, Taylor et al, 1994, Taylor and Harrison, 2002). Recently this species has been linked to sub-lethal damage to the digestive system of oysters through unknown toxins (Keppler et al, 2005). Outbreaks of the diatoms Chaetoceros concavicorne and C. convolutus can harm cultured salmon, even when present in low abundance (Sutherland, 1988, Taylor et al. 1991, Taylor and Harrison, 2002). The autumn phytoplankton blooms, when composed of diatoms such as Leptocylindrus danicus and Skeletonema costatum, have been reported to be harmful to fish 5 aquaculture elsewhere (Fryxell and Hasle, 2003). However, small diatoms such as these can be beneficial for shellfish cultures, as they provide high abundance of food. Other species that can produce toxins in the study area are dinoflagellates of the genus Dinophysis, which can cause Diarrheic Shellfish Poisoning (DSP), and Protoceratium reticulatum, a producer of yessotoxin. This toxin has similar effects to DSP when injected in mice (Satake et al. 1997, Seamer et al. 2000, Taylor et al, 2003, Samdal et al. 2004), and may cause damage to cultured marine animals (this study). Furthermore, this species' cysts can be found in estuarine environments around the world, being one of the most abundant since the last glaciation (Dobell, 1978). These and other phytoplanktonic species have been identified as the cause of massive mortalities of marine animals by means of toxins (Shumway et al, 1990, Landsberg, 2002). Hanriful algae can also cause depletion of the oxygen in the water; as the bloom decays, killing any animal life in the affected area. Examples of the algal species involved in this kind of harmful blooms are Gonyaulax polygramma and Noctiluca scintillans (Grindley and Taylor, 1964, La Barbera-Sanchez and Ferraz-Reyes, 1993) Negative effects for benthic organisms have been associated with many other algal blooms, like the "brown tides" of the phytoplanktonic pelagophyte Aureococcus anophagefferens (Pitcher et al, 1999). Some species of the raphidophyte Chattonella (Barraza et al 2004) and flagellates of the genus Chysocromulina (Moestrup, 2004) are not only fish killers, but also have caused massive mortalities of benthic organisms. The dinoflagellates Ceratium fusus and Akashiwo sanguinea (as Gymnodinium splendens or as G. sanguineum) (Cardwell et al, 1979, Bricelj et al, 1992), and Karenia mikimotoi (as Gymnodinium mikimotoi) (Wear, 1999), have caused losses to aquaculture operations and massive mortalities in estuarine environments. 6 Phytoplankton has been documented as the cause of "summer mortality" of juveniles and adult cultured oysters only in a few cases. In 1992, Bricelj et ah, found that a bloom of Akashiwo sanguined (as Gymnodinium sanguineum) was related to the massive mortality of hatchery-reared juveniles of Crassostrea virginica. Cardwell (1978) and Cardwell et al. (1979) recorded a bloom of Ceratium fusus as one of the probable causes for a massive die-off of cultured Pacific oysters. The main attributed cause, though, was the more commonly cited abnormally hot weather and high water temperature, and high abundance of Akashiwo sanguinea (as Gymnodinium splendens), but not reaching any definitive conclusions on the effects of the algae on the oysters during this study. Several methods have been tried for the characterization and quantification of the response of oysters and other shellfish to different phytoplanktonic species, such as, clearance rate, special flow-through individual chambers (Wildish et al., 1998), endoscopy, heart rate, ciliary movement, particle intake speed and handling time (Dwivedy, 1973, Ramesh, 1973, Ward et al, 1991, 1992, 2003, Milke and Ward, 2003), stress (Friedman et al, 1997), byssus production, or burial response, most of them are possible only on adult or larval subjects (Gainey and Shumway, 1988). The early juvenile stage is the least studied. Most of these methods rely on the clearance rate of the particles from the medium, lacking information on the immediate effect and handling of the particles (Baldwin and Newell, 1995, Barille et al, 1997). These methods do not cover the rapid response exhibited by the oysters, when subjected to high abundances of nutritive but potentially harmful species, or to combinations of them in different proportions. Quantification of the rapid response often proved impossible, but detailed direct observation and recording of the general behaviour were good tools for qualitative and semi-quantitative observations. 7 1.4. Massive mortality of juvenile oysters during the summer of 2001 at Sykes Island Repeated occurrence of summer mortalities of juvenile Pacific oysters had been detected since Pearl Seaproducts established an oyster farm in the vicinity of Sykes Island, located at the confluence of Jervis and Sechelt Inlets. These yearly mortality episodes normally affected the smaller juveniles and early seed introduced during spring. During 2001 and 2002, the mortality reached catastrophic levels, annihilating up to 80% of the oysters 3 to 15mm in shell length. As a result of the summer mortalities detected during the year 2000, several environmental parameters and phytoplankton were monitored at the site during 2001 and 2002. During the summer of 2001, massive mortalities of pacific oyster (£rassostrea gigas) spat and early juveniles were reported at a floating upwelling intensive nursery raft system. These mortalities accounted for about 80% of the oysters of some batches and size classes. The mortalities were first detected on August 15, 2001. They consisted mainly of small juveniles of 1 to 4.8mm in shell length. They were affected severely during most of August. Lower mortalities were registered for this size class during September (Fig. 1.4). The mortalities also affected the bigger size class of juveniles, of 5 to 10mm in shell length, detected since August 17 t h, 2001. During August they exhibited their highest mortalities, with much reduced die-offs during September. The larger than 10mm size class experienced no mortalities during most part of August, and only showed relatively small peaks during September, with the highest values observed on September 9*h and 13 th (Fig. 1.4). The adult oysters maintained in a grow-out operation at the site did not show any deleterious effects or mortalities during the period analyzed. 8 JO 3 > T3 •D (A (/> ra r o E (A o 1e + 6 8e + 5 6e + 5 4e + 5 2e + 5 0 J a - r - r ^ Size class T o t a l 1 -5mm JL I I 5-10mr > 10mm T i f T f i I i i f f f / Date Figure 1.4: Oyster mortalities (individuals) by size class detected during the summer of 2001. The number of early juveniles lost during August and September of 2001 was close to 3.5 million oyster seed, with 1.8 million seed of the smaller size class, 1.2 million of 5 to 10mm shell length oysters and 300x103 early juveniles larger than 10mm (Fig. 1.4). Of the total that died, 76% of the oyster seed was lost during August. Only 24% of the total mortality was detected during September. In search for a cause to these mortalities, some environmental parameters and the phytoplankton were monitored at the site. 9 1.5. Objectives The purpose of the present study was to investigate the mortality of juvenile Pacific oysters observed near Sykes Island during the summer of 2001. This using a combination of field and laboratory techniques, including a new methodology especially designed to study the rapid reactions of small size oysters when faced with different species of algae, including toxin producers and potentially harmful species. Our working hypothesis was that these mortalities were caused by the presence of harmful algae in the phytoplankton. In order to test its validity, we considered the following: 1s t: Were known harmful algae present and/or abundant in the local phytoplankton at the time of the mortalities? This involved the sampling of the phytoplankton at the farm site, and the determination of the species composition by microscopy. 2 n d : If harmful species were present, we obtained isolates and establish batch cultures, either from existing culture collections or by isolation from local field samples. 3 r d: To further test the hypothesis, juvenile Pacific oysters were exposed to various potentially harmful algae, under controlled experimental laboratory conditions. This should confirm if the suspected species had negative effects on juvenile oysters. This would involve both qualitative and quantitative methods, including the observation and measurement of the rapid response behaviour of the juvenile oysters, and the clearance rate of food particles from the water. The results from the above may be used to design possible strategies to help oyster farmers reduce the effect of harmful algal blooms on their aquacultural practices. 10 2. MATERIALS AND METHODS 2.1. Oyster mortality The mortalities of juvenile Pacific oysters were recorded during weekly sizing and grading operations conducted in a Floating Upweller System (FLUPSY) (Ralonde, 1998), by means of nested sieving through consecutive screens. Oyster seed of similar size are sorted into different batches, before being placed on high density forced flow trays. Most of the oyster spat that died was obtained from Taylor Sea Farms. -128'55' -127*30' -IZB'OS* -124'40 , -123'IS 1 -124'10" -124'00' -123'50' -123"40" -123'3ff -124'10' -124'OT -123"50' -123'40' -123'30' Figure 2.1: Jervis Inlet system and location of sampled stations. 2.2. Environmental parameters A sampling station was maintained at the mouth of Sechelt Inlet, part of Jervis inlet, British Columbia, from April 2001 until April 2002 (site 3). Two other sites were sampled (sites 11 1 and 2), but were not used for any commercial purpose by our industry partner during the period covered by this study, and only a preliminary analysis was performed in these samples (Fig. 2.1). Environmental parameters were measured as part of routine monitoring of farming conditions at the site of the FLUPSY. This included water temperature, salinity and transparency, measured with a thermometer, a manual refractometer, and a Secchi disk, respectively. The light penetration measurements were expressed as Secchi depth. 2.3. Phytoplankton At the farm's intensive nursery raft, water samples from three depths (1, 5 and 15m) were obtained weekly during the summer and biweekly during the winter months by means of Niskin sampling bottles. These samples were analysed quantitatively for microplankton, following the Utermohl method with the modifications described by Hasle (1978). Gently homogenized water samples were placed in 10, 25 or 50mL settling chambers, for 24 hours. Integrated water column microphytoplankton (>20um) samples were obtained with vertical hauls of a 20pm mesh size net. The detailed composition of these samples was obtained by means of observation, identification, and enumeration of the settled particles under a Zeiss Axiovert 10 inverted microscope. The abundance of these algae was compared with oyster mortality events during the summer of 2001, to estimate potentially harmful algal species to be targeted for isolation and culture for the instant response and clearance rate experiments. Carbon biomass per cell estimates were based on determinations of biovolume by Rowan Haigh (1992), who measured a number of individual cells of planktonic species present in Sechelt Inlet, and calculated average volume per cell by equating their shape to geometrical 12 figures. These biovolume measurements were used to obtain carbon estimations by using Strathman's equation (1967) for diatoms, and Montagnes' (2001) for other groups (Table 1). The different algal groups and species have marked differences in size and carbon content, with most of the large diatoms cells being filled up with vacuoles (Mullin et al. 1966). This method allows a better estimate of the actual contribution of each species to the total of the phytoplanktonic community, rather than their numeric abundance. Further analysis for taxonomical determinations was achieved by means of Scanning Electron Microscopy (SEM). For this, a lmL sub-sample of the qualitative net phytoplankton collected during the highest abundance of the desired species, was filtered through a 0.45pm membrane filter, buffered with a seawater solution of 2% cacodylate, dehydrated in a graded ethanol series, mounted on an S E M stub, critical point dried and coated with gold/palladium in an ion sputter, prior to observation, as described by Leander et al. (2002) and Truby (1997). The samples prepared by these procedures were observed under a Hitachi S4700 S E M to obtain the most detailed and clear images possible of the plate pattern, sutures and other taxonomically important details. The management and enhancement of S E M imagery was done using Adobe® Photoshop® 7.0, while the contour graphs were obtained utilizing SigmaPlot® 8.02 and Golden Software® Surfer® 7.0. Fluorescence microscopy was used for the identification and characterization of certain thecate dinoflagellate species. This was done with the help of cellofluor white, used in a freshly prepared solution containing 10mg/l and a microscope fitted with fluorescence equipment. 13 2.4. Isolation and culture of potentially harmful species The potentially harmful algal species (pHABs) determined during the in situ sampling, and others of known toxin production and deleterious effects in aquaculture, were used for the instant response and clearance rate experiments. Diverse species of autotrophic and heterotrophic algae were successfully cultured in batches for these experiments. These included the dinoflagellates Amphidinium carterae, Alexandrium tamarense, Gonyaulax spinifera, Karenia mikimotoi, Protoceratium reticulatum, the diatoms Chaetoceros calcitrans, Pseudo-nitszchia pseudodelicatissima, and Thalassiosira weisflogii, the chloromonad Dunaliella tertiolecta, the raphidophite Heterosigma akashiwo, and the prymnesiophite Isochrysis galbana. These organisms were obtained from the Canadian Culture Collection of Microorganisms (CCCM) of the University of British Columbia. Local strains isolated included a coccoid cyanobacterium, the diatom Phaeodactylum tricornutum, and Katodinium fungiforme, and a heterotrophic non-toxic Pfiesteria- like dinoflagellate. These species were isolated by means of micropippeting and serial dilution techniques; described in the UNESCO Phytoplankton Manual (Hasle, 1978) and in the Handbook of Phycological Methods (Stein, 1973). The Pseudo-nitzschia pseudodelicatissima strains isolated by Brian Bi l l were kindly provided by Vera L . Trainer from the Northwest Fisheries Science Center Culture Collection (Table 2). Several algal species were used as the main food source for the maintenance of the oysters, including D. tertiolecta, I. galbana, C. calcitrans, and, to a lesser degree, T. weisflogii. A l l the isolates were maintained and cultured in batches in HESNW medium prepared according to the recipe by Harrison et al. 1980, in a controlled environment chamber with a temperature of 18°C and a 14:10 L:D light cycle, following the recommendations by Guillard (1995). 14 2.5. Rapid response of juvenile oysters to potentially harmful algae The rapid response that 5mm shell length oysters exhibited, were obtained by placing one in a lOmL phytoplankton settling chamber, and observing its behaviour under an inverted or dissecting microscope. The oyster was submerged in 5mL of .45pm filtered seawater. Then 2mL of concentrated cultures of the well accepted species D. tertiolecta, P. tricornutum or I. galbana was added. When the oyster started feeding normally on this alga, lmL of the potentially harmful algae were placed on the surface, or in the feeding current of the oyster, using micropipettes. The chambers were observed with the inverted microscope, and the feeding behaviour of the oyster was recorded for ten minutes after the introduction of the target species by means of a Sony Trinicon video camera attached to an S-VHS recorder. This method allowed for detailed observation of the rapid behavioural response exhibited by the oysters. Directed introduction of small numbers of pHAB cells through a micropipette into the feeding current enabled the study of the instantaneous response, even to single cells. The following parameters were considered in a qualitative and semi-quantitative way: • Production of pseudo-feces: the production of pseudo- feces is normal, when the oyster is faced with excessive abundance of food, silt, and wrong-sized particles. A qualitative scale of 0 to 3 was used to describe the amount of pseudo- feces produced, with the lowest indicating no pseudo- feces containing the pHAB species, while the highest indicated the strong production of pseudo-feces. • Production of feces: relative abundance of the potentially harmful algae (pHAB) in the final product of the digestion of algal cells and other food particles (Fig. 1.3a). 15 • FeedhiR speed: the speed of the particles in the feeding currents is known to change according to food quality and quantity (Baldwin and Newell, 1995). In this case, a qualitative scale of four levels was used: • 0: No feeding currents observable; usually oyster is closed. • 1: Limited feeding activity, low speed feeding currents and frequent clapping, oyster is normally open. • 2: Medium to high speed feeding currents interrupted by clapping. • 3: High-speed continuous flow of particles into the pallial cavity. • State of digestion of the feces: Normally the ingested particles are not completely digested (Fig. 1.3b), and some dinoflagellate species can survive protected in temporary cysts (Laabdir and Gentien, 1999). An incrementing qualitative scale was used to describe the state of digestion, with the lowest assigned to algal cells that remained protected in a temporary cyst, remaining viable after digestion. • Clapping of the valves: The clapping of the valves has been indicated as a response mechanism used by the oysters to rid the pallial cavity of irritant particles (Lassus et al, 1999) and large bundles of pseudo-feces. An incrementing qualitative scale was used to describe their frequency. From the normal 1-5 claps per minute observed when feeding continually, incrementing with misshapen species, to the few fast claps seen before total closure with toxic taxa. • Total closure: When the oysters cannot actively deal with harsh environmental conditions, and clapping the valves won't suffice, they close their valves hermetically, sealing themselves from the irritant particles. 16 2.6. Quantitative response of juvenile oysters to potentially harmful algae A quantitative approach was achieved by means of clearance rate experiments, in which a 5mm shell length oyster was placed in 300mL of .45pm filtered seawater, innoculated with known abundances of selected species of microalgae. The clearance rate was obtained by the quantification of the abundance of the different algae through time, using Coughlan's (1969). Where 'm' is the filtering rate, ' M ' is the volume of suspension, 'n ' is the number of experimental animals, in this case a single 5mm shell length oyster, ' f is the time passed since the start of the experiment, 'concrr is the initial concentration of particles in suspension, while 'conc t' is the concentration of particles at time 't'. A control beaker was kept without the oyster, used to correct for the reproduction of the algae during the experiments. The oysters were acclimated to the experimental condition for one week, being fed a combination of well accepted algal species. These experiments were maintained for 48 to 72 hours in a controlled environmental chamber with a 14:10 L:D light cycle, and a constant temperature of 18°C. No aeration or artificial turbulence was provided to avoid mechanical damage to dinoflagellate cells (Taylor, 1987), dieing off and accumulating in the corners at the bottom of the flasks (Sullivan et al., 2003, Monica Bricelj, pers. comm.). This problem was avoided by using cylindrical flasks and a gentle homogenization with a spiral glass rod, prior to sampling every two hours during the light period. The oysters did not show adverse effects due 17 to the lack of aeration, with healthy specimens observed after five days under the experimental conditions, when maintained with a mixture of food species. These experiments were done in triplicate, plus a control, without the oyster. The abundance of the algae was measured using a Coulter Counter Z2 version 1.02, with different size settings for the feeding experiments with two algal species. Three measurements were done on each sample, and their average was taken as the final result. Simple standard deviation was used to calculate the variance between the triplicates, with 95% of confidence. 2.7. Yessotoxin Toxin analysis on Protoceratium reticulatum was performed by William Hardstaff and Michael Quillam of the Canadian National Research Council on a centrifugally concentrated pellet obtained from 4.5 litres of high abundance P. reticulatum culture. 18 3. RESULTS 3.1. Environmental parameters Only a few environmental parameters were measured by farm personnel at the site of the oyster mortality. The temperature values obtained during 2001 and 2002 (Fig. 3.2a) show that the waters around Sykes Island follow the normal annual cycle observed for British Columbian fjords (Thomson, 1981). During the summer, high temperatures between 24.4 and 17°C prevail on the surface layer of the water, while the subsurface layer remains at lower temperatures of 14 and 15°C. A strong thermocline can be observed at a depth between 8 and 10m (Fig. 3.2a). A fast reduction in temperature was observed between the 10 t h and 17 th of October, from a surface temperature of 16°C and 12°C at 15m, to only 12.5°C in the whole water column (Fig. 3.2a). The lowest values were reported at the surface on the first days of February of 2002, between 2.5 and 4.5°C. The salinity present in the area followed a similar pattern to that of the temperature (Fig. 3.2b). During the summer the water column had low salinity in the surface layer of around 20 PSU, while higher values were detected below the halocline closer to 29 PSU. A nixed water column was evident during fall and winter. This pattern was interrupted by several invasions of low salinity water, which normally affected the first 10m for varying periods of time. The water column was dominated by a strong intrusion the first week of July. This warm, low salinity water brought the halocline down from 8m to 14m. That was followed by a smaller episode, observed within the first 4m during the second week of August. Normal stratified conditions persisted during the last week of August and the first weeks of September. These were interrupted by a strong influence of high salinity Strait of Georgia water, which was detected on the 19 t h of 19 September (Fig. 3.2b). During the fell and winter the water column was dominated by mixed waters with salinity near 27 PSU, with the water near the surface about 1 to 2 units lower. a) Temperature ( C) 4 b) Salinity (PSU) 2 21 24 27 IZZI 30 c) T r a n s p a r e n c y (m) 5 • 28/6/01 26/7/01 23/8/01 20/9/01 18/10/01 15/11/0113/12/01 10/1/02 7/2/02 7/3/02 Date Figure 3.2: (a) Temperature (°C), (b) salinity (PSU) and (c) transparency (m) registered during summer, fell and winter of 2001. Secchi depth is a crude indicator of the concentration of particles in the upper water column. It varied from approximately 7m during the summer, to 19m in the winter. During the autumn the transparency increased gradually between September and December (Fig. 3.2c). A minimum value of 5m was detected during the last week of July. Near the end of the summer 20 period, a sudden increase in light penetration of up to around 20m was detected, comparable to winter values. This maximum value coincided with a brief incursion of the upper water column by cold and saline water (Fig. 3.2c). 3.2. Plankton composition In total, 55 species of diatoms, 31 dinoflagellate species, and 15 other microplanktonic taxa, were identified during the studied period (Table 3). The phytoplankton observed in the samples from the area followed normal seasonal fluctuations in species and group successions as described by Harrison et al. (1983), Sancetta (1989), Haigh et al. (1992), Y i n et al. (1997), L i et al. (2000), among others, with a spring bloom of diatoms. During the summer, dominance by motile organisms, including various flagellates, dinoflagellates and ciliates was observed. This period ended with an autumn bloom of diatoms and very low abundance of flagellates and diatoms during the winter (Fig. 3.3). During the summer, a small number of species of dinoflagellates reached their peak abundance. The main contributors were the autotrophic species Protoceratium reticulatum, Alexandrium tamarense, A. catenella and Ceratium fusus, and the heterotrophic and mixotrophic dinoflagellates of the genus Gyrodinium, Protoperidinium and Dinophysis. The highest abundances for a single species were achieved by the diatoms Leptocylindrus danicus (814xl0 3 cells L" 1), Skeletonema costatum (287xl03 cells L 1 ) and Eucampia zodiacus (247x103 cells L" 1), the dinoflagellate Protoceratium reticulatum (198xl0 3 cells L" 1) and the silicoflagellate Dictyocha speculum in its skeletonized and skeleton-less forms (140xl03 cells L" 21 m a a u D i a t o m s 1 5 m D i n o f l a g e l l a t e s 1 5 m C=3 O t h e r 1 5 m D a t e Figure 3.3: Depth profile of abundance (cells L"1) of the main phytoplanktonic groups: diatoms, dinoflagellates, and other planktonic groups present during the summer and fall of 2001. 3.2.1. Diatoms Of the 55 species of diatoms identified (Table 3), only 5 have been related to damage to other organisms (Table 4). These species have been related to mechanical damage to cultured fish, and to the production of domoic acid, involved in Amnesic Shellfish Poisoning (ASP) accumulated by shellfish (Taylor and Harrison, 2002, Fryxell and Hasle, 2003) (Table 4). The harmful diatoms were never present in high abundances. Chaetoceros convolutus never reached more than 6000 cells L" 1, while Pseudo-nitszchia reached only a brief high abundance of around 45x103 cells L"1 during the fall bloom. 22 The diatoms present in the area showed three peaks in abundance during the study period (Fig. 3.3). The first of these diatom blooms appeared at the end of June, and only reached total abundances of 240xl0 3 cells L"1. The second was observed co-occurring in part with the P. reticulatum bloom, reaching only around lOOxlO3 cells L" 1. The third diatom rich peak occurred around the first two weeks of October, and reached abundances of up to 160xl0 3 cells L" 1 following a period of high water transparency. Two of these peaks reached high biomass of single or small groups of species, while a smaller but highly diverse assemblage appeared during the summer. 3.2.2 Dinoflagellates Of the 30 dinoflagellate species observed (table 3), 10 have been identified to produce either toxins or harm to cultured marine organisms (Table 4). The genus Alexandrium is known to produce saxitoxins, the causative agent of Paralytic Shellfish Poisoning (PSP). Protoceratium reticulatum (Fig. 3.4) has been demonstrated to produce yessotoxin, which has yet poorly understood effects on humans and the marine ecosystem (Ogino et al, 1998, Satake et al, 1997, Seamer et al, 2000, Boni et al, 2000, Samdal et al, 2004). Dinoflagellates of the genus Dinophysis produce okadaic acid and dinophysis-toxins that are responsible for Diarrheic Shellfish Poisoning (DSP) (Taylor et al, 2003). The latter toxins have been detected in other estuarine environments around the world (Mufloz and Avaria, 1997, Taylor et al, 2003), and in British Columbia (K. Schallie, pers. comm.) (Table 4). In addition to the toxin-producing dinoflagellates, which normally appeared in our samples in concentrations of hundreds to thousands of cells per litre, there were numerous taxa that can produce deleterious effects on marine fauna when present in high abundance (Table 4), 23 often reaching over l x l O 6 cells per litre or more (Grindley and Taylor, 1964, Taylor, 1987, Landsberg, 2002). Dinoflagellates of the genus Cochlodinium can be a fish-killer. Ceratium fusus (Fig. 3.5) may cause damage to oysters by an unknown mechanism (Cardwell et al., 1979, Thomas, 1997, Moestrup, 2004, Landsberg, 2002). Figure 3.4: Scanning electron micrograph of Protoceratium reticulatum, (size bar = 20pm) Dinoflagellates had three peaks of abundance during the summer of 2001. The first of these was observed during the first days of July. It was composed of diverse heterotrophic species, and of the autotrophic Alexandrium tamarense, reaching only about 104 cells L" 1 . The second peak was mainly composed of Protoceratium reticulatum (Fig. 3.4), spreading both July and August. The main bloom of P. reticulatum reached a peak abundance of near 198xl0 3 cells L" 1 on August 13 t h, at a depth of 5m. Three other species of dinoflagellates were present during the bloom: Ceratium fusus, Alexandrium tamarense and a heterotrophic Gyrodinium sp. The abundances reached by these secondary species never surpassed 50xl0 3 cells L" 1 each. 24 The third peak was detected during the last week of August, and had a similar abundance and composition to the first peak. The toxic Alexandrium catenella was detected mainly at 5m, with abundances of up to 8,300 cells L" 1 on August 13 t h. The non toxic Ceratium fusus (Fig. 3.5), but suspected of being harmful to oyster spat (Cardwell, 1978), showed much higher abundances of up to 44x103 cells L" 1 . A small population of the heterotrophic genera Gyrodinium and Protoperidinium reached an abundance of 104 cells L" 1. Mixotrophic species ofDinophysis were also present in low numbers. Individuals of NoctUuca scinUllans, Cochlodinium sp, Prorocentrum gracile, Prorocentrum micans, Oxyphysis oxitoxoides, and Gymnodinium spp. were observed for short periods. The appearances of the heterotrophic species can be explained by the higher abundances of their prey, such as diatoms, dinoflagellates, or tintinnids (Taylor, 1987). These results suggested that Alexandrium catenella and A. tamarense, Protoceratium reticulatum, and Ceratium fusus may have been possible causes of the massive die-offs of juvenile Pacific oysters detected during 2001. Figure 3.5: Scanning electron micrograph of a Protoceratium reticulatum (Pr) bloom, with a cell of Ceratium fusus (Cf) and skeletons of Dictyocha speculum (Ds) (Size bar = lOOum). 25 3.2.3 Silicoflagellates and other organisms The silicoflagellates Dirtyocha fibula and D. speculum (Fig 3.5), and other groups of microplankters had medium to high abundances during the summer (Fig 3.3). The first peak in their numbers was observed during the decline of the first bloom of diatoms and the beginning of the dinoflagellate dominance, in July. In this period they constituted 73% of the total microplankton in the top five meters, with a total abundance of 70xl0 3 cells L" 1 . The second peak of silicoflagellates coincided with the P. reticulatum bloom, with its peak abundance occurring in August, when they represented 27% of the total microplankton cell count with almost 160xl0 3 cells L" 1 . Other organisms present in the samples included the autotrophic ciliate Myrionecta rubra (formally known as Mesodinium rubrum), normally in low to medium abundances of up to 7200 cells L " 1 during the summer and autumn. The high abundance of Dictyocha speculum during the oyster mortalities, and previous observations of their possible relation with damage to cultured fish (lochem and Babenerd, 1989, Henriksen et al, 1993) makes D. speculum one of the suspect species of being the cause of these die-offs, especially in their skeleton-less form, which was detected in high abundances during the summer. 3.2.4. Biomass The trends observed for carbon estimates followed the cell concentration during the summer, which was dominated by dinoflagellates, ciliates and other flagellates. This relationship was not maintained during early summer and the fall, when a higher abundance of diatoms with vacuoles was detected. The highest carbon values were observed during July and August, reaching a total for the water column near 1.8xl0 1 0 pgC L" ! and 1.4xl0 1 0 pgC L" 1, respectively (Fig. 3.6). Normally the diatoms were present with low values around l x l O 6 pgC L"1, but had 26 three maxima of carbon marking early summer, mid-summer, and during the fall. The first two had values near l x l O 7 pgC L"1, while during the fall bloom Leptocylindrus danicus reached l x l O ^ g C L " 1 (Fig. 3.6). 25/06/01 1 6/07/01 06/08/01 27/08/01 17/09/01 08/10/01 29/10/01 D a t e Figure 3.6: Estimated carbon (pgC L" 1) concentration in seawater from diatoms (a), dinoflagellates (b), other planktonic groups (c), and total biomass (d) during the summer and fall of 2001. 27 The dinoflagellates had two major peaks in carbon, constituting a high percentage of the total biomass estimated. The first occurred during July, while the second peak occurred during August (Fig. 3.6). The high amounts of carbon produced by a wide variety of dinoflagellate species during the summer in the top 8m ( lx lO 9 pgC L" 1), gradually decreased over time, with values near l x l O 7 pgC L"1 in September and only l x l O 6 pgC L"1 in October (Fig. 3.6). The carbon corresponding to other organisms observed in the plankton was normally only in background abundances between l x l O 7 and l x l O 6 pgC L" 1 . During early July they reached peak values of near l x l O 8 pgC L"1 in the upper 10m of the water column (Fig. 3.6). 3.3. Oyster mortality and environmental parameters When the oyster juvenile mortality data was compared to the abundance of different algal species, no clear correlation was obtained due to the time lag that exists in the normal sizing operations in the shellfish farms (Fig. 3.7). Most of the mortalities were detected during the first grading of the 2001 seed batch. Thus the data represent only the cumulative mortality of the previous weeks. In addition to this, the different batches of oyster juveniles were mixed during the grading several times, as they reached the different size classes, thus making it impossible to follow the mortality of any given sub-group. Several phytoplankton species were found to be in medium and high abundance in the water column during the oyster die-off (Fig. 3.7). The dinoflagellate Protoceratium reticulatum had a bloom that preceded and included the most severe part of the mortality (Fig. 3.8a). This species was first detected on June 25 t h , but reached peak abundances between July 23 r d and t Vi August 20 , remaining as part of the phytoplankton in lower abundances until early September. 28 The P. reticulatum bloom was mainly at 5m (Fig. 3.8b), which is the depth from which the water is drawn into the FLUPSY intensive nursery raft (Ralonde, 1998). 25/06/01 09/07/01 23/07/01 06/08/01 20/08/01 03/09/01 17/09/01 01/10/01 Date Figure 3.7: Total abundance (cells L"1) of selected dinoflagellate species and total oyster mortality during the summer of 2001. The main secondary species present during the peak of the P. reticulatum bloom was the dinoflagellate Ceratium fusus. This species has been identified as a possible cause of mechanical and associated bacteriological damage to oyster juveniles (Cardwell et al, 1979). In Puget Sound, it caused severe mortalities in cultivated oysters in conjunction with high abundances of other dinoflagellates, although the mechanisms involved in the mortality remain unknown. The diatoms present at the time of the die-off were mainly of Skeletonema costatum and a variety of large species of Chaetoceros that have shown no harmful effect to juvenile oysters. 2 9 ( A ) 0 2 Protoceratium reticulatum^ a b u n d a n c e (B) O y s t e r m o r t a l i t y by s i z e c l a s s » O 8 e + 5 6 e + 5 4 e + 5 2 e + 5 0 3 . 0 0 e + 3 3 . 0 0 e + 3 _ 3 . 3 0 6 + 4 I I 6 . 3 0 e + 4 9 . 3 0 e + 4 1 . 2 3 e + 5 1 . 5 3 e + 5 1 . 8 3 e + 5 P. reticulatum abundance ( c e l l s L " 1 ) 1 - 5 m m 5 - 1 0 mm > 1 0 m m O y s t e r j u v e n i l e s i z e c l a s s r.nl r_ 2 3 / 7 / 0 1 6 / 8 / 0 1 2 0 / 8 / 0 1 3 / 9 / 0 1 D a t e 1 7 / 9 / 0 1 J _ J 1 / 1 0 / 0 1 Figure 3.8: (A) Depth distribution of P. reticulatum cells (cells L ) in the water column. (B) Size composition of dead juvenile oysters. 1/T (a) re 1e + 6 3 •o > 8e + 5 c 6e + 5 rtali' 4e + 5 o E 2e + 5 _ t i >. 0 2 O _, 4 _L 6 £ 8 g- 10 ° 12 14 (b) 6/8/01 • Oyster mortality - P. reticulatum total abundance |_ 2.5e + 5 - T ransparency 5 a o u 25 20 15 10 5 0 5 10 I I 1 5 I I 20 25 y c <D ra a i/> c re T e m p e r a t u r e (C ) Figure 3.9: Oyster mortalities during the summer of 2001 and environmental parameters: (a) Oyster mortality (individuals), Protoceratium reticulatum total abundance (cells L" 1), transparency (m) and (b) temperature (°C). 30 The start of the die-offs was also coincidental with one of the short periods of high temperatures and low salinity registered in the top 8 meters of the water column. During August, peak temperatures of up to 24.4°C were in obserwd near surface waters (Fig. 3.9). High temperatures have been cited as possible causes of environmental stress that could lead to oyster mortalities (Cheney et ah, 1999), but also have been identified as the best for their growth (Pauley et al., 1988). These sustained high temperatures and higher fresh water run-off are also ideal for the creation of a strong pycnocline, decline of diatoms in the top layer and development of dinoflagellate blooms. 3.4. Rapid response of juvenile oysters when exposed to algal cultures The responses of oyster juveniles observed experimentally ranged from full acceptance, to a strong rejection of the algal cells by "clapping" of the valves, retraction of the mantle and tentacles, increased production of pseudo-feces, followed by total closure and/or diminished feeding activity (Tables 5 and 6). These reactions were seen with large spiny phytoplankton, inorganic detritus, and more violently, with most toxin producing species. In contrast, ether algae were eaten by the oysters through high speed continuous feeding currents, even at extremely high concentrations (6xl0 5 cells mL"1). Experimental observations made on the rapid response behaviour of juvenile oysters suggest that medium to high abundances of toxic algae (+/- 3xl0 6 cells mL"1) could disrupt the feeding activity and contribute to starvation, even when presented concomitantly with a high abundance of certain species accepted by the oyster. This was the case of Alexandrium tamarense and Protoceratium reticulatum. Both elicited strong rejection reactions even in very low abundances. The placement of 3 to 6 cells in the feeding current interrupted it and induced 31 violent clapping. These cells were so irritating to the oysters that the sensory tentacles retracted at the contact of even one cell. This reaction was instantaneous, and so cell surface recognition could have been involved. Gonyaulax spinifera was similarly irritant to the oysters, but only when present in higher abundances of tens to hundreds of cells in the vicinity of the oyster. Amphidinium carterae was taken in the feeding currents, only to be expelled rapidly in large bundles of pseudo-feces. When this species was in high abundance, it disrupted the normal feeding activity, as evidenced by the small production of feces. To test the reaction of the juvenile oysters to the variety of organisms present in natural microplankton, a 20pm mesh size net was used to obtain a live sample at Jericho beach, Vancouver. This sample contained several species of large diatoms, like Coscinodiscus spp., including those with long projections, such as Chaetoceros spp. and Rhizosolenia setigera, and medium abundance of heterotrophic dinoflagellates and tintinnids. This combination of organisms elicited a mixed reaction; the large and spiny phytoplankton was taken into the pallial cavity and rapidly ejected in large bundles of pseudo-feces, while the smaller particles were ingested and produced low amounts of feces. During the experiments with natural microplankton, the feeding currents were intermittent, but of high speed when active, being interrupted by frequent claps of the valves, mainly used to expel the large bundles of pseudo-feces. Most of the microzooplankton captured in the feeding currents, was eliminated in large bundles of pseudo-feces actively by means of claps. Cultures of Pseudo-nitszchia pseudodelicatissima were rejected in the same manner as large and spiny phytoplankton. The reaction of the oysters to a coccoid cyanobacterium isolated from Jericho Beach, and batch cultured to high abundances, was of an instant feeding arrest. When this alga is in high abundance, the oyster produced very slow and intermittent feeding 32 currents, but it didn't clap or close down. This reaction was also observed with Heterosigma akashiwo which arrested feeding when the cultures where senescent, but logarithmically growing cultures of this alga were well accepted, having a normal feeding response. The ichthyotoxic dinoflagellate Karenia mikimotoi elicited a normal feeding reaction, but its cells appeared only partially digested in the feces. No abnormal behaviour was apparent, and only moderate clapping was observed, even when this species was present in high abundance. The well accepted "food" species Dunaliella tertiolecta, Isochrysis galbana, Chaetoceros calcitrans and Phaeodactylum tricornutum elicited high speed continuous feeding currents conveying high amounts of cells into the pallial cavity. The production of feces and pseudo- feces was normal, and very few claps were observed. The heterotrophic dinoflagellate Katodinium fungiforme was ingested in high-speed feeding currents, occasionally being expelled in pseudo-feces but, in general, it was accepted like a "food" species. Similar to other small heterotrophic dinoflagellates, this organism relied on temporary cysts for protection when ingested by filter feeding bivalves (Cassis and Taylor, 2003). The rapid reactions elicited by all these algal species were classified into a scale (Table 7), depending on the level of acceptance shown by the oyster (Tables 5 and 6). Those algae that were lethal when in high abundance were categorized as level 1. The taxa under this classification also produced an intense rejection reaction. Those algal species, to which the oyster reacted violently, impeding all feeding activity, even in low abundance, were classified into level 2. These algae did not cause mortality after a 24 hour exposure to high abundances. The algae that were actively rejected into high amounts of pseudo-feces, were assigned to level 3, while those that caused feeding arrest, to group 4, although this group also contained some algae with mixed responses. The well accepted "food" algae that elicited normal feeding activity 33 were grouped in level 5. A special case was Katodinium fungiforme. It was ingested, but not digested, and was classified into its own level 6. This simple classification was used to characterize the response behaviour elicited by microalgae, from the toxic and highly irritant species of groups 1 and 2, that can kill the oysters, and interfere in their feeding, even when present in low abundance over long periods of time, to the "food" species of group 5, used in hatcheries, known for their good acceptance and nutritional value. 3.5. Rejection locus A scale based on rejection locus was constructed to characterize the behavioural response of the oysters to different algal species (Fig. 3.10). The rejection site of the pHAB particles can be located outside of the oyster, thus indicating a dissolved feeding deterrent which induces a feeding arrest, as in the case of cultures of a coccoid cyanobacterium, and high abundance of senescent Heterosigma akashiwo. The sensory response was also located on the front edge of the gills and sensory tentacles. This indicates that the recognition lies on the cell surface. These more intense defensive reactions were provoked even by a small number of cells of the known toxin producers Protoceratium reticulatum and Alexandrium tamarense. The oysters can also exhibit a response originated in the labial palps and general area of the gills, signalling that the particle is normally inert but awkward in shape, size, quality, or that it produces a mild toxin released after some handling by the oyster (Fig. 3.10). This is the case of diatoms with long projections, silt, and the toxin-producing Amphidinium carterae. A final group can be constituted with the well accepted species, and those that resulted immune to the digestion process of the oyster. Cells of this group of algae were expelled at the end of the digestive system, in the form of feces, or encapsulated in a temporary cyst 34 "Food" and Indigestible species: in feces or protected in temporary cysts Feeding arrest: possible dissolved feeding deterrent Cyanobacteria Senescent Heterosigma akashiwo Active rejection: (into pseudo-feces) gills and labial palps Amphidinium carterae Large/spiny phytoplankton Microzooplankton Silt Toxic and/or acute reaction: tentacles and mantle Alexandrium tamarense Protoceratium reticulatum Gonyaulax spinifera Figure 3.10: Rejection sites of some algal species observed within the oysters during the rapid response experiments. 3.6. Single-species clearance rate In most cases, the clearance rate experiments confirmed the rapid response results, with the food species showing significant decreases in their numbers, when compared to the control, while most of the potentially harmful species were not eaten by the oysters, which remained closed in the most extreme cases (Figs. 3.11-3.15). The algae classified during the rapid response experiments in groups 1 and 2, as the most violently rejected by the oysters, maintained the initial abundance. There was no filtration and the oysters remained closed for the duration of the experiments. 35 In this study, Alexandrium tamarense proved to be harmful to the oysters, when this dinoflagellate is in an abundance of around 1500 cells mL"1. It killed 30% of 5mm shell length juveniles when exposed for over 24 hours (data not shown), although the mechanism involved is not clear as the oysters remained closed. Unknown compounds, or the saxitoxin produced by this dinoflagellate, could be involved although adults are not killed (Gainey and Shumway, 1987) (Fig. 3.11a). 1800 0 2 4 6 H o u r s Figure 3.11: Abundance (cells L" ) of (a) Alexandrium tamarense and (b) Protoceratium reticulatum during clearance rate experiments. Solid line represents the control, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates. Protoceratium reticulatum (Fig. 3.11b) had the same effect as A. tamarense on the feeding response of the oysters, with no clearance of the particles being observed. The oyster remained closed for the duration of the experiment. However, no mortalities were observed within 24 hours. No feces or pseudo-feces production was observed during the experiments. 36 These toxic species effectively prevented all feeding activity in the oysters, which recovered rapidly when introduced in high abundance of well accepted species. The toxin-producing dinoflagellates Karenia mikimotoi and Amphidinium carterae elicited two distinctive feeding responses in the oysters. The first was cleared from the water in very much the same way as other "food" algal species during the first six hours, with much lower clearance rates after 24 hours (Fig. 3.12a). Cells of this species were observed partially digested in the resulting feces. A. carterae was not eaten by the oysters and, even though a very low clearance rate was observed during these experiments, the algal cells were mainly directed into pseudo-feces, not being observed in the real feces (Fig. 3.12b). E <n "55 a u c (0 •a c 3 .Q < (b) 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0 0 i •—• • i Karenia mikimotoi Control 10 15 H o u r s Amphidinium carterae Control 20 25 30 Figure 3.12: Abundance (cells L" ) of (a) Karenia mikimoti and (b) Amphidinium carterae during clearance rate experiments. The solid line represents the control, without the 5mm shell length oyster, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates. 37 The potentially harmful alga Heterosigma akashiwo was cleared from the water, when in logarithmic growth, as i f it was a "food" species, even when present in medium and high initial abundance (Fig. 3.13). This microalga produces massive blooms every year in the fjords and inlets in British Columbia (Taylor and Haigh, 1992). It has the capacity to produce an unknown toxin that can affect fish trapped by these blooms. Nevertheless, this species behaved like an alga well suited for hatchery usage in our feeding experiments. However, it is possible that it may be toxic under other conditions, damaging the digestive system of oysters (Keppler et ah, 2005). Senescent cultures of H. akashiwo elicited strong feeding arrest behaviour in the rapid response experiments, while cultures in exponential phase were well accepted by the oysters in the rapid response experiments. The latter kind of response was observed during the clearance rate experiments conducted. _ 2 5 0 0 £ 500 -< j -0 -1 *—T-*—'—~*—' I ' 1 1 1 I 0 5 10 15 20 25 30 H o u r s Figure 3.13: Abundance (cells L"1) of Heterosigma akashiwo during clearance rate experiments. The solid line represents the control, while the bars represent the abundance of the pHABs when the oyster was present. Error bars indicate the standard deviation between the triplicates. The abundance of the conventional "food" species, Dunaliella tertiolecta and Isochrysis galbana, diminished drastically when an oyster was present in the flask, while showing a normal 38 growth rate in the control (Fig. 3.14). The abundance of these algae, measured during the clearance rate experiments presented an interesting pattern; during the day a decrease in the algal concentration was evident, as the oyster fed. In the mornings, the abundance was much higher, indicating that, either the algae reproduced during the night, or that the oyster fed slower or did. not feed at all during the night (Fig. 3.14). E u> 0) o c n T3 c 3 < 6 0 0 0 ( a ) 5 0 0 0 4 0 0 0 3 0 0 0 2 0 0 0 10 0 0 18 0 0 ( b ) 1 6 0 0 14 0 0 12 0 0 10 0 0 8 0 0 6 0 0 4 0 0 2 0 0 0 Isochrysis galbana Control yyyyyyyyyyyyyyym ^ Dark period / / / , Vyyyyyyyyyyyyyj X i C o n t r o l Dunaliella tertiolecta X yyyyyyyyyyyyyyy^ ^ Dark period VyyyyyyyyyyyyyyM. 10 15 H o u r s 2 0 2 5 X 3 0 Figure 3.14: Abundance (cells L"1) of (a) Isochrysis galbana and (b) Dunaliella teriolecta during clearance rate experiments. The solid line represents the control, while the bars represent the abundance of the "food" species when the oyster was present. Error bars indicate the standard deviation between the triplicates. Coughlan's (1969) formula, was used to calculate the average clearance rate of the juvenile oysters during the first six hours after introduction. The results expressed in Table 8 indicate that D. tertiolecta and I. galbana were filtered rapidly from the medium, thus making them an ideal food species. The clearance rate reached with D. tertiolecta was around 40mL hr"1. The oysters cleared the potentially harmful algae H. akashiwo as if it was a "food" alga, while 39 the pHAB species K. mikimotoi and A. carterae were treated in different manner. The first one was well accepted at first, only to be rejected later. The alga A. carterae was rejected after some degree of handling in the pallial cavity of the oyster juveniles, being sent into pseudo-feces (Table 8). 3.7. Two-species clearance rate The yessotoxin-producing dinoflagellate Protoceratium reticulatum demonstrated its ability to disrupt the normal feeding activity of juvenile oysters, even when present in low proportions, decreasing the clearance rate to less than a 25% that of pure cultures of well accepted species. This was observed even in situations in which this toxic species accounted for less than 10%> of the total particles present. The clearance rate observed on Dunaliella tertiolecta during two-species experiments was adversely affected during the experiments with D. tertiolecta and P. reticulatum in different proportions, showing slow filtration at 10:1, and being completely hhibited at a ratio of 3:1 (Figs. 3.15 and 3.16, Table 9). When the oysters were maintained with both species for a period of three days at the latter ratio, they showed signs of debilitation, but were still alive and able to feed immediately when placed in filtered seawater with high abundance of well accepted species. The response of the oysters to P. reticulatum varied between individuals. In general, the slightly larger oyster juveniles were able to bear the high abundance of the toxic alga better than the smaller ones. The 10mm shell length juvenile oysters had slightly higher clearance rates relative to their smaller 4-7mm long counterparts, and did not succumb to high abundance of Alexandrium tamarense (data not shown). 40 Dunaliella tertiolecta and Protoceratium reticulatum 10:1 35000 -r 0 I 111 I I I II I , , II I II II, I , I I M I I U | 0 10 20 30 40 50 60 Hours Figure 3.15: Abundance (cells L"1) of Dunaliella tertiolecta during two-species clearance rate experiments, when D. tertiolecta and P. reticulatum were introduced in a ratio of 10:1. Solid line represents control, while the bars represent the abundance of the algae when the oyster was present. Error bars indicate the standard deviation between the triplicates. The initial proportion between the two species of algae was not maintained for long during the experiments, as D. tertiolecta had much higher division rates than P. reticulatum. The dinoflagellate maintained almost the same abundance throughout the shorter experiments, while reaching only double the initial abundance at the end of 60 hours. The original proportion only lasted for the first 8 hrs, after which the exponential growth of D. tertiolecta and the slow increase of P. reticulatum, led to increasingly higher proportions of the former. When the oysters were exposed to D. tertiolecta and P. reticulatum at a ratio of 3 to 1 (Fig. 3.16), the clearance rate diminished drastically; from 40mL hr"1 exhibited with pure cultures ofD. tertiolecta, to near-zero or negative values (Table 9). While at a ratio of 10:1 (Fig. 41 3.16), the clearance rate reached almost 9 mL hr"1, which represents bss than a fourth of the clearance of pure D. tertiolecta cultures (Table 8). Dunaliella tertiolecta and Protoceratium reticulatum 3:1 5 0 0 0 H 0 1 i 1 1 1 1 1 , , L-LJ-I , , L J - I 0 10 20 30 40 50 H o u r s Figure 3.16: Abundance (cells L"1) of Dunaliella tertiolecta during two-species clearance rate experiments, when D. tertiolecta and P. reticulatum were introduced in a ratio of 3:1. Solid line represents control, while the bars represent the abundance of the algae when the oyster was present. Error bars indicate the standard deviation between the triplicates. 3.8. Yessotoxinproduction Toxin analysis of local strains of Protoceratium reticulatum isolated from Jericho beach was performed by Bi l l Hardstaff and Mike Quilliam, of the Canadian National Research Council. They asserted that the pellet obtained by us from 4.5 litres of concentrated algal culture contained 10 mg mL"1 of yessotoxin. These results indicated for the first time that the local strain of P. reticulatum was an active producer of yessotoxin. The average yessotoxin content of P. reticulatum cells was estimated by Stobo et al. (2003), at 0.3 pg cell"1. When related to the abundance of this alga in the water column one can obtain a theoretical concentration of the 42 toxin during the algal bloom (Fig. 3.17). The peak values calculated followed the abundance's, although MacKenzie et al. (2004) have pointed out that toxins produced by P. reticulatum remain in high concentrations in the water column after the causative organism has disappeared. Figure 3.17: Theoretical concentration of yessotoxin (mg L ) at different depths, during the summer of 2001. 43 4. DISCUSSION The results of our analyses seem to implicate the yessotoxin-producing dinoflagellate Protoceratium reticulatum, as the main factor in the mass mortality that repeatedly affected cultured juvenile Pacific oysters rear Sykes Island. This species bloomed before and during the die-offs. The experimental oysters showed strong rejection behaviour and reduced feeding capacity, even to medium and low abundances of this alga. Other known harmful and potentially harmful species could have been implicated, or worked synergistically to produce the mass mortality. Alexandrium tamarense was able to kill the smallest juvenile oysters experimentally, and was present in the water during the die-offs, but only in medium and low abundance of less thanlO 4 cells L ' 1 . There have been numerous reports of massive summer mortalities of adult and juvenile farmed Pacific oysters on the coasts of the Pacific Ocean (Cheney et al., 1999, Chew, 1996, Landsberg, 2002). Several hypotheses have been developed to explain this recurring phenomenon. They include environmental stress due to high temperatures, the high energy expended during their gametogenic cycle, and opportunistic diseases and parasitism (Cheney et al. 1998 and 2000, Meyers and Short, 1990, Perdue et al, 1981, Pauley et al, 1988, Ray, 2002). Juveniles are thought to share most of the morphological characteristics of the adults (Barre et al., 2002), although their reduced size and energy reserves make them a more likely target fir diseases and pathogens. On the other hand, oyster juveniles are not subject to exhaustion associated with an excessive reproductive effort. The mortality of the juveniles could have been produced by starvation, or by toxins released by the dinoflagellates that dominated the phytoplankton during the summer. 44 In the case of the Sykes Island mortality of juvenile oysters, the P. reticulatum bloom lasted for a whole month. Furthermore, a high transparency period was observed shortly after the P. reticulatum bloom. Consequently the nutritive particles were available only in low abundance for a long period of time during the summer. This might have been long enough for a number of even the larger oyster juveniles to die of starvation (Fig. 4.1). The debilitating effect produced by this dinoflagellate was worsened due to the FLUPSY system obtaining its water from 5m deep, where the maximum abundance of P. reticulatum was recorded. C L 20 A Z 10 H Clearance rate = 0. Feeding activity probably affected by P. reticulatum, 25/O6/01 16/07/01 06/08/01 27/08/01 17/09/01 08/1 cj a nd other pHABs Date Clearance rate = 25% of normal. I Figure 4.1: Percentage of the total microplanktonic abundance represented by P. reticulatum, and levels of deleterious effects on the feeding response of juvenile oysters, due to potentially harmful microalgae. The harmful effects of P. reticulatum could have been extended in time and severity by other potentially harmful algae, like Alexandrium catenella, A. tamarense, and Ceratium fusus, 4 5 which were present in low and medium abundances during the summer. During most of the summer, the dominant species within the diatoms were the large and spinous Chaetoceros spp. These dinoflagellates and diatoms were accompanied by high abundance of the fish-killing silicoflagellate Dictyocha speculum in both morphotypes. The total abundance of the phytoplankton during much of the summer was very low. This, compounded with the pHAB effects, could have extended the starvation and impeded feeding period for over two months (Fig. 4.1). The few environmental parameters measured, remained within normal boundaries for oyster development. The temperature and salinity followed the usual cycle observed in British Columbia coastal waters. The high temperature (24 C°) detected during the summer could have contributed to the mortality indirectly, by increasing the metabolism of the oysters, demanding more of their reserves, accelerating their death through starvation. Other causes that could have contributed to the mortality of juvenile oysters in the vicinity of Sykes Island were: • The late introduction of the seed to the nursery raft in April, after the spring bloom, thus depriving the oysters of the necessary reserves to survive the summer conditions. • Parasites and diseases attacked the oysters once the bloom debilitated them. Hinge-ligament disease was found the weakened oyster juveniles to be an easy prey (James Manders, pers. comm.). • Overstocking of the intensive nursery raft could have been one of the factors involved, 46 especially when the amount of nutritive particles in the water diminished drastically during the summer (James Manders, pers. comm.). • The nutritional status and general quality of the seed introduced in 2001 was low. Jeff Babuin (pers. comm.) observed a low condition index in the oyster juveniles acquired that year. This low quality seed could have been affected more easily by adverse environmental factors, pathogens and long periods of obstructed feeding. 47 5. CONCLUSIONS The dinoflagellate P. reticulatum is a well known cosmopolitan species found in all temperate coastal waters around the world, and regular summer blooms of it can be seen in the Strait of Georgia (F.J.R. Taylor, pers. comm.). This species has been known as a yessotoxin producer, which has caused DSP-like toxicity in shellfish in recent years. However, this is the first time that P. reticulatum has been implicated as a cause of severe mortalities of cultured juvenile oysters. This study combined detailed qualitative natural food analysis from field samples, with observation of the rapid behavioural response of juvenile Pacific oysters, when exposed to medium and high abundances of various species of algae. The rapid responses observed experimentally varied from a severe rejection of the particles, as observed with P. reticulatum, and mortality, with Alexandrium tamarense, to a full acceptance of well accepted "food" algae. The quantitative clearance rate experiments were consistent with those of the rapid response experiments. The dinoflagellate Protoceratium reticulatum was most probably the main causative agent in the summer mortality of juvenile Pacific oysters observed at Sykes Island. This dinoflagellate species may be harmful to shellfish aquaculture in British Columbia by causing mortalities of juvenile oysters of less than 1cm in shell length, i f they were introduced to the farm site after the diatom spring bloom. The timing of the introduction of the hatchery seed to sea, and the size of this seed, are critical factors to consider in preventing future summer die-offs of juveniles. The introduction 48 should be as early in the spring, and with the biggest seeds possible. Bigger initial size will render them less susceptible to the effects of harmful algae, and able to feed in a slightly larger size range of organisms. Since toxic dinoflagellates such as Alexandrium, and especially P. reticulatum, bloom every summer in the Strait of Georgia, shellfish farms should implement a harmful phytoplankton monitoring programme, similar to that already in place at fish farms. 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Influence of diet on pre-ingestive particle processing in bivalves I: transport velocities on the ctenidium. Journal of Experimental Marine Biology and Ecology 293: 129-149. 61 Wear, R.G. 1999. New Zealand, Mortality among marine life in Wellington Harbor. Harmful Algae News 18: 16. Wildish, D , Lassus, P , Martin, J , Saulnier, A . and M . Bardouil. 1998. Effect of the PSP-causing dinoflagellate, Alexandrium sp. on the initial feeding response of Crassostrea gigas. Aquatic Living Resources 11: 35-43. Yin , K , Harrison, P . J , Goldblatt, R . H , St.John, M . A . and R.J. Beamish. 1997. Factors controlling the timing of the spring bloom in the Strait of Georgia, British Columbia, Canada. Canadian Journal of Fisheries and Aquatic Science 54: 1985-1995. 62 7. TABLES Table 1: Carbon per cell (pgC cell-1) estimations modified from Haigh (1992), used for calculations of total biomass for the species identified during 2001-2002. Diatoms pgC cell"1 pgC cell"1 Actinoptychus senarius 1418 Coscinodiscus radiatus 1034 Asterionellopsis glacialis 117 Coscinodiscus sp. 12173 Cerataulina pelagica 553 Cylindrotheca closterium 25 Chaetoceros affinis 179 Dactylosolen fragilissimus 311 Chaetoceros anastomosans 40 Detonula pumila 382 Chaetoceros compressus 98 Ditylum brightwellii 1926 Chaetoceros convolutus 248 Eucampia zodiacus 233 Chaetoceros constrictus 206 Grammatophora marina 226 Chaetoceros curvisetus 142 Leptocylindrus danicus 159 Chaetoceros decipiens 331 Leptocylindrus minimus 21 Chaetoceros diadema 197 Licmophora sp. 446 Chaetoceros didymus 93 Melosira numuloides 648 Chaetoceros lasciniosus 127 Melosira varians 648 Chaetoceros lorenzianus 255 Navicula spp. 75 Chaetoceros similis 78 Odontella longicruris 2283 Chaetoceros pseudocurvisetus 132 Pseudo-nitzschia cf. delicatula 28 Chaetoceros radicans 52 Pseudo-nitzschia granii 28 Chaetoceros spp. Small 18 Pseudo-nitzschia cf. seriata 161 Chaetoceros spp. 114 Rhizosolenia setigera 1005 Corethron criophilum 1437 Skeletonema costatum 48 63 Table 1 (cont'd): Carbon per cell (pgC cell"1) estimations modified from Haigh, 1992, used for calculations of total biomass for the species identified during 2001-2002. Diatoms pgC cell"1 pgC cell"1 Synedra sp. 116 Noctiluca scintillans 3755949 Thalassionema frauenfeldii 119 Oxyphysis oxytoxoides 1565 Thalassionema nitzschioides 119 Phalacroma rotundatum 5768 Thalassiosira (medium) 986 Prorocentrum gracile 455 Thalassiosira (small) 659 Prorocentrum micans 1699 Thalassiosira (very small) 24 Protoceratium reticulatum 5111 Thalassiosira gravida/nordenskioeldii 539 Protoperidinium bipes 251 Dinoflagellates Protoperidinium claudicans 14006 Alexandrium tamarense 3237 Protoperidinium conicum 25061 Ceratium fusus 3276 Protoperidinium oceanicum 17834 Cochlodinium sp. 1940 Protoperidinium pellucidum 2302 Dinophysis acuta 4591 Protoperidinium pentagonum 28191 Dinophysis acuminata 1512 Protoperidinium sp. 810 Dinophysis norvegica 3019 Pyrophacus horologicum 13422 Dinophysis sp. 2915 Scrippsiella trochoidea 1036 Gymnodinium sp. 2 754 Other Gymnodinium sp. 5306 Dictyocha fibula 4074 Gyrodinium sp. (small) 36 Dictyocha speculum 620 Heterocapsa triquetra 708 Myrionecta rubra big 1156 64 Table 1 (cont'd): Carbon per cell (pgC cell"1) estimations modified from Haigh, 1992, used for calculations of total biomass for the species identified during 2001-2002. Other pgC cell"1 Myrionecta rubra small 89 Sommes w/o pellicle 2890 Euglenoids 101 Favella sp. 18405 Helicostomella sp. 841 Table 2: Algal species used for rapid response experiments and their source. Alexandrium tamarense 742 C C C M at UBC Amphidinium carterae 692 C C C M at UBC Coccoid cyanobacteria Isolated at Jericho beach, Vancouver Chaetoceros calcitrans 590 C C C M at UBC Dunaliella tertiolecta 1 C C C M at UBC Heterosigma akashiwo 560 C C C M at UBC Gonyaulax spinifera 850 C C C M at UBC Katodinium fungiforme Isolated at Jericho beach, Vancouver Isochysis galbana 601 C C C M at UBC Karenia mikimotoi 665 C C C M at UBC Protoceratium reticulatum 838 C C C M at UBC Phaedactylum tricornutum Isolated at Jericho beach, Vancouver Pseudo-nitzschia pseudodelicatissima PP95 Northwest Fisheries Science Center Thalassiossira weisflogii 741 C C C M at UBC Thalassiossira sp. Isolated at Jericho beach, Vancouver 65 Table 3: Planktonic species and groups identified during 2001 and 2002. Diatoms Chaetoceros radicans Schutt Actinoptychus senarius (Ehrenberg) Ehrenberg Chaetoceros spp. Amphiprora sp. Chaetoceros vistulae Apstein Asterionellopsis glacialis (Castracane) Round Corethron criophilum Castracane Bacillaria paxillifera (Muller) Hendey Coscinodiscus radiatus Ehrenberg Cerataulina pelagica (Cleve) Hendey Coscinodiscus sp. Chaetoceros affinis Lauder Cylindrotheca closterium (Ehrenberg) Reimann & Lewin) Chaetoceros anastomosans Grunow Dactylosolen fragilissimus (Bergon) Hasle Chaetoceros atlanticus Cleve Detonula pumila (Castracane) Gran Chaetoceros compressus Lauder Ditylum brightwellii (West) Grunow, Van Heurck Chaetoceros convolutus Castracane Eucampia zodiacus Ehrenberg Chaetoceros constrictus Gran Grammatophora marina Lyngbye Chaetoceros curvisetus Cleve Guinardia delicatula (Cleve) Hasle Chaetoceros decipiens Cleve Gyrosigma sp./ Pleurosygma sp. Chaetoceros diadema (Ehrenberg) Cleve Leptocylindrus danicus Cleve Chaetoceros didymus Ehrenberg Leptocylindrus minimus Gran Chaetoceros lasciniosus Schutt Licmophora sp. Chaetoceros lorenzianus Grunow Melosira nummuloides (Dillwyn) Agardh Chaetoceros neglectus Karsten Melosira varians Agardh Chaetoceros simile Cleve Navicula sp. Chaetoceros pseudocurvisetus Mangin Odontella longicruris (Greville) Hoban 66 Table 3 (cont'd): Planktonic species and groups observed and identified during 2001 and 2002. Diatoms Ceratium fusus (Ehrenberg) Dujardin Pseudo-nitzschia cf. delicatissima (Cleve) heiden & Kolbe Cochlodinium sp. Pseudo-nitzschia granii Hasle Dinophysis acuta Ehrenberg Pseudo-nitzschia cf. seriata (Cleve) Peregallo Dinophysis acuminata Claparede & Lachmann Rhizosolenia setigera Brightwell Dinophysis norvegica Claparede & Lachmann Skeletonema costatum (Greville) Cleve Dinophysis sacculus Hallegraeff & Lucas Synedra sp. Dinophysis sp. Thalassionema frauenfeldii (Grunow) Hallegraeff Gymnodinium sp. 2 (small spindle-like) Thalassionema nitzschioides (Grunow) Mereschkowsky Gymnodinium sp. (naked large brown gymnodinioid) Thalassiosira sp. (medium) Gyrodinium sp. (small) Thalassiosira lineata (small) Heterocapsa triquetra (Ehrenberg) Balech Thalassiosira sp. (very small) Noctiluca scintillans (Macartney) Kofoid & Swezy Thalassiosira gravida/nordenskioeldii Oxyphysis oxytoxoides Kofoid Unidentified pennate diatoms Phalacroma rotundatum (Claparede & Lachmann) Kofoid and Michener Dinoflagellates Prorocentrum gracile Schiitt Alexandrium catenella (Whedon & Kofoid) Balech Prorocentrum micans Ehrenberg Alexandrium tamarense (Lebour) Balech Protoceratium reticulatum (Claparede & Lachmann) Biitschli 67 Table 3 (cont'd): Planktonic species and groups observed and identified during 2001 and 2002. Dinoflagellates Other Protoperidinium (Minuscula) bipes (Paulsen) Balech Dictyocha fibula Ehremberg Protoperidinium claudicans (Paulsen) Balech Dictyocha speculum Ehremberg Protoperidinium conicum (Gran) Balech Dictyocha speculum askeletal Protoperidinium oceanicum (VanHQffen) Balech Myrionecta rubra big (ex Mesodinium rubrurri) (Lohmann) Jankowski Protoperidinium parthenopes Zingone & Montresor Myrionecta rubra small Protoperidinium pellucidum Berg Sommes w/o pellicle (species without recognizable shape in fixed samples) Protoperidinium pentagonum (Gran) Balech Euglenoids Protoperidinum punctulatum (Paulsen) Balech Tintinnids Protoperidinium sp. Favella sp. Pyrocystis lunula (Schutt) Schutt Helicostomella sp. Pyrophacus horologicum Stein Copepods Scrippsiella trochoidea (Stein) Balech Chattonella aff. g/obosa Hara & Chihara P. reticulatum cysts stage 1 identical to Operculodinium israelianum (Rossignol) Wal l P. reticulatum cysts stage 2 identical to Operculodinium centrocarpum (Deflandre and Cookson) Wal l Cysts 68 Table 4: Harmful planktonic species identified during the sampling period, and their possible effect on other organisms (modified from Landsberg, 2002, Taylor et al. 1994, Moestrup, 2004, Fryxell and Hasle, 2003). Diatoms Harmful effect Chaetoceros convolutus, C. concavicornis Mechanical harm to cultured salmon Pseudo-nitzschia cf. delicatula Domoic acid (ASP a.k.a. D.A.P.) Pseudo-nitzschia cf. seriata Domoic acid (ASP) Skeletonema costatum Mechanical/chemical harm to cultured fish, oxygen depletion (in Asia) Dinoflagellates Alexandrium tamarense and A. catenella Saxitoxins (PSP) Ceratium fusus Possible mechanical or bacteriological damage to shellfish Cochlodinium sp. Fish killer Dinophysis acuta, D. acuminate, D. norvegica and D. sacculus Okadaic acid and DTXs (DSP) Noctiluca scintillans Red tides, oxygen depletion, ammonia Prorocentrum micans Red tides, oxygen depletion Protoceratium reticulatum Yessotoxin Other Dictyocha speculum Fish killer Myrionecta rubra Red tides 69 Table 5: Rapid feeding responses observed in 5mm shell length oysters to selected algal species: pseudo-feces production, feces production, feeding current speed, and digested state in incrementing qualitative scale (see Materials and Methods for detailed scale). Species Pseudo- feces production Feces production Feeding speed Digested state Alexandrium tamarense 0 N 0 n/a Protoceratium reticulatum 0 N 0 n/a Gonyaulax spinifera 0 N 1 n/a Amphidinium carterae 3 N 1 n/a Large phytoplankton and small zooplankton 3 N7 Y 2 n/a Silt 3 N 2 n/a Cyanophyte (Isolate 4) 1 N 1 n/a Pseudo-nitzschia pseudodelicatissima 3 N 1 n/a Heterosigma akashiwo 1 N7 Y 0 / 2 n/a/3 Karenia mikimotoi 1 Y 3 2 Chaetoceros calcitrans 1 Y 3 3 Dunaliella tertiolecta 1 Y 3 3 Isochrysis galbana 1 Y 3 3 Phaeodactylum tricornutum 1 Y 3 3 Katodinium fungiforme 1 Y 3 0 70 Table 6: Rapid responses observed in 5mm shell length oysters to selected algal species: clapping frequency shown in an incrementing qualitative scale (see Materials and Methods for detailed scale). Species Clapping Total closure Alexandrium tamarense 3 Y Protoceratium reticulatum 3 Y Gonyaulax spinifera 2 Y Amphidinium carterae 2 Y / N Large phytoplankton and small zooplankton 2 N Silt 2 N Cyanophyte (Isolate 4) 1 N Pseudo-nitzschia pseudodelicatissima 2 N Heterosigma akashiwo 1 N Karenia mikimotoi 1 N Chaetoceros calcitrans Normal N Dunaliella tertiolecta Normal N Isochrysis galbana Normal N Phaeodactylum tricornutum Normal N Katodinium fungiforme Normal N 71 Table 7: Algal species and their grouping according to their trophic acceptance (or palatability) by 5mm shell length oysters, during the rapid response experiments. Species Group Alexandrium tamarense 112 Lethal Protoceratium reticulatum 2 Acute reaction Gonyaulax spinifera 2 /3 Acute reaction Amphidinium carterae 3 Active rejection Large phytoplankton and small zooplankton 3 Active rejection Silt and dust 3 Active rejection Cyanophyte (Isolate 4) 4 Feeding arrest Pseudo-nitzschia pseudodelicatissima 4 Feeding arrest Heterosigma akashiwo 4 / 5 Feeding arrest Karenia mikimotoi 4 / 5 Feeding arrest Chaetoceros calcitrans 5 Food species Dunaliella tertiolecta 5 Food species Isochrysis galbana 5 Food species Phaeodactylum tricornutum 5 Food species Katodinium fungiforme 6 Non-digestible 72 Table 8: Average clearance rate (mL hr ) measured during the first six hours after introduction into medium containing high abundance of the selected species. Species Six hour average clearance rate (mL hr"l) Dunaliella tertiolecta 40 +/- 7.57 Isochrysis galbana 34 +/- 7.25 Heterosigma akashiwo 28-30 +/- 3.84 Karenia mikimotoi 17 +/- 5.05 (partially digested and decreasing rapidly) Amphidinium carterae 12 +/- 5.28 (into pseudo-feces) Protoceratium reticulatum -Alexandrium tamarense -Table 9: Average clearance rate (mL hr ) measured during the first six hours after introduction into medium containing different proportions of Dunaliella tertiolecta and Protoceratium reticulatum. D. tertiolecta : P. reticulatum proportion D. tertiolecta clearance rate (mL hr"1) 3 : 1 -2.47 +/- 3.01 3 : 1 (corrected for D. tertiolecta growth) -5.36+/- 3.48 10 : 1 8.77 +/- 0.24 10 : 1 (corrected for P. reticulatum growth) 8.87 +/- 0.46 73 

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