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Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters
Harmful Algae 10 (2011) 629–635
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Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters Aoife Nı´ Rathaille, Robin Raine * Martin Ryan Institute, National University of Ireland, Galway, University Road, Galway, Ireland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 November 2010 Received in revised form 22 April 2011 Accepted 22 April 2011 Available online 30 April 2011
Excystment experiments were carried out on cysts of Alexandrium minutum and A. tamarense (Group III) taken from Cork Harbour, Ireland. Freshly sampled cysts were isolated into well plates and their excystment was monitored over a 30 day period. A pronounced seasonality was observed in the excystment behaviour in both species when measurements were carried out seasonally. Between 80 and 100% of the isolated cysts excysted within 30 days when samples were taken between March and June. However, between only 0 and 15% excysted in samples taken between August and early February. This seasonal characteristic was observed repeatedly over a 3 year period (2004–2007). The effect was observed in cysts which had been sampled from both inter-tidal and sub-tidal locations. No endogenous clock was evident in the germination of A. tamarense and A. minutum cysts taken from Cork Harbour which had been stored cool and in the dark under anoxic conditions for up to 18 months. The seasonal effect observed was independent of water temperature, although temperature did affect the rate of excystment. Temperature also affected the maturation of cysts which had been freshly formed in the laboratory. The significance of incorporating seasonality in excystment of Alexandrium into models describing its growth is also discussed. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Alexandrium Excystment Ireland
1. Introduction The life cycle of the dinoflagellate Alexandrium is well understood in general terms. In spring or early summer, planktonic vegetative cells arise from diploid resting cysts which have remained dormant through the winter in the sediment. After an unspecified period, usually a few weeks, the haploid cells become gametes which fuse to form a short-lived (Turpin et al., 1978) diploid cell, known as a planozygote as it remains motile. This then forms a pellicle, transforming into a non-motile cyst (zygote). The cyst settles into the sediment where it matures for a mandatory period before it can excyst (Dale, 1977; Anderson, 1980), restarting the cycle. The details and the dynamics of the intermediary stages between the resting cyst and vegetative planktonic cell (the haploid state) are, however, less well known. For example, the transitions between planktonic forms – the vegetative cell, motile gamete and planozygote – are not well studied principally due to the morphological similarity between the gamete and planktonic cell. Furthermore, the small amount of available literature on this subject has indicated that under culture conditions transitions between the zygote and planktonic cell are far more complex than originally thought. For example, the planozygote of A. tayloryi is
* Corresponding author. Tel.: +353 91 492271; fax: +353 91 525005. E-mail address: [email protected] (R. Raine). 1568-9883/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2011.04.015
capable of by-passing the cyst stage through division in the mobile stage (Figueroa et al., 2006). Some studies exist, however, which have involved examination of the dynamics of the transition between the cyst (zygote) and planktonic phase. For example, an endogenous clock has been observed in Alexandrium cysts sampled from deep water (Anderson and Keafer, 1987; Matrai et al., 2005) and temperature effects on excystment have been observed (e.g. Anderson, 1998). Knowledge of the dynamics of excystment is necessary in order to predict the initiation of Alexandrium blooms, a capability that is highly desirable since many of the species of this genus produce potent toxins which can give rise to paralytic shellfish poisoning (PSP) symptoms if contaminated filter-feeding bivalves such as oysters and mussels are consumed by humans (Hallegraeff, 2003). Around Ireland, the only location that has a history of repetitive PSP contamination of shellfish is Cork Harbour (Silke et al., 2006). Here, there is a resident population of Alexandrium minutum (Touzet et al., 2008). Shellfish harvesting from this estuary has been prohibited in most summers due to contamination with the gonyautoxins GTX2 and GTX3 which are typical of the toxin profile of A. minutum (Touzet et al., 2008). The timing of the blooms occurs in June and July, with planktonic cell densities usually >105 L1. Blooms of smaller intensity can occur later in the year, for example one achieving cell densities of 20,000 cells L1 occurred in September 2004 (Nı´ Rathaille, 2007).
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The present study comprised field and laboratory investigations of the excystment dynamics of A. minutum from Cork Harbour. Within the estuary, there is also a population of non-toxic A. tamarense. This form belongs to a group of five cryptic species designated by Scholin et al. (1994) as the Western European clade, now re-defined as in Group III of the species complex (Lilly et al., 2007). It co-exists with A. minutum in Cork Harbour, and the excystment dynamics of this species were also included in the study. 2. Methods 2.1. Seasonal regulation of excystment The method applied to investigate seasonal variation in excystment was a modified version of that used by Itakura and Yamaguchi (2001). On 12 occasions between March 2004 and May 2007, intertidal sediment was sampled from the North Channel of Cork Harbour (Fig. 1) using manually operated modified syringes as corers. On the day of sampling, the surface 10 mm layer was extruded, homogenised thoroughly and approximately 30 cysts of each species were extracted and isolated into individual wells of a 96-well plate with 200 mL of fresh sterile-filtered seawater in each well. The plate was incubated at a temperature as close (within 1.5 8C) to the in situ temperature of the seawater as possible. Irradiance levels in the incubator were approximately 15 mE m2 s1 and a 14:10 h light to dark cycle was used. Plates were examined for signs of excystment on a regular basis over a period of 30 days. Signs of excystment included the presence of at least one vegetative cell or the presence of an empty cyst. On two occasions (October 25th 2005
and March 28th 2006) sediment from a sub-tidal station (Fig. 1) was also sampled in order to compare the excystment pattern observed for inter-tidal cysts with that of sub-tidal cysts. On two occasions (October 7th 2006 and May 4th 2007), excystment measurements were supplemented by observations using sediment cores, thus eliminating the need to extract the cysts from the sediment. Three fresh sediment cores were taken from the intertidal sampling site. The top 1 cm layer of each core was removed, placed individually into the bottom of one of three 50 mL centrifuge tubes and 20 mL of fresh sterile filtered seawater was carefully added to each tube. The centrifuge tubes were incubated at the same temperature and light conditions as the 96-well plate, but in a Styrofoam holder such that only the top layer of the sediment was exposed to the light. Every 24 h the 20 mL of seawater was replaced with fresh sterile filtered seawater. The 20 mL that was removed was placed in an Utermo¨hl’s settling chamber, preserved with formalin and the entire contents examined for the presence of vegetative cells of Alexandrium sp. This continued every 24 h until three consecutive samples gave a zero result. 2.2. Endogenous clock regulation An investigation into the presence of an endogenous clock in Alexandrium cysts from Cork Harbour was initiated in 2002 using a method similar to that described in the experiments of Anderson and Keafer (1987) and Matrai et al. (2005) and described in detail in Anderson et al. (2003). Approximately 2 L of sediment known to contain Alexandrium spp. cysts was collected from the surface 5 cm at the intertidal station (Fig. 1) in the North Channel in early
Fig. 1. (a) Map of Cork Harbour, Ireland showing the location of the North Channel. (b) The North Channel, showing the location of the intertidal (A) and sub-tidal (B) sampling stations. Isobaths are in metres. The outline of land is the mean high tide level, whilst the mean low tide level is shown in (b) as the ‘0’ isobath, indicating the extensive area of intertidal mud-flats in the North Channel.
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October 2002. This sediment was homogenised and sub-divided into one hundred 9 mL bijou bottles which were stored in anoxic conditions in the dark at 4 8C. In January 2003, after a 4 month maturation period, one of the bijou bottles was removed from storage. Approximately 40 cysts of Alexandrium tamarense were isolated from the sediment and placed into individual wells of a 96well plate, with each well containing 200 mL f/2-Si medium (Guillard, 1975). The well plate was incubated at 15 8C for 30 days in an illuminated incubator (15 mE m2 s1 and a 14:10 h light to dark cycle) in similar fashion to the seasonal regulation experiment. Cysts in the plate were checked for signs of excystment on a weekly basis over the 30 day period using an Olympus CKX41 inverted binocular microscope. This procedure was repeated approximately every 2 weeks for an 18 month period, each time removing a new bijou bottle from the dark, anoxic storage conditions. The cysts isolated during this experiment varied in condition, but were always partially filled (in addition to the characteristic orange-red accumulation body). For the last 6 months of this period, the experiment was expanded to include cysts of A. minutum. 2.3. Temperature regulation of excystment The effect of temperature on the excystment dynamics of A. tamarense and A. minutum was investigated on cysts taken from the North Channel of Cork Harbour. Approximately 30 mature cysts of each species were isolated into each of five 96-well plates from the material stored for the endogenous clock experiment. This was carried out during February 2004. Each of these plates was then incubated at a specific temperature for a period of 30 days. The temperatures chosen were 5 8C, 10 8C, 15 8C, 20 8C and 25 8C which reflect the annual temperature range found in the North Channel. The light levels that the plates were exposed to were low, approximately 15 mE m2 s1, and the light:dark photoperiod was 14:10 h. Each plate was checked for signs of excystment, i.e. the presence of at least one vegetative cell or the presence of an empty cyst, every second day for the first 2 weeks, and approximately every 7 days for the remaining 2 weeks. Excystment rates for each species at each temperature were measured using Eq. (1), where N0 was the initial concentration of cysts, Nt was the concentration of cysts remaining at time t and k was the specific germination rate with units of day1 (Anderson et al., 1987). Nt ¼ N 0 expðktÞ
(1)
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2.4. Cyst maturation Cyst maturation times were investigated on newly formed cysts of both A. tamarense and A. minutum. Newly formed cysts were obtained by filling a 25 L drum with fresh seawater collected from the north Channel of Cork Harbour during the peak of the June 2004 bloom. To this was added plankton material obtained with a 20 mm mesh phytoplankton net, and the mixture was then spiked with f/80-Si medium (Guillard, 1975). The seawater drum was kept out of direct sunlight and at room temperature for 20 days until fresh resting cysts were observed to have formed and settled to the bottom of the drum. These cysts were collected and stored using the methods outlined in Anderson (1980) and Anderson et al. (2003). Aliquots of cysts were put into 9 mL bijou bottles. A layer of 20 mm mesh was put over the neck of the bijou bottle and the lid, with a small circular hole in it, was screwed on tightly. Sixty such bijou bottles were prepared and placed in anoxic conditions by submerging them in a jar of anoxic mud taken from a location (Galway Bay) where no cysts had been observed; 15 each at 5 8C, 10 8C, 15 8C and 20 8C. Every 3 weeks one bijou bottle was removed from each temperature condition and 30 cysts of each species were isolated from each bottle. The cysts were removed to a 96-well plate and incubated in 200 mL of f/2-Si medium (Guillard, 1975) at 15 8C. The plates were then periodically checked for signs of excystment. Cyst maturation time was defined as the time needed for 50% of the cysts to germinate (Binder and Anderson, 1987). 3. Results 3.1. Seasonal regulation of excystment Results showing the excystment success of freshly sampled cysts of A. tamarense and A. minutum incubated at in situ temperatures over a 3 year period are shown in Fig. 2. The general pattern was the same for both species showing clear spring maxima and autumn minima. Maximum values of between 73– 96% success for A. tamarense and 60–100% success for A. minutum were obtained between March and June over the 30 day incubation period used. Minimum values of 0–14% were obtained for both species between the months of August and October. The spring maxima coincided with a water column temperature of approximately 10 8C. Minimum excystment success occurred during the time that the water column temperature was decreasing from its annual maximum (20 8C) in August to 10 8C about 4 months later.
Fig. 2. Seasonal variation in the excystment success of freshly sampled A. tamarense (*) and A. minutum (*) cysts from the North Channel of Cork Harbour over the period March 2004 to May 2007. The seawater temperature (8C) recorded over the same time period is shown as the background hairline. Sampling locations are given in Fig. 1.
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Table 1 A comparison of the excystment success of A. tamarense and A. minutum from intertidal and subtidal sediments sampled during October 2005 and March 2006. The location of sampling stations is shown in Fig. 1. Date
Species
% Excystment Intertidal
Subtidal
25 October 2005
A. tamarense A. minutum
7 (n = 30) 0 (n = 25)
0 (n = 30) 0 (n = 27)
28 March 2006
A. tamarense A. minutum
70 (n = 30) 60 (n = 25)
76 (n = 30) 75 (n = 24)
These results strongly indicate that the seasonal patterns for both species appear to be independent of a temperature function. The seasonal signal in excystment did not appear to be due to either the intertidal nature of the samples or the cyst isolation process. When excystment success was compared between intertidal samples and sub-tidal samples at times when minimum and maximum success was expected in October and March, respectively, success rates were very similar (Table 1). In October 2005, 0% excystment was observed for A. minutum in both intertidal and sub-tidal samples. The pattern for A. tamarense was very similar, with 7% excystment in the inter-tidal sample and 0% excystment in the sub-tidal sample. In March 2006, 60% excystment was observed for A. minutum in the inter-tidal sample with 75% excystment in the sub-tidal sample. The pattern for A. tamarense was again similar, with 70% excystment in the intertidal sample and 76% excystment in the sub-tidal sample. These results suggest that there is no difference between the excystment dynamics of cysts from inter-tidal sediment and those from subtidal sediments. The seasonal trend in excystment was also observed in incubations with untreated sediment. No vegetative cells were observed in the overlying water of sediment samples taken and incubated in October 2006. In May 2007, however, excystment from freshly sampled sediment occurred over a period of 2 weeks. The cumulative numbers of vegetative cells observed in the overlying water of sediment samples at this time is shown in Fig. 3. These results further substantiate the original observations of seasonality in excystment.
Fig. 4. Excystment success of mature cysts of (a) A. tamarense (filled circles) and (b) A. minutum (open circles) stored in cold, dark anoxic conditions as a function of storage time. Each month, 30 cysts were isolated and incubated at 15 8C. Trendlines show a linear regression analysis of the data. The arrow marks the date that the temperature effect experiment described in Section 2.3 took place.
3.2. Endogenous clock regulation Results of investigations into the presence of an endogenous clock in cysts of A. tamarense and A. minutum from Cork Harbour are shown in Fig. 4. These results spanned an 18 month period from
Fig. 3. The cumulative numbers of vegetative cells of Alexandrium spp. observed with time in the water overlying sediment samples taken in May 2007. Results are presented for each of the triplicate tubes used in the experiment (Tube 1: filled circles; Tube 2: open circles; Tube 3: half-filled circles). Experimental details are given in the text.
Fig. 5. Excystment dynamics of (a) A. tamarense and (b) A. minutum over the range of temperatures encountered in the North Channel of Cork Harbour, Ireland. (c) The excystment success of A. tamarense and A. minutum after 30 days as a function of temperature derived from the results shown in (a) and (b).
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January 2003 to July 2004 for A. tamarense and a 6 month period from January 2004 to July 2004 for A. minutum. There was no evidence of a seasonal pattern of excystment that would have confirmed the presence of an endogenous clock for either species, although the data for A. minutum cover too short a time-span to be conclusive. The average excystment success for the experiment was 74%. For the 2003 data alone (i.e. the first 12 months of the experiment), the average excystment success was 79% for A. tamarense. The lowest value recorded was 40% excystment in July 2004. 84% of all the values recorded showed excystment success greater than 60%. Average excystment success for A. minutum was 87%. Regression analysis of both data sets indicated a general decrease in excystment success as the experiment progressed. 3.3. Temperature regulation of excystment Fig. 5a and b shows the effect of temperature on the rate of excystment of A. tamarense and A. minutum, respectively. The cysts were taken from the stock used for the endogenous clock experiment (see Fig. 4). Results indicated that excystment for each species occurred earlier over the upper range of temperatures although at the highest temperature of 25 8C, the excystment of A. tamarense was slightly reduced from that measured at 20 8C. No effect above 15 8C was observed on the excystment of A. minutum. When the excystment success of each species is plotted against temperature (Fig. 5c), then A. tamarense shows a maximum (77%) at 15 8C, with reduced success at the higher and lower temperatures, whereas that of A. minutum was more or less equal over the entire range of temperatures investigated. Table 2 shows the calculated excystment rates for each species at each of the temperatures investigated. Generally excystment rates of A. minutum (0.05–0.38 day1) were higher than those of A. tamarense (0.01–0.18 day1). A. tamarense excystment rates showed an optimum at 15 8C (0.18 day1), whereas the A. minutum excystment rates increased linearly with temperature. 3.4. Cyst maturation Fig. 6 shows a summary of the relationship between maturation times of newly formed cysts plotted against storage temperature. It can be seen that the maturation time was 66 days for newly formed cysts of A. tamarense stored at 5 8C, 58 days for those stored at 10 8C and for those stored at 20 8C, maturation time was 22 days. The maturation time was longer for newly formed cysts of A. minutum stored at lower temperatures, 112 days at 5 8C and 83 days for those stored at 10 8C. An additional value of up to 20 days could be added to these values. This is because there was a period of 20 days between the time when f/80-Si medium was added to the drum and the day (day 0 on Fig. 6) when the cysts were collected from it.
Fig. 6. Maturation time of freshly formed A. tamarense (filled circles) and A. minutum (open circles) cysts as a function of storage temperature. Maturation time is defined as the time taken for 50% of the cysts to excyst.
4. Discussion The results presented have shown a pronounced seasonality in the excystment behaviour of A. minutum and A. tamarense, an effect which was independent of temperature. No endogenous clock was evident in the germination of either species. Temperature did, however, affect the rate of excystment, and also affected the maturation rate of cysts which had been freshly formed in the laboratory. Excystment of Alexandrium spp. cysts from Cork Harbour was not inhibited over the annual range of in situ temperatures observed. This would suggest that the local cysts are adapted to excyst at any time of the year, even if conditions for growth are not suitable. Similar results were found for A. minutum cysts from the Fal Estuary in England (Perez-Blanco, 2005). Excystment experiments conducted on these cysts which had been stored at 4 8C for at least 1 month resulted in no inhibition of excystment at temperatures between 4 8C and 24 8C. Cysts of A. tamarense sampled in April (1991) from the St. Lawrebce estuary and stored for 2 weeks in the dark excysted over the entire range of temperatures (1–16 8C) subsequently applied. Anderson (1998) presents evidence for a temperature window for excystment of A. tamarense cysts from Perch Pond, USA. This window (5–21 8C) represents the range of temperatures within which excystment will occur but outside of which temperature completely inhibits excystment causing mature cysts to remain in a quiescent state. Other experiments on A. tamarense cysts from Perch Pond have shown that cysts stored at extremes of this window at 5 8C and 22 8C will excyst only when the temperature is raised or cooled, respectively (Anderson and Morel, 1979).
Table 2 Excystment rates for A. tamarense and A. minutum at each of the 5 temperatures investigated and the data used to calculate them (data in parentheses are the maximum percentage germination recorded during the experiment for the species at that temperature and the day this maximum was recorded. These data were not used in the calculation of excystment rate). Temperature (8C) A. tamarense N0 % germination t (d) Nt k (d1) A. minutum N0 % germination t (d) Nt k (d1)
5
10
15
20
35
29 28 (28) 30 (30) 21 0.01
29 47 (55) 16 (30) 15 0.04
30 77 (77) 8 (8) 7 0.18
30 60 (63) 8 (10) 12 0.11
30 40 (43) 4 (8) 18 0.13
30 67 (77) 22 (30) 10 0.05
30 70 (77) 10 (22) 9 0.12
30 87 (87) 6 (6) 4 0.34
30 86 (90) 6 (8) 4 0.33
30 90 (93) 6 (6) 3 0.38
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Extrapolation of laboratory results of temperature effects on excystment to natural conditions has been discussed by several authors. One chief problem has been the term applied to excystment. A common measure has been excystment success, which represents the proportion of a cyst population that will excyst in a given time period. This term does not, however, take into account the speed or rate of excystment. A problem making direct comparisons of excystment success from the literature is that investigations often differ in the incubation time applied for the experiment. Changes in incubation time can influence the result. For example, it is quite probable that in the methods applied here, increasing the incubation time would have yielded a higher excystment success at 5 8C for A. tamarense. Excystment success of cysts of A. tamarense from Cork Harbour indicated a temperature effect with the greatest proportion of cysts (77%) excysting at 15 8C after a 30 day incubation period. Excystment success was lowest at the temperature extremes (28% at 5 8C and 43% at 25 8C). Anderson (1998) reported equal success (almost 100%) for cysts of A. tamarense from Perch Pond at temperatures between 9 8C and 21 8C, using an incubation time of 3 weeks. At temperatures between 6 8C and 8 8C, excystment success was somewhat lower (50%). Castell Perez et al. (1998) reported equal success (>80%) for cysts of A. tamarense from the Lower St. Lawrence Estuary at temperatures between 2 8C and 16 8C, with slightly lower success (50%) at 1 8C. In that investigation, cysts were incubated for 38 days. Excystment of cysts of A. minutum presented here showed no temperature effect with almost equal success (>75%) at each temperature. These results contrast with data for the species from Australia (Cannon, 1993), where a marked temperature effect was observed with maximum percentage excystment (55%) occurring at 16 8C and lower excystment success (15% and 23%) occurring at 12 8C and 20 8C, respectively, after a 10 day incubation time. On the other hand, the results found here compare well with experiments carried out on specimens taken from the Fal Estuary, England, where excystment success after 15 days showed no signs of being affected by temperature (Perez-Blanco, 2005). High percentage excystment (>80%) was noted by Perez-Blanco (2005) by the fourth day of the experiment. All these experiments were carried out on cysts which had been stored for periods in excess of 2 weeks in cool (4 8C) and dark conditions. Given the inherent problems when comparing excystment success data with different incubation times when no data showing the progression of the experiment with time has been presented, calculating the excystment rate at each temperature would be a more reliable way to compare excystment dynamics between one area and another. Using the data for A. minutum cysts from Cork Harbour as an example, excystment success showed no temperature effect; however, excystment rates based on the maximum percentage excystment and time when maximum percentage excystment was recorded, revealed a distinct relationship with temperature. Lower temperatures (5 8C) resulted in lower excystment rates (0.05 day1) and higher temperatures (25 8C) resulted in higher excystment rates (0.38 day1). Similarly, Blanco et al. (2009) reported for A. minutum a slightly longer time taken for 50% germination to occur at 4 8C than at 24 8C (1.5 days compared with 1.1 days), despite the excystment success being equal at all temperatures. The results of the experiments showing the effects of temperature on Alexandrium spp. excystment suggest that these cysts are adapted to excyst at any time of the year. However, when fresh cysts were isolated and incubated at in situ temperatures, a clear seasonal pattern was observed over a 3 year period. This pattern showed seasonal maxima occurring in late spring and seasonal minima occurring in late autumn when water temperatures were not very different. Similar seasonal patterns have been
reported for A. tamarense cysts from Hiroshima Bay, Japan (Itakura and Yamaguchi, 2001) and Masan Bay, Korea (Kim et al., 2002). There are many similarities between the results of the Masan and Hiroshima Bay studies and those obtained from Cork Harbour. These include the temperature pattern and the timing of the vegetative cell blooms which occurred in late spring or early summer when water temperatures reach 15 8C. The time difference between the occurrence of maximum excystment and the occurrence of maximum vegetative cell densities differed slightly, being approximately 2 months in the case of Hiroshima Bay and Cork Harbour, but closer to 4 months in the case of Masan Bay. In Hiroshima Bay, highest excystment success (>50%) occurred between December and April as water temperatures decreased from 16.5 8C to the annual minimum of 10 8C. Low excystment success (<50%) occurred between May and November with no excystment occurring when water temperatures were at their annual maximum (ca. 23.5 8C) in September. In Masan Bay, similarly high excystment success (>50%) also occurred as water temperatures decreased to the annual minimum 6 8C between November and February. Excystment success was low during March and April when water temperatures began to increase, and was almost negligible between May and October, as water temperatures increased towards the annual maximum of 22 8C. In the cases of both Hiroshma Bay (Itakura and Yamaguchi, 2001) and Masan Bay (Kim et al., 2002), the authors proposed a temperature window for germination between 10 8C and 15 8C coupled with the mandatory dormancy of newly formed cysts to explain their observations. No data for the mandatory dormancy period of local cysts were presented in either case. In this study there is certainly a strong argument for mandatory dormancy preventing excystment during the period immediately after bloom decline. The dormancy period for Cork Harbour cysts would, however, only seem to be sufficient to explain the low excystment success for a period of about 2 months after the demise of the observed blooms (end of June). This would imply that by September, most cysts should be through their mandatory dormancy period and ready to excyst once again. This, however, did not occur, despite favourable water temperatures and the ability of cysts of both A. tamarense and A. minutum to excyst over the entire temperature range experienced in Cork Harbour. No seasonal signal that would have indicated endogenous control of excystment was observed in either A. tamarense or A. minutum cysts taken from Cork Harbour. Endogenous control of excystment has been reported for A. tamarense from the Gulf of Maine (Anderson and Keafer, 1987; Matrai et al., 2005). It has also been suggested for cysts of A. tamarense from the Lower St. Lawrence estuary in Canada (Castell Perez et al., 1998), but further work was required there to confirm its presence. No endogenous control of excystment has been reported for cysts of A. minutum. Endogenous control of excystment would be a distinct ecological advantage for cysts that occur in deeper areas of the ocean where environmental cues such as temperature and photoperiod do not exist. A biological clock would be able to cue such cysts to excyst at a time of year when surface water temperature and irradiance levels are suitable for growth. Both the Gulf of Maine cysts and the St. Lawrence Estuary cysts were sampled from such depths (60–160 m in the case of the Gulf of Maine). By contrast, the Cork Harbour cysts that were used in this investigation were sampled from intertidal sediment of the North Channel, a shallow water area less than 10 m deep. These cysts are exposed to a very broad range of environmental variables such as temperature and irradiance and, as such, would not benefit from the presence of an endogenous clock. Anderson and Keafer (1987) have also reported the lack of an endogenous clock for cysts from a shallow estuary. Given the seasonal pattern observed over a 3 year
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period, despite the lack of an endogenous clock, other factor(s) must therefore be controlling cyst germination in Cork Harbour. Secondary dormancy which prevents germination during periods that are favourable for germination but are not favourable for growth has been reported to occur in the seeds of higher plants (Vleeshouwers et al., 1995; Bewley, 1997). This results in seeds being alternately responsive and unresponsive to the same environmental conditions. Such a phenomenon, or another as yet undescribed process which results in the same effect, would explain why cysts that germinate in Cork Harbour in the environmental conditions of spring are prevented from germinating in autumn. This rhythm generally disappears with storage in constant conditions (Karssen, 1980/81) which would be one possible reason why it did not emerge during the endogenous clock experiment, where sediment had been stored in constant conditions for a period of 4 months prior to the beginning of the experiment. Anderson and Keafer (1987) discounted the secondary dormancy hypothesis for deep water cysts of A. tamarense from Cape Cod due to the multi-year persistence of the germination cycle under constant storage conditions, instead proposing the endogenous clock theory to explain the seasonal germination pattern. In contrast to the deep water environment which has a nearly invariant nature, environmental signals vary considerably in shallow systems. Shallow water cysts might be better served by germination cued to the environment through secondary dormancy. It is highly desirable to predict the occurrence of toxic dinoflagellate blooms in order allow mitigation of their economically damaging effects to aquaculture. Although many harmful algal blooms are caused by transport of toxic populations into areas of shellfish production, the Alexandrium population in the North Channel of Cork Harbour is a resident population, first reported by Gross (1988). Blooms of planktonic toxin-producing cells of A. minutum logically initiate from excystment of mature cysts. Blooms are therefore less likely to arise during seasonal minima in the excystment of this species. Marked seasonality in the germination of toxic dinoflagellates must be incorporated into coupled physical biological models designed to predict blooms. Thus the endogenous clock observed in cysts from the Gulf of Maine is incorporated into the Alexandrium model described by McGillicuddy et al. (2005). The seasonality in excystment observed in Cork Harbour should likewise be incorporated into models describing its growth here. Acknowledgements The authors wish to acknowledge the assistance of David HughJones, Brian Byrne and Donie Geary for the use of their facilites and for the provision of boats during the fieldwork carried out in this study. Thanks are also due to Georgina McDermott, Mairead Murphy and Nicolas Touzet for their help in the laboratory. This work was funded by an RTDI Scholarship awarded to Aoife Nı´ Rathaille by the Marine Institute under the National Development Plan (2000–2006) and also by the EC 6th Framework Programme (SEED project: GOCE-CT-2005-003375). [SS] References Anderson, D.M., 1980. Effects of temperature conditioning on development and germination of Gonyaulax tamarensis (Dinophyceae) hypnozygotes. Journal of Phycology 16, 166–172. Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transitions. In: Anderson, D.M., Cembella, A.D.,
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Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. NATO ASI Series G41. Springer-Verlag, Berlin/Heidelberg, pp. 29–48. Anderson, D.M., Morel, F.M.M., 1979. The seeding of two red tide blooms by the germination of benthic Gonyaulax tamarensis hypnocysts. Estuarine and Coastal Marine Science 8, 279–293. Anderson, D.M., Keafer, B.A., 1987. An endogenous annual clock in the toxic marine dinoflagellate Gonyaulax tamarensis. Nature 325, 616–617. Anderson, D.M., Taylor, C.D., Armbrust, V.E., 1987. The effects of darkness and anaerobiosis on dinoflagellate cyst germination. Limnology and Oceanography 32 (2), 340–351. Anderson, D.M., Fukuyo, Y., Matsuoka, K., 2003. Cyst methodologies. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual on Harmful Marine Microalgae. IOC Monographs on Oceanographic Methodology, vol. 11. UNESCO Publishing, Paris, pp. 165–189. Bewley, J.D., 1997. Seed germination and dormancy. The Plant Cell 9, 1055–1066. Binder, B.J., Anderson, D.M., 1987. Physiological and environmental control of germination in Scrippsiella trochoidea (Dinophyceae) resting cysts. Journal of Phycology 23, 99–107. Blanco, E.P., Lewis, J., Aldriedge, J., 2009. Germination characteristics of Alexandrium minutum (Dinophyceae), a toxic dinoflagellate from the Fal estuary (UK). Harmful Algae 8, 518–522. Cannon, J.A., 1993. Germination of the toxic dinoflagellate Alexandrium minutum from sediments in the Port River, South Australia. In: Smayda, T.J., Shimizu, Y. (Eds.), Toxic Phytoplankton Blooms in the Sea. Elsevier, Amsterdam, pp. 103–107. Castell Perez, C., Roy, S., Levasseur, M., Anderson, D.M., 1998. Control of germination of Alexandrium tamarense (Dinophyceae) cysts from the lower St. Lawrence estuary (Canada). Journal of Phycology 34, 242–249. Dale, B., 1977. Cysts of the toxic red-tide dinoflagellate Gonyaulax excavata (Braarud) Balech from Oslofjorden, Norway. Sarsia 63, 29–34. Figueroa, R.I., Bravo, I., Garces, E., 2006. Multiple routes of sexuality in Alexandrium taylori (Dinophyceae) in culture. Journal of Phycology 42, 1028–1039. Gross, J., 1988. A red tide outbreak in Cork Harbour, July, 1987. Red Tide Newsletter 1 (1), 4. Guillard, R., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W., Chanley, M. (Eds.), Culture of Marine Invertebrate Animals. Plenum Press, New York, pp. 29–60. Hallegraeff, G.M., 2003. Harmful algal blooms: a global overview. In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.), Manual on Harmful Marine Microalgae. IOC Monographs on Oceanographic Methodology, vol. 11. UNESCO Publishing, Paris, pp. 25–49. Itakura, S., Yamaguchi, M., 2001. Germination characteristics of naturally occurring cysts of Alexandrium tamarense (Dinophyceae) in Hiroshima Bay, Inland Sea of Japan. Phycologia 40 (3), 263–267. Karssen, C.M., 1980/81. Environmental conditions and endogenous mechanisms involved in secondary dormancy of seeds. Israel Journal of Botany 29, 45–64. Kim, Y.-O., Park, M.-H., Han, M.-S., 2002. Role of cyst germination in the bloom initiation of Alexandrium tamarense (dinophyceae) in Masan Bay, Korea. Aquatic Microbial Ecology 29, 279–286. Lilly, E.L., Halanych, K.M., Anderson, D.M., 2007. Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae). Journal of Phycology 43, 1329–1338. Matrai, P., Thompson, B., Keller, M.D., 2005. Circannual excystment of resting cysts of Alexandrium spp. from eastern Gulf of Maine populations. Deep-Sea Research II 52, 2560–2568. McGillicuddy Jr., D.J., Anderson, D.M., Lynch, D.R., Townsend, D.W., 2005. Mechanisms regulating large-scale seasonal fluctuations in Alexandrium fundyense populations in the Gulf of Maine: results from a physical–biological model. Deep-Sea Research II 52, 2698–2714. Nı´ Rathaille, A., 2007. Modelling Alexandrium bloom dynamics in Cork Harbour, Ireland. Ph.D. Thesis, National University of Ireland, Galway, 277 pp. Perez-Blanco, E., 2005. A study of the distribution and germination dynamics of alexandrium cysts in U.K. sediments. Ph.D. Thesis, University of Westminster, U.K. Scholin, C.A., Herzog, M., Sogin, M., Anderson, D.M., 1994. Identification of groupand strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae): dispersal in the North American and west Pacific regions. Phycologia 34, 472–485. Silke, J., McMahon, T., Hess, P., 2006. Irish shellfish biotoxin monitoring programme. In: Deegan, B., Butler, C., Cusack, C., Henshilwood, K., Hess, P., Keaveney, S., McMahon, T., O’Cinneide, M., Silke, J. (Eds.), Molluscan Shellfish Safety. The Marine Institute, Rinville, Galway, Ireland. Touzet, N., Franco, J.M., Raine, R., 2008. PSP toxin analysis and discrimination of the naturally co-occurring Alexandrium tamarense and A. minutum in Cork Harbour, Ireland. Aquatic Microbial Ecology 51, 285–299. Turpin, D.H., Dobell, P.E.R., Taylor, F.R.J., 1978. Sexuality and cyst formation in Pacific strains of the toxic dinoflagellate Gonyaulax tamarensis. Journal of Phycology 14, 235–238. Vleeshouwers, L.M., Bouwmeester, H.J., Karssen, C.M., 1995. Redefining seed dormancy: an attempt to integrate physiology and ecology. Journal of Ecology 83, 1031–1037.
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Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters Aoife Nı´ Rathaille, Robin Raine * Martin Ryan Institute, National University of Ireland, Galway, University Road, Galway, Ireland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 November 2010 Received in revised form 22 April 2011 Accepted 22 April 2011 Available online 30 April 2011
Excystment experiments were carried out on cysts of Alexandrium minutum and A. tamarense (Group III) taken from Cork Harbour, Ireland. Freshly sampled cysts were isolated into well plates and their excystment was monitored over a 30 day period. A pronounced seasonality was observed in the excystment behaviour in both species when measurements were carried out seasonally. Between 80 and 100% of the isolated cysts excysted within 30 days when samples were taken between March and June. However, between only 0 and 15% excysted in samples taken between August and early February. This seasonal characteristic was observed repeatedly over a 3 year period (2004–2007). The effect was observed in cysts which had been sampled from both inter-tidal and sub-tidal locations. No endogenous clock was evident in the germination of A. tamarense and A. minutum cysts taken from Cork Harbour which had been stored cool and in the dark under anoxic conditions for up to 18 months. The seasonal effect observed was independent of water temperature, although temperature did affect the rate of excystment. Temperature also affected the maturation of cysts which had been freshly formed in the laboratory. The significance of incorporating seasonality in excystment of Alexandrium into models describing its growth is also discussed. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Alexandrium Excystment Ireland
1. Introduction The life cycle of the dinoflagellate Alexandrium is well understood in general terms. In spring or early summer, planktonic vegetative cells arise from diploid resting cysts which have remained dormant through the winter in the sediment. After an unspecified period, usually a few weeks, the haploid cells become gametes which fuse to form a short-lived (Turpin et al., 1978) diploid cell, known as a planozygote as it remains motile. This then forms a pellicle, transforming into a non-motile cyst (zygote). The cyst settles into the sediment where it matures for a mandatory period before it can excyst (Dale, 1977; Anderson, 1980), restarting the cycle. The details and the dynamics of the intermediary stages between the resting cyst and vegetative planktonic cell (the haploid state) are, however, less well known. For example, the transitions between planktonic forms – the vegetative cell, motile gamete and planozygote – are not well studied principally due to the morphological similarity between the gamete and planktonic cell. Furthermore, the small amount of available literature on this subject has indicated that under culture conditions transitions between the zygote and planktonic cell are far more complex than originally thought. For example, the planozygote of A. tayloryi is
* Corresponding author. Tel.: +353 91 492271; fax: +353 91 525005. E-mail address: [email protected] (R. Raine). 1568-9883/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2011.04.015
capable of by-passing the cyst stage through division in the mobile stage (Figueroa et al., 2006). Some studies exist, however, which have involved examination of the dynamics of the transition between the cyst (zygote) and planktonic phase. For example, an endogenous clock has been observed in Alexandrium cysts sampled from deep water (Anderson and Keafer, 1987; Matrai et al., 2005) and temperature effects on excystment have been observed (e.g. Anderson, 1998). Knowledge of the dynamics of excystment is necessary in order to predict the initiation of Alexandrium blooms, a capability that is highly desirable since many of the species of this genus produce potent toxins which can give rise to paralytic shellfish poisoning (PSP) symptoms if contaminated filter-feeding bivalves such as oysters and mussels are consumed by humans (Hallegraeff, 2003). Around Ireland, the only location that has a history of repetitive PSP contamination of shellfish is Cork Harbour (Silke et al., 2006). Here, there is a resident population of Alexandrium minutum (Touzet et al., 2008). Shellfish harvesting from this estuary has been prohibited in most summers due to contamination with the gonyautoxins GTX2 and GTX3 which are typical of the toxin profile of A. minutum (Touzet et al., 2008). The timing of the blooms occurs in June and July, with planktonic cell densities usually >105 L1. Blooms of smaller intensity can occur later in the year, for example one achieving cell densities of 20,000 cells L1 occurred in September 2004 (Nı´ Rathaille, 2007).
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The present study comprised field and laboratory investigations of the excystment dynamics of A. minutum from Cork Harbour. Within the estuary, there is also a population of non-toxic A. tamarense. This form belongs to a group of five cryptic species designated by Scholin et al. (1994) as the Western European clade, now re-defined as in Group III of the species complex (Lilly et al., 2007). It co-exists with A. minutum in Cork Harbour, and the excystment dynamics of this species were also included in the study. 2. Methods 2.1. Seasonal regulation of excystment The method applied to investigate seasonal variation in excystment was a modified version of that used by Itakura and Yamaguchi (2001). On 12 occasions between March 2004 and May 2007, intertidal sediment was sampled from the North Channel of Cork Harbour (Fig. 1) using manually operated modified syringes as corers. On the day of sampling, the surface 10 mm layer was extruded, homogenised thoroughly and approximately 30 cysts of each species were extracted and isolated into individual wells of a 96-well plate with 200 mL of fresh sterile-filtered seawater in each well. The plate was incubated at a temperature as close (within 1.5 8C) to the in situ temperature of the seawater as possible. Irradiance levels in the incubator were approximately 15 mE m2 s1 and a 14:10 h light to dark cycle was used. Plates were examined for signs of excystment on a regular basis over a period of 30 days. Signs of excystment included the presence of at least one vegetative cell or the presence of an empty cyst. On two occasions (October 25th 2005
and March 28th 2006) sediment from a sub-tidal station (Fig. 1) was also sampled in order to compare the excystment pattern observed for inter-tidal cysts with that of sub-tidal cysts. On two occasions (October 7th 2006 and May 4th 2007), excystment measurements were supplemented by observations using sediment cores, thus eliminating the need to extract the cysts from the sediment. Three fresh sediment cores were taken from the intertidal sampling site. The top 1 cm layer of each core was removed, placed individually into the bottom of one of three 50 mL centrifuge tubes and 20 mL of fresh sterile filtered seawater was carefully added to each tube. The centrifuge tubes were incubated at the same temperature and light conditions as the 96-well plate, but in a Styrofoam holder such that only the top layer of the sediment was exposed to the light. Every 24 h the 20 mL of seawater was replaced with fresh sterile filtered seawater. The 20 mL that was removed was placed in an Utermo¨hl’s settling chamber, preserved with formalin and the entire contents examined for the presence of vegetative cells of Alexandrium sp. This continued every 24 h until three consecutive samples gave a zero result. 2.2. Endogenous clock regulation An investigation into the presence of an endogenous clock in Alexandrium cysts from Cork Harbour was initiated in 2002 using a method similar to that described in the experiments of Anderson and Keafer (1987) and Matrai et al. (2005) and described in detail in Anderson et al. (2003). Approximately 2 L of sediment known to contain Alexandrium spp. cysts was collected from the surface 5 cm at the intertidal station (Fig. 1) in the North Channel in early
Fig. 1. (a) Map of Cork Harbour, Ireland showing the location of the North Channel. (b) The North Channel, showing the location of the intertidal (A) and sub-tidal (B) sampling stations. Isobaths are in metres. The outline of land is the mean high tide level, whilst the mean low tide level is shown in (b) as the ‘0’ isobath, indicating the extensive area of intertidal mud-flats in the North Channel.
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October 2002. This sediment was homogenised and sub-divided into one hundred 9 mL bijou bottles which were stored in anoxic conditions in the dark at 4 8C. In January 2003, after a 4 month maturation period, one of the bijou bottles was removed from storage. Approximately 40 cysts of Alexandrium tamarense were isolated from the sediment and placed into individual wells of a 96well plate, with each well containing 200 mL f/2-Si medium (Guillard, 1975). The well plate was incubated at 15 8C for 30 days in an illuminated incubator (15 mE m2 s1 and a 14:10 h light to dark cycle) in similar fashion to the seasonal regulation experiment. Cysts in the plate were checked for signs of excystment on a weekly basis over the 30 day period using an Olympus CKX41 inverted binocular microscope. This procedure was repeated approximately every 2 weeks for an 18 month period, each time removing a new bijou bottle from the dark, anoxic storage conditions. The cysts isolated during this experiment varied in condition, but were always partially filled (in addition to the characteristic orange-red accumulation body). For the last 6 months of this period, the experiment was expanded to include cysts of A. minutum. 2.3. Temperature regulation of excystment The effect of temperature on the excystment dynamics of A. tamarense and A. minutum was investigated on cysts taken from the North Channel of Cork Harbour. Approximately 30 mature cysts of each species were isolated into each of five 96-well plates from the material stored for the endogenous clock experiment. This was carried out during February 2004. Each of these plates was then incubated at a specific temperature for a period of 30 days. The temperatures chosen were 5 8C, 10 8C, 15 8C, 20 8C and 25 8C which reflect the annual temperature range found in the North Channel. The light levels that the plates were exposed to were low, approximately 15 mE m2 s1, and the light:dark photoperiod was 14:10 h. Each plate was checked for signs of excystment, i.e. the presence of at least one vegetative cell or the presence of an empty cyst, every second day for the first 2 weeks, and approximately every 7 days for the remaining 2 weeks. Excystment rates for each species at each temperature were measured using Eq. (1), where N0 was the initial concentration of cysts, Nt was the concentration of cysts remaining at time t and k was the specific germination rate with units of day1 (Anderson et al., 1987). Nt ¼ N 0 expðktÞ
(1)
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2.4. Cyst maturation Cyst maturation times were investigated on newly formed cysts of both A. tamarense and A. minutum. Newly formed cysts were obtained by filling a 25 L drum with fresh seawater collected from the north Channel of Cork Harbour during the peak of the June 2004 bloom. To this was added plankton material obtained with a 20 mm mesh phytoplankton net, and the mixture was then spiked with f/80-Si medium (Guillard, 1975). The seawater drum was kept out of direct sunlight and at room temperature for 20 days until fresh resting cysts were observed to have formed and settled to the bottom of the drum. These cysts were collected and stored using the methods outlined in Anderson (1980) and Anderson et al. (2003). Aliquots of cysts were put into 9 mL bijou bottles. A layer of 20 mm mesh was put over the neck of the bijou bottle and the lid, with a small circular hole in it, was screwed on tightly. Sixty such bijou bottles were prepared and placed in anoxic conditions by submerging them in a jar of anoxic mud taken from a location (Galway Bay) where no cysts had been observed; 15 each at 5 8C, 10 8C, 15 8C and 20 8C. Every 3 weeks one bijou bottle was removed from each temperature condition and 30 cysts of each species were isolated from each bottle. The cysts were removed to a 96-well plate and incubated in 200 mL of f/2-Si medium (Guillard, 1975) at 15 8C. The plates were then periodically checked for signs of excystment. Cyst maturation time was defined as the time needed for 50% of the cysts to germinate (Binder and Anderson, 1987). 3. Results 3.1. Seasonal regulation of excystment Results showing the excystment success of freshly sampled cysts of A. tamarense and A. minutum incubated at in situ temperatures over a 3 year period are shown in Fig. 2. The general pattern was the same for both species showing clear spring maxima and autumn minima. Maximum values of between 73– 96% success for A. tamarense and 60–100% success for A. minutum were obtained between March and June over the 30 day incubation period used. Minimum values of 0–14% were obtained for both species between the months of August and October. The spring maxima coincided with a water column temperature of approximately 10 8C. Minimum excystment success occurred during the time that the water column temperature was decreasing from its annual maximum (20 8C) in August to 10 8C about 4 months later.
Fig. 2. Seasonal variation in the excystment success of freshly sampled A. tamarense (*) and A. minutum (*) cysts from the North Channel of Cork Harbour over the period March 2004 to May 2007. The seawater temperature (8C) recorded over the same time period is shown as the background hairline. Sampling locations are given in Fig. 1.
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Table 1 A comparison of the excystment success of A. tamarense and A. minutum from intertidal and subtidal sediments sampled during October 2005 and March 2006. The location of sampling stations is shown in Fig. 1. Date
Species
% Excystment Intertidal
Subtidal
25 October 2005
A. tamarense A. minutum
7 (n = 30) 0 (n = 25)
0 (n = 30) 0 (n = 27)
28 March 2006
A. tamarense A. minutum
70 (n = 30) 60 (n = 25)
76 (n = 30) 75 (n = 24)
These results strongly indicate that the seasonal patterns for both species appear to be independent of a temperature function. The seasonal signal in excystment did not appear to be due to either the intertidal nature of the samples or the cyst isolation process. When excystment success was compared between intertidal samples and sub-tidal samples at times when minimum and maximum success was expected in October and March, respectively, success rates were very similar (Table 1). In October 2005, 0% excystment was observed for A. minutum in both intertidal and sub-tidal samples. The pattern for A. tamarense was very similar, with 7% excystment in the inter-tidal sample and 0% excystment in the sub-tidal sample. In March 2006, 60% excystment was observed for A. minutum in the inter-tidal sample with 75% excystment in the sub-tidal sample. The pattern for A. tamarense was again similar, with 70% excystment in the intertidal sample and 76% excystment in the sub-tidal sample. These results suggest that there is no difference between the excystment dynamics of cysts from inter-tidal sediment and those from subtidal sediments. The seasonal trend in excystment was also observed in incubations with untreated sediment. No vegetative cells were observed in the overlying water of sediment samples taken and incubated in October 2006. In May 2007, however, excystment from freshly sampled sediment occurred over a period of 2 weeks. The cumulative numbers of vegetative cells observed in the overlying water of sediment samples at this time is shown in Fig. 3. These results further substantiate the original observations of seasonality in excystment.
Fig. 4. Excystment success of mature cysts of (a) A. tamarense (filled circles) and (b) A. minutum (open circles) stored in cold, dark anoxic conditions as a function of storage time. Each month, 30 cysts were isolated and incubated at 15 8C. Trendlines show a linear regression analysis of the data. The arrow marks the date that the temperature effect experiment described in Section 2.3 took place.
3.2. Endogenous clock regulation Results of investigations into the presence of an endogenous clock in cysts of A. tamarense and A. minutum from Cork Harbour are shown in Fig. 4. These results spanned an 18 month period from
Fig. 3. The cumulative numbers of vegetative cells of Alexandrium spp. observed with time in the water overlying sediment samples taken in May 2007. Results are presented for each of the triplicate tubes used in the experiment (Tube 1: filled circles; Tube 2: open circles; Tube 3: half-filled circles). Experimental details are given in the text.
Fig. 5. Excystment dynamics of (a) A. tamarense and (b) A. minutum over the range of temperatures encountered in the North Channel of Cork Harbour, Ireland. (c) The excystment success of A. tamarense and A. minutum after 30 days as a function of temperature derived from the results shown in (a) and (b).
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January 2003 to July 2004 for A. tamarense and a 6 month period from January 2004 to July 2004 for A. minutum. There was no evidence of a seasonal pattern of excystment that would have confirmed the presence of an endogenous clock for either species, although the data for A. minutum cover too short a time-span to be conclusive. The average excystment success for the experiment was 74%. For the 2003 data alone (i.e. the first 12 months of the experiment), the average excystment success was 79% for A. tamarense. The lowest value recorded was 40% excystment in July 2004. 84% of all the values recorded showed excystment success greater than 60%. Average excystment success for A. minutum was 87%. Regression analysis of both data sets indicated a general decrease in excystment success as the experiment progressed. 3.3. Temperature regulation of excystment Fig. 5a and b shows the effect of temperature on the rate of excystment of A. tamarense and A. minutum, respectively. The cysts were taken from the stock used for the endogenous clock experiment (see Fig. 4). Results indicated that excystment for each species occurred earlier over the upper range of temperatures although at the highest temperature of 25 8C, the excystment of A. tamarense was slightly reduced from that measured at 20 8C. No effect above 15 8C was observed on the excystment of A. minutum. When the excystment success of each species is plotted against temperature (Fig. 5c), then A. tamarense shows a maximum (77%) at 15 8C, with reduced success at the higher and lower temperatures, whereas that of A. minutum was more or less equal over the entire range of temperatures investigated. Table 2 shows the calculated excystment rates for each species at each of the temperatures investigated. Generally excystment rates of A. minutum (0.05–0.38 day1) were higher than those of A. tamarense (0.01–0.18 day1). A. tamarense excystment rates showed an optimum at 15 8C (0.18 day1), whereas the A. minutum excystment rates increased linearly with temperature. 3.4. Cyst maturation Fig. 6 shows a summary of the relationship between maturation times of newly formed cysts plotted against storage temperature. It can be seen that the maturation time was 66 days for newly formed cysts of A. tamarense stored at 5 8C, 58 days for those stored at 10 8C and for those stored at 20 8C, maturation time was 22 days. The maturation time was longer for newly formed cysts of A. minutum stored at lower temperatures, 112 days at 5 8C and 83 days for those stored at 10 8C. An additional value of up to 20 days could be added to these values. This is because there was a period of 20 days between the time when f/80-Si medium was added to the drum and the day (day 0 on Fig. 6) when the cysts were collected from it.
Fig. 6. Maturation time of freshly formed A. tamarense (filled circles) and A. minutum (open circles) cysts as a function of storage temperature. Maturation time is defined as the time taken for 50% of the cysts to excyst.
4. Discussion The results presented have shown a pronounced seasonality in the excystment behaviour of A. minutum and A. tamarense, an effect which was independent of temperature. No endogenous clock was evident in the germination of either species. Temperature did, however, affect the rate of excystment, and also affected the maturation rate of cysts which had been freshly formed in the laboratory. Excystment of Alexandrium spp. cysts from Cork Harbour was not inhibited over the annual range of in situ temperatures observed. This would suggest that the local cysts are adapted to excyst at any time of the year, even if conditions for growth are not suitable. Similar results were found for A. minutum cysts from the Fal Estuary in England (Perez-Blanco, 2005). Excystment experiments conducted on these cysts which had been stored at 4 8C for at least 1 month resulted in no inhibition of excystment at temperatures between 4 8C and 24 8C. Cysts of A. tamarense sampled in April (1991) from the St. Lawrebce estuary and stored for 2 weeks in the dark excysted over the entire range of temperatures (1–16 8C) subsequently applied. Anderson (1998) presents evidence for a temperature window for excystment of A. tamarense cysts from Perch Pond, USA. This window (5–21 8C) represents the range of temperatures within which excystment will occur but outside of which temperature completely inhibits excystment causing mature cysts to remain in a quiescent state. Other experiments on A. tamarense cysts from Perch Pond have shown that cysts stored at extremes of this window at 5 8C and 22 8C will excyst only when the temperature is raised or cooled, respectively (Anderson and Morel, 1979).
Table 2 Excystment rates for A. tamarense and A. minutum at each of the 5 temperatures investigated and the data used to calculate them (data in parentheses are the maximum percentage germination recorded during the experiment for the species at that temperature and the day this maximum was recorded. These data were not used in the calculation of excystment rate). Temperature (8C) A. tamarense N0 % germination t (d) Nt k (d1) A. minutum N0 % germination t (d) Nt k (d1)
5
10
15
20
35
29 28 (28) 30 (30) 21 0.01
29 47 (55) 16 (30) 15 0.04
30 77 (77) 8 (8) 7 0.18
30 60 (63) 8 (10) 12 0.11
30 40 (43) 4 (8) 18 0.13
30 67 (77) 22 (30) 10 0.05
30 70 (77) 10 (22) 9 0.12
30 87 (87) 6 (6) 4 0.34
30 86 (90) 6 (8) 4 0.33
30 90 (93) 6 (6) 3 0.38
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Extrapolation of laboratory results of temperature effects on excystment to natural conditions has been discussed by several authors. One chief problem has been the term applied to excystment. A common measure has been excystment success, which represents the proportion of a cyst population that will excyst in a given time period. This term does not, however, take into account the speed or rate of excystment. A problem making direct comparisons of excystment success from the literature is that investigations often differ in the incubation time applied for the experiment. Changes in incubation time can influence the result. For example, it is quite probable that in the methods applied here, increasing the incubation time would have yielded a higher excystment success at 5 8C for A. tamarense. Excystment success of cysts of A. tamarense from Cork Harbour indicated a temperature effect with the greatest proportion of cysts (77%) excysting at 15 8C after a 30 day incubation period. Excystment success was lowest at the temperature extremes (28% at 5 8C and 43% at 25 8C). Anderson (1998) reported equal success (almost 100%) for cysts of A. tamarense from Perch Pond at temperatures between 9 8C and 21 8C, using an incubation time of 3 weeks. At temperatures between 6 8C and 8 8C, excystment success was somewhat lower (50%). Castell Perez et al. (1998) reported equal success (>80%) for cysts of A. tamarense from the Lower St. Lawrence Estuary at temperatures between 2 8C and 16 8C, with slightly lower success (50%) at 1 8C. In that investigation, cysts were incubated for 38 days. Excystment of cysts of A. minutum presented here showed no temperature effect with almost equal success (>75%) at each temperature. These results contrast with data for the species from Australia (Cannon, 1993), where a marked temperature effect was observed with maximum percentage excystment (55%) occurring at 16 8C and lower excystment success (15% and 23%) occurring at 12 8C and 20 8C, respectively, after a 10 day incubation time. On the other hand, the results found here compare well with experiments carried out on specimens taken from the Fal Estuary, England, where excystment success after 15 days showed no signs of being affected by temperature (Perez-Blanco, 2005). High percentage excystment (>80%) was noted by Perez-Blanco (2005) by the fourth day of the experiment. All these experiments were carried out on cysts which had been stored for periods in excess of 2 weeks in cool (4 8C) and dark conditions. Given the inherent problems when comparing excystment success data with different incubation times when no data showing the progression of the experiment with time has been presented, calculating the excystment rate at each temperature would be a more reliable way to compare excystment dynamics between one area and another. Using the data for A. minutum cysts from Cork Harbour as an example, excystment success showed no temperature effect; however, excystment rates based on the maximum percentage excystment and time when maximum percentage excystment was recorded, revealed a distinct relationship with temperature. Lower temperatures (5 8C) resulted in lower excystment rates (0.05 day1) and higher temperatures (25 8C) resulted in higher excystment rates (0.38 day1). Similarly, Blanco et al. (2009) reported for A. minutum a slightly longer time taken for 50% germination to occur at 4 8C than at 24 8C (1.5 days compared with 1.1 days), despite the excystment success being equal at all temperatures. The results of the experiments showing the effects of temperature on Alexandrium spp. excystment suggest that these cysts are adapted to excyst at any time of the year. However, when fresh cysts were isolated and incubated at in situ temperatures, a clear seasonal pattern was observed over a 3 year period. This pattern showed seasonal maxima occurring in late spring and seasonal minima occurring in late autumn when water temperatures were not very different. Similar seasonal patterns have been
reported for A. tamarense cysts from Hiroshima Bay, Japan (Itakura and Yamaguchi, 2001) and Masan Bay, Korea (Kim et al., 2002). There are many similarities between the results of the Masan and Hiroshima Bay studies and those obtained from Cork Harbour. These include the temperature pattern and the timing of the vegetative cell blooms which occurred in late spring or early summer when water temperatures reach 15 8C. The time difference between the occurrence of maximum excystment and the occurrence of maximum vegetative cell densities differed slightly, being approximately 2 months in the case of Hiroshima Bay and Cork Harbour, but closer to 4 months in the case of Masan Bay. In Hiroshima Bay, highest excystment success (>50%) occurred between December and April as water temperatures decreased from 16.5 8C to the annual minimum of 10 8C. Low excystment success (<50%) occurred between May and November with no excystment occurring when water temperatures were at their annual maximum (ca. 23.5 8C) in September. In Masan Bay, similarly high excystment success (>50%) also occurred as water temperatures decreased to the annual minimum 6 8C between November and February. Excystment success was low during March and April when water temperatures began to increase, and was almost negligible between May and October, as water temperatures increased towards the annual maximum of 22 8C. In the cases of both Hiroshma Bay (Itakura and Yamaguchi, 2001) and Masan Bay (Kim et al., 2002), the authors proposed a temperature window for germination between 10 8C and 15 8C coupled with the mandatory dormancy of newly formed cysts to explain their observations. No data for the mandatory dormancy period of local cysts were presented in either case. In this study there is certainly a strong argument for mandatory dormancy preventing excystment during the period immediately after bloom decline. The dormancy period for Cork Harbour cysts would, however, only seem to be sufficient to explain the low excystment success for a period of about 2 months after the demise of the observed blooms (end of June). This would imply that by September, most cysts should be through their mandatory dormancy period and ready to excyst once again. This, however, did not occur, despite favourable water temperatures and the ability of cysts of both A. tamarense and A. minutum to excyst over the entire temperature range experienced in Cork Harbour. No seasonal signal that would have indicated endogenous control of excystment was observed in either A. tamarense or A. minutum cysts taken from Cork Harbour. Endogenous control of excystment has been reported for A. tamarense from the Gulf of Maine (Anderson and Keafer, 1987; Matrai et al., 2005). It has also been suggested for cysts of A. tamarense from the Lower St. Lawrence estuary in Canada (Castell Perez et al., 1998), but further work was required there to confirm its presence. No endogenous control of excystment has been reported for cysts of A. minutum. Endogenous control of excystment would be a distinct ecological advantage for cysts that occur in deeper areas of the ocean where environmental cues such as temperature and photoperiod do not exist. A biological clock would be able to cue such cysts to excyst at a time of year when surface water temperature and irradiance levels are suitable for growth. Both the Gulf of Maine cysts and the St. Lawrence Estuary cysts were sampled from such depths (60–160 m in the case of the Gulf of Maine). By contrast, the Cork Harbour cysts that were used in this investigation were sampled from intertidal sediment of the North Channel, a shallow water area less than 10 m deep. These cysts are exposed to a very broad range of environmental variables such as temperature and irradiance and, as such, would not benefit from the presence of an endogenous clock. Anderson and Keafer (1987) have also reported the lack of an endogenous clock for cysts from a shallow estuary. Given the seasonal pattern observed over a 3 year
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period, despite the lack of an endogenous clock, other factor(s) must therefore be controlling cyst germination in Cork Harbour. Secondary dormancy which prevents germination during periods that are favourable for germination but are not favourable for growth has been reported to occur in the seeds of higher plants (Vleeshouwers et al., 1995; Bewley, 1997). This results in seeds being alternately responsive and unresponsive to the same environmental conditions. Such a phenomenon, or another as yet undescribed process which results in the same effect, would explain why cysts that germinate in Cork Harbour in the environmental conditions of spring are prevented from germinating in autumn. This rhythm generally disappears with storage in constant conditions (Karssen, 1980/81) which would be one possible reason why it did not emerge during the endogenous clock experiment, where sediment had been stored in constant conditions for a period of 4 months prior to the beginning of the experiment. Anderson and Keafer (1987) discounted the secondary dormancy hypothesis for deep water cysts of A. tamarense from Cape Cod due to the multi-year persistence of the germination cycle under constant storage conditions, instead proposing the endogenous clock theory to explain the seasonal germination pattern. In contrast to the deep water environment which has a nearly invariant nature, environmental signals vary considerably in shallow systems. Shallow water cysts might be better served by germination cued to the environment through secondary dormancy. It is highly desirable to predict the occurrence of toxic dinoflagellate blooms in order allow mitigation of their economically damaging effects to aquaculture. Although many harmful algal blooms are caused by transport of toxic populations into areas of shellfish production, the Alexandrium population in the North Channel of Cork Harbour is a resident population, first reported by Gross (1988). Blooms of planktonic toxin-producing cells of A. minutum logically initiate from excystment of mature cysts. Blooms are therefore less likely to arise during seasonal minima in the excystment of this species. Marked seasonality in the germination of toxic dinoflagellates must be incorporated into coupled physical biological models designed to predict blooms. Thus the endogenous clock observed in cysts from the Gulf of Maine is incorporated into the Alexandrium model described by McGillicuddy et al. (2005). The seasonality in excystment observed in Cork Harbour should likewise be incorporated into models describing its growth here. Acknowledgements The authors wish to acknowledge the assistance of David HughJones, Brian Byrne and Donie Geary for the use of their facilites and for the provision of boats during the fieldwork carried out in this study. Thanks are also due to Georgina McDermott, Mairead Murphy and Nicolas Touzet for their help in the laboratory. This work was funded by an RTDI Scholarship awarded to Aoife Nı´ Rathaille by the Marine Institute under the National Development Plan (2000–2006) and also by the EC 6th Framework Programme (SEED project: GOCE-CT-2005-003375). [SS] References Anderson, D.M., 1980. Effects of temperature conditioning on development and germination of Gonyaulax tamarensis (Dinophyceae) hypnozygotes. Journal of Phycology 16, 166–172. Anderson, D.M., 1998. Physiology and bloom dynamics of toxic Alexandrium species, with emphasis on life cycle transitions. In: Anderson, D.M., Cembella, A.D.,
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