Hydrobiologia 453/454: 107–120, 2001. R.M. Lopes, J.W. Reid & C.E.F. Rocha (eds), Copepoda: Developments in Ecology, Biology and Systematics. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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Feeding, egg production, and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens Jean A. Lincoln1, Jefferson T. Turner1,∗ , Stephen S. Bates2 , Claude L´eger2 & David A. Gauthier1 1 Department
of Biology and Center for Marine Science and Technology, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, U.S.A. E-mail:
[email protected] 2 Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick, Canada E1C 9B6 (∗ Author for correspondence) Key words: copepod feeding, domoic acid, Pseudo-nitzschia multiseries
Abstract In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudonitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female−1 d−1 . There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.
Introduction The possibility of a global increase in toxic marine phytoplankton blooms (Anderson, 1989; Smayda, 1989; Hallegraeff, 1993) has focused increased interest on effects of phytoplankton toxins in food webs. Accumulation of phytoplankton toxins in shellfish
with potential poisoning of humans (Shumway, 1990) and fish kills (Steidinger, 1983) are well known. Less well understood are interactions between toxic phytoplankton and their primary grazers, the zooplankton (reviewed by Turner & Tester, 1997; Turner et al., 1998a). Zooplankton can provide the entry point for trophic transport of phytoplankton toxins through con-
108 sumer food webs, with vectorial intoxication and mortality of fish, marine mammals and seabirds (White, 1979, 1981; Anderson & White, 1992). Variations in zooplankton population grazing pressure can also be important in allowing development or termination of toxic phytoplankton blooms (Turner & Anderson, 1983; Uye, 1986). If phytoplankton toxins deter potential grazers such as copepods, then these toxins might promote blooms of toxic phytoplankton species. Effects of phytoplankton toxins on copepods and other zooplankters are varied and situation-specific (Uye & Takamatsu, 1990; Teegarden & Cembella, 1996; Turner et al., 1998b). Some grazers are deleteriously affected by phytoplankton toxins, whereas for others there are no apparent effects (Turner & Tester, 1989). Potential adverse effects of phytoplankton toxins on copepods might include reduced grazing, egg production, egg hatching and survival. Recent studies have revealed contradictions, such as the results of Turriff et al. (1995) showing that Calanus finmarchicus ingested few of the toxic Alexandrium excavatum, yet the copepod still accumulated toxins from this dinoflagellate. However, there has been comparatively little investigation of such interactions with other copepods and toxic phytoplankton. The phytoplankters most often associated with toxicity have been dinoflagellates or other phytoflagellates. Diatoms were not known to be toxic until 1987, when a bloom of the diatom Pseudo-nitzschia multiseries (formerly known as Nitzschia pungens f. multiseries) was found responsible for a shellfish toxicity event at Prince Edward Island (P.E.I.), Canada. Approximately 150 people became ill, and 5 people died after the consumption of tainted mussels (Perl et al., 1990). Symptoms from the shellfish toxicity included vomiting and diarrhea, followed in some cases by confusion, disorientation and short-term memory loss (Todd, 1993). Such symptoms helped to coin the term, ‘amnesic shellfish poisoning.’ The neurotoxin responsible was identified as domoic acid (DA) (Wright et al., 1989), which can be responsible for degeneration of the hippocampal regions of the brain (Debonnel et al., 1989). Other DA outbreaks have subsequently occurred since the 1987 P.E.I. episode (Villac et al., 1993), and closures of shellfish harvesting areas as recently as 1994 indicate that the problem is still present. Another DA-producing bloom at P.E.I. occurred in 1988 (Smith et al., 1990), and subsequent DA incidents in Monterey Bay, California, U. S. A. caused the deaths of pelicans and cormorants in September, 1991 (Fritz
et al., 1992; Work et al., 1992), and sea lions in May and June, 1998 (Scholin et al., 2000). Mortality of the birds and sea lions was traced to the ingestion of anchovies, which had been feeding on Pseudonitzschia australis, a similar toxic species to the one causing the P.E.I. toxicity events. However, no human fatalities were reported. These episodes were the only examples of transmission of toxic levels of domoic acid to vertebrates through a planktivorous fish vector. Unlike the many grazing studies conducted with toxic dinoflagellates, there has been almost no investigation of copepod grazing on toxic diatoms of the genus Pseudo-nitzschia. Bates et al. (1989) suggested that the neurotoxic properties of domoic acid (Maeda et al., 1984) may have been a contributing factor to the longevity of the bloom. Domoic acid is highly toxic to mammals, and is used as an insecticide in Japan (Wood & Shapiro, 1992). Thus, domoic acid may function as a grazing deterrent to zooplankton. Conversely, if domoic acid is not a deterrent to zooplankton grazing, zooplankton may facilitate vectorial transmission of the toxin, which could have severe impacts on the marine ecosystem, as well as on the maritime economy and public health. The non-toxic species Pseudo-nitzschia pungens is morphologically similar to, and coexists with the toxic Pseudo-nitzschia multiseries in coastal waters of Atlantic Canada, but P. pungens does not produce domoic acid. These toxic and non-toxic species are ideal for zooplankton feeding studies, since both diatoms are the same size, thereby reducing complications from preferential or discriminatory size-selective grazing (Windust, 1992). Windust (1992) exploited the similarity between the toxic and non-toxic species, and explored the hypothesis that domoic acid inhibits grazing using the copepods Temora longicornis, Pseudocalanus acuspes and Calanus glacialis. Windust used domoic acid concentrations three orders of magnitude higher than the highest domoic acid concentrations of the 1987– 1988 P.E.I. bloom. Although such high concentrations may be considered ecologically irrelevant, ingestion of high doses, surprisingly, had no deleterious effects on copepod feeding rates, behaviour, or survival. Thus, Windust’s work did not support the hypothesis that phytoplankton toxins repel grazing. However, the copepods sequestered the toxin in the tissues long after gut evacuation. Toxins concentrated in the copepods may potentially be transferred to other organisms at higher trophic levels.
109 The results of Windust (1992) suggest that domoic acid has no obvious, adverse effects on the adult populations of species of Temora, Pseudocalanus or Calanus. However, if copepod fecundity is related to quantity and quality of food, a toxic diatom diet might affect the reproductive phase of the life cycle. Thus, ingestion of toxic diatoms (as well as of any other toxic phytoplankton species) may interfere with reproduction, thereby reducing the next generations of grazers. This could be caused by phytoplankton toxins reducing egg production, or by the production of non-viable eggs. Most studies reporting decreases in egg production in relation to toxic phytoplankton suggest that decreases in egg production are an effect of low ingestion rates (Huntley et al., 1986; Uye, 1986; Buskey & Stockwell, 1993). Few studies have examined relationships between ingestion of toxic phytoplankton and low copepod egg hatching rates (Turner, Tester & Hansen, 1998; Turner, Lincoln & Cembella, 1998). This is despite indications as reviewed by Ianora (1998), that chemicals in certain diatom diets, while allowing normal rates of copepod egg production, can cause reduced success of copepod egg hatching. This investigation addressed the following questions: 1. does toxicity in Pseudo-nitzschia multiseries function as a grazing deterrent, thereby reducing the ingestion rates of the coastal calanoid copepods Acartia tonsa and Temora longicornis, and; 2. upon ingestion, is toxic Pseudo-nitzschia multiseries able to impair the reproductive efforts of A. tonsa and T. longicornis?
Materials and methods Experiments were conducted at the Gulf Fisheries Centre, Department of Fisheries and Oceans, Moncton, New Brunswick, Canada, in July 1994, June 1995, August 1995 and August 1996. Phytoplankton cultures and domoic acid analyses All isolates of P. multiseries and P. pungens were cultured and maintained in the laboratory, using procedures of Bates et al. (1991), Bates et al. (1993a), Bates et al. (1993b), Bates et al. (1995) and Bates et al. (1996). At the start of each experimental series, domoic acid levels in P. multiseries cultures were quantified. Since domoic acid leaches from cells into
culture medium, it was necessary to distinguish domoic acid in cells from domoic acid in solution in the culture medium. Thus, in addition to freezing 15 ml of culture for analyses of intracellular domoic acid plus dissolved domoic acid which had leaked into the culture medium (‘whole culture’ measurements), 100 ml of each culture was filtered onto 3.0 µm pore Nucleopore filters, rinsed with filtered seawater and frozen for later domoic acid analyses (‘intracellular’ or ‘filtered’ measurements). Since leakage of domoic acid from cells into cultures increases with time, levels of intracellular domoic acid can vary greatly with culture age. Toxin analyses by High Performance Liquid Chromatography (HPLC) were performed following the protocols of Pocklington et al. (1989) and Bates et al. (1991). Concentrations of cells in cultures were determined by microscopic counting with a SedgwickRafter cell. We determined domoic acid concentrations per cell for suspensions of P. multiseries used in feeding experiments, knowing the total amount of domoic acid in samples of cells from 100 ml of culture, and the number of cells ml−1 in the same culture. Similar analyses were performed on cultures of P. pungens, to confirm that they produced no domoic acid. Zooplankton collection and preparation Zooplankton were collected with 363 µm-mesh nets in Shediac Bay and the Northumberland Strait, approximately 35 km northeast of Moncton. Nets were towed for 1–2 min, and live zooplankton samples were transported to the laboratory within 2 h of tow time. The dominant copepod in most tows throughout summer sampling in 1994–1996 was Acartia tonsa, an abundant omnivorous coastal species in nearshore warmer waters of Atlantic Canada during the summer (Scott, 1907; McAlice, 1981; Citarella, 1982). Another abundant species was Temora longicornis, an omnivorous coastal copepod, which was used in 1994 and 1995. Adult female copepods were sorted from net tows with pipettes using a Wild M5 dissecting microscope. The copepods were then placed into 1-l plastic jars with algal food suspensions made from dilutions of unialgal cultures to approximate various experimental concentrations. The concentrations used in experiments were intended to bracket the naturallyocurring bloom concentrations of Pseudo-nitzschia spp. Cell concentrations in the food suspensions were
110 Table 1. Acartia tonsa grazing experiments, 1994–1996, for diets of toxic and non-toxic diatom cultures, with food concentrations that were not significantly different (p-value for t-test comparing food concentrations >0.05). F is filtration rate (ml copepod−1 h−1 ) and I is ingestion rate (cells copepod−1 h−1 ). Each F and I value is a mean of triplicate replicates, ± standard deviation. If the p-value for the t-test comparing ingestion rates was >0.05, then ingestion rates were significantly different for toxic and non-toxic diets. In the 25 July, 1994 experiment, cultures were added to natural sea water (NSW), whereas for all others cultures were unialgal Date
Food concentration cells l−1 Toxic Non-toxic
P-value for Toxic T-test of food value (standard deviation) concentrations F I
3.25×105 + 2.70×105 + 0.087 NSW NSW 4.07×105 0.142 7 Jun 95 3.65×105 4.34×106 0.262 29 Aug 95 3.45×106 26 Aug 96 1.64×106 2.29×106 0.211 25 Jul 94
0.48 (±0.10) 169.12 (±32.93)
Non-toxic value (standard deviation) F I
P-value for T-test of ingestion rates
0.12(±0.09)
0.012a
32.06(±24.65)
0.25 (±0.13) 65.58 (±27.50) 0.35 (±0.03) 100.40 (±5.45) 0.212 0.40 (±0.09) 925.51 (±137.40) 0.15 (±0.01) 540.89 (±39.18) 0.051 0.17 (±0.07) 251.49 (±100.24) 0.017 (±0.01) 386.14 (±18.62) 0.242
a Significant difference.
Table 2. Temora longicornis grazing experiments, 1994, for diets of toxic and non-toxic diatom cultures, with food concentrations that were not significantly different (p-value for t-test comparing food concentrations >0.05). F is filtration rate (ml copepod−1 h−1 ) and I is ingestion rate (cells copepod−1 h−1 ). Each F and I value is a mean of triplicate replicates, ± standard deviation. If the p-value for the t-test comparing ingestion rates was >0.05, then ingestion rates were significantly different for toxic and non-toxic diets. In the 22 July, 1994 experiment, cultures were added to natural sea water (NSW), whereas for all others cultures were unialgal Date
Food concentration P-value for cells l−1 T-test of food Toxic Non-toxic concentrations F
22 Jul 94 1.21×105 22 Jul 94 1.93×106 25 Jul 94 3.25×105 +NSW
1.02×105 0.146 2.19×106 0.238 2.70×105 0.087 +NSW
Toxic value (standard deviation) I
F
Non-toxic value (standard deviation) I
P-value for T-test of ingestion rates
0.17 (±0.19) 17.76 (±19.54) 0.76 (±0.35) 58.81 (±24.73) 0.139 0.99 (±0.34) 2201.76 (±617.27) 0.87(±0.45) 1630.18 (±772.16) 0.459 0.11 (±0.09) 42.82 (±34.14) 0.11 (±0.08) 31.34 (±22.03) 0.804
dependent upon amounts of available culture, and are summarized in Tables 1 and 2. Experimental grazing protocols Copepods were preconditioned on experimental food suspensions for 13–22 h, usually overnight. Preconditioning was necessary because of the concurrent use of these specimens in egg production and hatching experiments. Tester & Turner (1990) found a lag of at least 9.5 h for A. tonsa to convert ingested food into egg production. Preconditioning and experimental incubations were at ambient field temperatures at times of collection, 19 ◦ C in June 1994, 14 ◦ C in June 1995, 17 ◦ C in August 199 and 20 ◦ C in August 1996. Temperatures were controlled in an incubation chamber. After preconditioning, diatom suspensions were prepared from cultures for use in experiments. Microscopic counts of algal cultures established initial cell concentrations, and these were diluted with filtered
seawater to make experimental unialgal food suspensions of P. pungens and P. multiseries bracketing natural bloom concentrations. In addition to unialgal diatom suspensions, we also performed mixed-diet treatments. Inocula of an intended food concentration of the toxic diatom and the non-toxic diatom were added to natural seawater with naturally occurring assemblages of phytoplankton. Essentially, the mixed-diet mean ingestion rates of the toxic diatom and non-toxic diatom were to be compared with the monospecific-diet mean ingestion rates. The intent of the mixture experiments was to test the null hypothesis that the ingestion rates on the toxic diet versus the non-toxic diet would be similar in the presence of alternative food sources compared to ingestion rates on a monospecific diet. The naturally occurring assemblages were not enumerated. Two independent experiments were prepared to separate effects on copepods of feeding on toxic P. multiseries and non-toxic P. pungens. The calculated cell concentrations were intended to provide equal
111 concentrations of toxic and non-toxic food suspensions. The food suspensions were placed into 1-l plastic jars. Each grazing experiment consisted of three treatments: one or two initial ungrazed jars, one or two control jars and three to five replicate grazing jars. The number of treatments varied from year to year according to availability of cultures. The initial ungrazed jar was preserved at the onset of the experiment with Utermöhl’s iodine solution (Guillard, 1973). The control jar allowed for the growth of Pseudo-nitszchia spp. during experimental incubations. The control contained only the designated food suspension without copepods, and was preserved simultaneously with the replicate grazing jars at the end of experimental incubations. The grazing jars contained either 5, 10 or 20 copepods per jar, depending on the sizes of the copepods and the volume of suspension (100–800 ml). All animals were sorted live with a Pasteur pipette, using a dissecting microscope. Only actively swimming individuals were selected for the grazing experiments. Active swimming was confirmed during both the initial sorting for preconditioning, and the final sorting for the timed grazing experiment. Mortality of females throughout the 14–26 h grazing experiments on toxic and non-toxic diets was (0–23.3% (mean = 10.4%). Animals that died during incubations were calculated to have fed for half of the experimental period. Jars were incubated on an orbital shaker at 43– 44 rpm in the dark during the experimental period, to prevent sedimentation of diatoms. Following the incubations for grazing experiments, aliquots were always visually inspected to note activity of the animals. At the conclusion of the grazing experiments, control and grazing containers were preserved with Utermöhl’s iodine solution, and concentrated for microscopic counting by gravimetric sedimentation for at least 72 h. The supernatant was frequently allowed to settle and examined for cells, and very few were found. Three replicate 1-ml subsamples were counted with a 1 ml Sedgwick-Rafter chamber. Cells were counted at 250 × magnification with an Olympus model BH-2 microscope. A total of 500 cells was always counted from each replicate, in an attempt to form a statistically robust estimate of the total number of phytoplankton cells in a sample, often necessary to detect small changes in cell concentrations due to grazing. These cell counts were slightly in excess of the 400-cell criterion based on the work of Gossett (1907), which yields estimates of the total phytoplank-
ton abundance of ±10% accuracy at the 95% confidence level for the total phytoplankton count (Guillard, 1973; Edler, 1978). However, more recent work by Edgar & Laird (1993) found that because some of the assumptions of the Poisson distribution are violated, particularly in the Sedgwick-Rafter counting chamber, the error may increase an additional 5–15%. Means were calculated from the triplicate counts of all the initial samples, controls and grazed samples. The means were then used to determine ingestion and filtration rates, according to the equations of Frost (1972). Egg production and egg hatching success experimental protocols All egg experiment jars contained the same diatom concentrations as the grazing experiment jars for the diatom diets, since both sets of diatom food mixtures were prepared simultaneously. Egg production and egg hatching success experiments were carried out concurrently with the grazing experiments, in order to have animals subjected to similar laboratory conditions (e.g. temperature, length of captivity). These experiments were carried out in June and August 1995 with A. tonsa and T. longicornis, and in August 1996 with A. tonsa only. No egg production experiments were conducted in 1994. Females (usually 5, 10 or 20) plus males (1 or 2) were placed in 100 ml capacity clear plastic jars for 14–24 h. Males were added to prevent decreased egg production in the absence of re-mating (Parrish & Wilson, 1978; Kiørboe, 1989). At the end of the experimental incubations, copepods were separated from the eggs by pouring suspensions into petri dishes. This was relatively easy, since the copepods swam toward the tops of the jars when laboratory lights were turned on. Any eggs that were inadvertently removed were pipetted back into the experimental egg containers. Eggs were seldom decanted, as they generally sink quickly. Once the copepods were removed with pipettes, mortality was assessed. Dead or moribund animals were counted in order to adjust the calculation of eggs produced per animal, assuming that they were alive for half the incubation period. All animals were then discarded. The eggs were counted immediately under a dissecting microscope, usually by viewing through the water column towards the bottom of the clear plastic jars, and left in the jars for hatching. After approximately 17–24 h, the numbers of unhatched eggs were coun-
112 ted and subtracted from the total, for determination of percent hatching. There were no screens in the experimental jars to separate adult copepods from liberated eggs, to prevent egg cannibalism. We decided not to use screens because of the possibility of damage to eggs by scraping as they were passing through the screen. Determination of egg hatching success, as well as rates of egg production, was a major goal of these experiments. Preparation of food suspensions Because Pseudo-nitzschia spp. cells in suspension are sorted during pipetting as chains with unequal numbers of cells per chain, rather than as individual cells, in many experiments the toxic versus non-toxic subsample cell counts of the initials were not the same. Such disparities complicate statistical comparisons of the ingestion rates on toxic versus non-toxic diets. Therefore, because of the mismatch in actual food concentrations, Student’s t-test was employed to test the null hypothesis that the toxic versus non-toxic initial sample means of the actual food treatment concentrations were similar (Sokal & Rohlf, 1995). If initial count means were found to be statistically similar at given paired food concentrations, a t-test was used to test the null hypothesis that the ingestion rates of copepods grazing on toxic versus non-toxic diatoms were similar. Prior to the t-test, an F-max test was used to determine whether the variances of the toxic versus non-toxic ingestion rates were similar, as was done when comparing the toxic and non-toxic actual food concentrations. The results of the t-tests appear in Table 1 for A. tonsa and Table 2 for T. longicornis. Alternatively, if initial count means between paired toxic and non-toxic cell concentrations were found to be significantly different, the data are not presented here. These data are provided by Lincoln (1998). Domoic acid uptake and depuration and survival by copepods For domoic acid analyses on copepods from feeding experiments, three replicates each of 16–19 copepods were screened onto 400 µm mesh, which would retain copepods but allow passage of P. multiseries cells. Domoic acid in copepods was determined with HPLC, using the same protocols as for the phytoplankton cultures. Domoic acid analyses were also performed on copepods from experiments using non-
toxic P. pungens, to confirm that there was no domoic acid uptake. On 20–21 July 1994, we simultaneously measured survival of, and uptake and depuration of domoic acid by A. tonsa females feeding on cultures of 5 × 106 cells l−1 of toxic P. multiseries (culture KP-82-2, domoic acid levels of 0.09–0.28 pg cell−1 ) and nontoxic P. pungens (culture C-92-10-2). Aliquots of 20 copepods each were removed by pipette after intervals of 1.2–1.6, 2.3–3.0, 4.6–5.0, 6.4–7.0, 16.2, 21.5 and 25.1–26.0 h, and the percentage of copepods still alive and the domoic acid content per copepod were determined. We also measured depuration and survival of aliquots of 42–60 T. longicornis females after feeding on toxic P. multiseries cultures (KP-82-3, domoic acid levels of 0.96–1.46 pg cell−1 ) for 10 h. These females were then transferred to filtered sea water and removed at intervals of 1.17–5.08 h and frozen for domoic acid analyses. Also in July 1994, we determined uptake of domoic acid in grazing experiments with triplicate replicates of either 17–19 Acartia tonsa females (18–19 July), or 20 Temora longicornis females (22–23 July), feeding on cultures of toxic P. multiseries (KP-82-2 for A. tonsa, and KP-82-1 for T. longicornis). In both accumulation experiments, three replicate experiments were performed with copepods feeding on similar concentrations of cultures (C-92-10) of non-toxic P. pungens.
Results Domoic acid in cultures Domoic acid levels in whole cultures and intracellular domoic acid levels were highly variable over time, both within and between cultures (Table 3). Generally after 11–13 days, most cultures declined in domoic acid, both in terms of intracellular content within cells, as well as intracellular plus dissolved domoic acid in the culture medium. In some cases, these changes were rapid, for instance the decline in domoic acid content on 20 July between 08:30 and 16:30 h. Domoic acid was never detected in cultures of non-toxic P. pungens.
113
Figure 1. Rates of ingestion (cells copepod−1 h−1 ) of toxic Pseudo-nitzschia multiseries and non-toxic P. pungens by Acartia tonsa for paired experiments with no significant difference between actual concentrations of toxic and non-toxic cells, in July, 1994, June 1995, August, 1995 and August, 1996 (see Table 1).
Figure 2. Rates of ingestion (cells copepod−1 h−1 ) of toxic Pseudo-nitzschia multiseries and non-toxic P. pungens by Temora longicornis for paired experiments with no significant difference between actual concentrations of toxic and non-toxic cells, in July, 1994 (see Table 2).
Ingestion rates Acartia tonsa At the initial concentrations of 2.70–3.26 × 105 cells l−1 plus natural seawater, A. tonsa feeding on the toxic diatom showed a significantly higher mean ingestion rate (169 cells copepod−1 h−1 ) than for feeding on the non-toxic diatom (32 cells copepod−1 h−1 ) (Table 1) (p = 0.012). This was the only significantly different p-value for toxic versus non-toxic diatoms found in all seven comparisons conducted with both copepods over all 3 years (Tables 1 and 2). Otherwise, for paired experiments in which there were no significant differences between initial cell concentrations of the toxic and non-toxic Pseudo-nitzschia species, either in monospecific cultures, or when cultures were added to natural sea water, there were no significant differences in rates of ingestion of the toxic versus the non-toxic Pseudo-nitzschia by A. tonsa (Fig. 1, Table 1). Temora longicornis For paired experiments in which there were no significant differences between initial cell concentrations of the toxic and non-toxic Pseudo-nitzschia species, either in monospecific cultures, or when cultures were added to natural sea water, there were no significant differences in rates of ingestion of the toxic versus the non-toxic Pseudo-nitzschia by T. longicornis (Fig. 2, Table 2).
Figure 3. Rates of egg production (eggs female−1 d−1 ) by Acartia tonsa feeding on toxic Pseudo-nitzschia multiseries and non-toxic P. pungens, natural sea water and filtered sea water in June, 1995 and August, 1995.
Egg production and hatching experiments Rates of egg production in these experiments were extremely low. Mean egg production values in all experiments ranged from 0 to 2.79 eggs copepod−1 d−1 . Although we intended to compare egg production rates of T. longicornis and A. tonsa when fed the toxic and non-toxic Pseudo-nitzschia spp. in June 1995, T. longicornis laid no eggs in either of the egg production experiments.
114 The mean egg production rates of A. tonsa when fed unialgal toxic and non-toxic Pseudo-nitzschia spp. diets at concentrations of 3.65 × 105 and 4.07 × 105 cells l−1 , respectively, in June 1995 were 0.22 eggs copepod−1 d−1 and 0.29 eggs copepod−1 d−1 , respectively (Fig. 3). Egg production experiments were also performed in June, 1995 with food suspensions of natural seawater with naturally occurring assemblages of phytoplankton and with filtered seawater. The mean egg production rate by A. tonsa was higher on the natural diet of phytoplankton with a mean egg production rate of 2.66 eggs copepod−1 d−1 than values of 0.24 eggs copepod−1 d−1 for filtered sea-water replicates (Fig. 3). In August 1995, mean egg production rates of A. tonsa feeding on unialgal toxic and non-toxic food concentrations, of 3.45 × 106 and 4.34 × 106 cells l−1 , respectively, were 2.79 eggs copepod−1 d−1 and 2.54 eggs copepod−1 d−1 , respectively (Fig. 3). Mean egg production rate of copepods with a natural diet of phytoplankton was 1.87 eggs copepod−1 d−1 , whereas the mean egg production rate of copepods in filtered sea-water was 0.58 eggs copepod−1 d−1 (Fig. 3). Copepod mortality and egg hatching success The mortality of females after feeding on the toxic and non-toxic food diets in June, 1995 was 7% for toxic diatoms and 0% for non-toxic diatoms. Hatching success after 24 h on the toxic and non-toxic Pseudo-nitzschia spp. was 22% for toxic diatoms and 61% for non-toxic diatoms. Mortality of female copepods feeding on naturally ocurring phytoplankton was 2%, and hatching success after 24 h was 51%. Female copepods with no food had a mortality of 15%, and a hatching success percentage of 65%. In August 1995, female mortality after feeding on the toxic and non-toxic Pseudo-nitzschia spp. was 4% and 10%, respectively. Hatching success after approximately 24 h on the toxic and non-toxic Pseudonitzschia spp. was 48% and 68%, respectively. Female mortality after feeding on natural sea-water phytoplankton was 18%; hatching success after 24 h was 96%. Mortality of female copepods with no food was 7% with a hatching success percentage of 85%. In August 1996, egg production rates of A. tonsa were compared when copepods were feeding on toxic and non-toxic Pseudo-nitzschia spp. at food concentrations of 1.64 × 106 and 2.29 × 106 cells l−1 , respectively, compared to a third diet of the nontoxic P. pungens (1.95 × 106 cells l−1 ) plus dissolved
Figure 4. Rates of egg production (eggs female−1 d−1 ) by Acartia tonsa feeding on toxic Pseudo-nitzschia multiseries and non-toxic P. pungens, and non-toxic P. pungens plus domoic acid (DA) at a concentration of 1.2 ng ml−1 in August, 1996.
domoic acid (Fig. 4). Concentrations of dissolved domoic acid were 1.2 ng ml −1 , similar to the amount calculated for similar experiments performed by Windust (1992). This value corresponds to the highest recorded level of domoic acid in seawater in Atlantic Canada. The mean egg production rates on the toxic and non-toxic diets were 0.78 eggs copepod−1 d−1 and 0.92 eggs copepod−1 d−1 , respectively, whereas the mean egg production rate on a diet of non-toxic culture plus dissolved domoic acid was 0.43 eggs copepod−1 d−1 (Fig. 4). Acartia tonsa produced no eggs with a diet of natural phytoplankton plus dissolved domoic acid, or with filtered seawater plus dissolved domoic acid. The mean egg production of copepods in filtered seawater without domoic acid was 0.01 eggs copepod−1 d−1 . Mortality of females after feeding on the toxic and non-toxic Pseudo-nitzschia spp. in August 1996 was 7% and 16%, respectively. Hatching success after approximately 24 h on the toxic and non-toxic diatom was 76% and 65%, respectively. Mortality of females after feeding on the natural seawater, filtered seawater, and the non-toxic Pseudo-nitzschia spp. (all treatments enriched with dissolved domoic acid) was 10%, 20% and 8%, respectively. Hatching success after approximately 24 h on these treatments was 0%, 0% and 37%, respectively. To summarize egg production, and egg hatching experiments, rates of egg production were low so values for egg hatching success were not statistically
115 robust. However, there were no apparent differences in these parameters for copepods feeding on diets of toxic versus non-toxic Pseudo-nitzschia. Domoic acid uptake, depuration and survival by copepods In the domoic acid accumulation experiments with A. tonsa and T. longicornis (Table 4), both species accumulated domoic acid. The accumulation was dosedependent in A. tonsa, in that levels increased with increasing food concentrations. Accumulation was less dose-dependent with T. longicornis, in that levels of domoic acid were much lower than in A. tonsa, and showed no clear increase with increasing food concentrations (Table 4). In the depuration and survival experiment with A. tonsa (Table 5), mortality of copepods was not appreciably different between toxic and non-toxic diets. Copepods accumulated domoic acid, and there was a slight decline in the domoic acid content of copepods over time. However, these values are near the limit of detection, so it was not clear whether this represented depuration or variability in measurement. No domoic acid was detected in any copepods fed on non-toxic cultures of P. pungens. In a similar experiment with T. longicornis feeding upon toxic P. multiseries (Table 6), and being allowed to depurate in filtered sea water for several hours, there was no copepod mortality, and no clear pattern of depuration. However, domoic acid values in copepods were considerably higher than in the depuration experiments with A. tonsa because the culture used in the T. longicornis depuration experiments (KP-82-3) had higher intracellular cell concentrations (0.96–1.46 pg cell−1 ) than the cultures (KP-82-2) in the experiments with A. tonsa, where intracellular domoic acid concentrations were only 0.09–0.28 pg cell−1 . Domoic acid values in T. longicornis in this (25–26 July) experiment were also higher than in the previous (22– 23 July) accumulation experiment with this species. This occurred, again because intracellular domoic acid levels were much higher in culture KP-82-3 used here, than in culture KP82-1 (0.05 pg cell−1 ) used in the previous T. longicornis accumulation experiments.
Discussion Our study revealed no differential grazing, egg production or mortality for copepods feeding on the
non-toxic versus the toxic diatoms. Windust (1992) reported similar results for other copepod species. However, ingestion and clearance rates for both A. tonsa and T. longicornis varied considerably (Tables 1 and 2). Of the four t-tests performed on ingestion rates with A. tonsa, one significant P value was found, revealing a higher ingestion rate for the toxic P. pungens. No significant P values were reported from the three t-tests on ingestion rates performed with T. longicornis. The absence of any apparent effect of domoic acid on copepod feeding behavior in the present study, is similar to results of studies with some toxic species of dinoflagellates (Turriff et al., 1995; Teegarden & Cembella, 1996; Dutz, 1998). Some other phytoplankton toxins have been shown to alter copepod feeding behavior. For instance, some species of Alexandrium produce various toxins which are water-soluble sodium-ion-channel blockers, and ingestion of these toxins eventually inhibits nerve transmission, relaxing smooth muscle and causing paralysis and respiratory failure in humans (Dale & Yentsch, 1978). Some copepods appear to exhibit similar impairment of neuromuscular activity. Gill & Harris (1987) quantified a significant reduction in frequency of limb beating of Temora longicornis within minutes of exposure to bloom-simulated levels of Alexandrium tamarense, resulting in decreased feeding activity soon after. Ives (1985) found a substantial decrease in ingestion rates of Calanus helgolandicus and Temora longicornis over time when they were fed increasing amounts of a toxic clone of A. tamarense, compared to a non-toxic clone, at toxicity levels of 0–40 pg cell−1 . Sykes & Huntley (1987) recorded elevated heart rates and loss of motor control for Calanus pacificus feeding on Ptychodiscus brevis, and regurgitation of Gonyaulax grindleyi. Such behavioural abnormalities associated with toxins produced by dinoflagellates were not observed in either the study of Windust (1992) or this one. It is possible that domoic acid may affect primarily the highly developed central nervous systems of vertebrates, rather than the more simple ones of invertebrates. While a case might be made for the antifeedant role of some toxins produced by dinoflagellates, domoic acid does not appear to have such an influence on copepods. We also examined the role of domoic acid and its relationship to copepod reproductive physiology. However, egg production rates for A. tonsa were very low, ranging between 0 and 2.79 eggs−1 d−1 , and T. longicornis did not produce any eggs, although they
116 Table 3. Whole culture (WC)a and intracellular levels in filtered (F) cellsb of domoic acid (DA), dates of measurement, cells ml−1 and culture age for cultures used in experiments Date
Culture
Cells ml−1
Culture age (days)
Whole culture DA (ng ml−1 )
Intracellular DA (pg cell−1 )
19 July 1994 20 July 1994 22 July 1994 25 July 1994 26 July 1994 20 July 1994 20 July 1994 22 July 1994 25 July 1994 26 July 1994 6 June 1995 7 June 1995 9 June 1995 12 June 1995 6 June 1995 7 June 1995 9 June 1995 12 June 1995 28 Aug 1995 29 Aug 1995 30 Aug 1995 28 Aug 1995 29 Aug 1995 30 Aug 1995 26 Aug 1996 26 Aug 1996 26 Aug 1996 27 Aug 1996 27 Aug 1996 27 Aug 1996
KP-82-2(WC) KP-82-2(WC) KP-82-1(WC) KP-82-3(WC) KP-82-3(WC) KP-82-2 (F) KP-82-2 (F) KP-82-1 (F) KP-82-3 (F) KP-82-3 (F) KP-82 (WC) KP-82 (WC) KP-82 (WC) KP-82 (WC) KP-82 (F) KP-82 (F) KP-82 (F) KP-82 (F) KP-82 (WC) KP-82 (WC) KP-82 (WC) KP-82 (F) KP-82 (F) KP-82 (F) KP-105 (F) KP-105 (F) KP-105 (F) KP-105 (F) KP-105 (F) KP-105 (F)
350×103 350×103 203×103 177×103 177×103 350×103 350×103 203×103 177×103 177×103 266×103 298×103 181×103 208×103 266×103 298×103 181×103 208×103 144×103 144×103 205×103 144×103 144×103 205×103 298×103 298×103 298×103 272×103 272×103 272×103
12 13 15 10 11 13 (08:30h) 13 (16:30h) 15 10 11 11 12 11 12 11 12 11 12 12 13 12 12 13 12 ? ? ? ? ? ?
2190.7 1992.0 1643.4 236.0 426.3 654.3 203.4 62.7 1132.7 1718.7 154.8 179.8 90.5 72.5 811.3 952.1 450.7 292.7 148.5 189.5 130.7 1248.6 1411.7 980.2 5129.3 5483.4 6356.1 4223.0 4650.3 5187.9
6.20 5.64 8.10 1.33 2.41 0.28 0.09 0.05 0.96 1.46 0.58 0.60 0.50 0.35 0.30 0.32 0.25 0.14 1.03 1.32 0.64 0.87 0.98 0.48 1.72 1.84 2.20 1.55 1.71 1.91
a Whole culture = domoic acid in cells plus culture medium, measured after sonicating cells in culture
medium. b Filtered cells = domoic acid in cells after concentrating 100 ml of culture by filtering onto a Nucleopore
filter, and quantitatively removing cells by washing with filtered sea water into a final volume of 10 ml.
Table 4. Accumulation of domoic acid (DA) by female Acartia tonsa and Temora longicornis feeding on cultures of Pseudo-nitzschia multiseries in various concentrations in grazing experiments in July, 1994 Copepod
Time (h:min)
P. multiseries (cells l−1 )
A. tonsa A. tonsa A. tonsa T. longicornis T. longicornis T. longicornis
21:56 21:43 19:17 14:46 14:27 14:18
3.25×105 4.67×105 1.31×106 1.21×105 3.25×105 1.93×106
DA in culture (ng ml−1 ) 1.97 6.50 13.60 0.61 1.15 0.99
DA accumulation (ng copepod−1 d−1 ) 1.01 3.38 8.46 0.40 0.76 0.66
117 Table 5. Depuration and survival experiment 21–22 July, 1994, with 20 Acartia tonsa females each grazing on toxic cultures (KP-82-2) of Pseudo-nitzschia multiseries or non-toxic cultures (C-92-10-1) of P. pungens. Survival is % of 20 copepods alive at the end of the experiment, and domoic acid (DA) is in ng copepod−1 Time (h)
0.0 1.2–1.6 2.3–3.0 4.6–5.0 6.4–7.0 16.2 21.5 25.1–26.0
Toxic culture % Survival DA (ng copepod−1 ) 0 100 90 100 80 – 85 75
0.55a 0.43 0.34 0.37 0.32 – 0.28 0.32
Non-toxic culture % Survival DA (ng copepod−1 ) 0 100 100 95 85 90 – 80
0 0 0 0 0 0 – 0
a Mean of values of 0.47 ng copepod−1 for 23 copepods dead after feeding on the toxic culture, but before the depuration and survival experiment began, and replicates at the start of the depuration and survival experiment of 0.37 ng copepod−1 for 20 copepods and 0.82 ng copepod−1 for 70 copepods.
ingested large numbers of cells. Although the time of year may not have been optimal for maximal fecundity, these calanoid copepods should have been capable of reproduction during the summer months. Similarly, low egg production and hatching success, coupled with adequate ingestion rates of the toxic Alexandrium tamarense by Centropages hamatus was reported by Turner et al. (1998b). Despite the low numbers of eggs produced, and subsequent calculations of percent hatching success from these numbers, domoic acid did not appear to definitively inhibit egg hatching. Percent hatching of A. tonsa eggs on diets of the toxic P. multiseries at various food concentrations from 1995 to 1996 ranged between 22.2% and 76.2%, with a mean percent hatch of 45%. Although these rates may seem low, if the toxin served primarily to debilitate the hatching of the next generation of grazers, one would expect a much lower hatching success of eggs. Likewise, percent hatching on the non-toxic P. pungens ranged between 38.9% and 65.1%, with a mean percent hatch of 58%. Statistical analyses were not performed because of small sample sizes. During the 1996 experiments conducted in this study, experimental treatments of filtered seawater, natural seawater and the non-toxic P. multiseries had dissolved domoic acid added to them in order to examine effects of domoic acid on egg production rates. Concentrations were based on the amounts calculated for similar experiments performed by Windust (1992).
Although the treatments with dissolved domoic acid resulted in slightly lower egg production rates (Fig. 4), the overall egg production rates were too low to allow realistic comparisons. Comparisons between egg production rates on diets of the toxic versus the non-toxic diatoms revealed no differential egg production rates. Similarly, egg hatching success revealed no differences for females feeding on toxic versus non-toxic diets. However, because of the generally low numbers of eggs produced, it is difficult to conclude whether domoic acid inhibited egg production. There were low chlorophyll levels in the natural seawater (
