Anabaena flos-aquae. Susceptibility of planktonic rotifers to a toxic strain of

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1 Limnol. Oceanogr., 39(6), 1994, , by the American Society of Limnology and Oceanography, Inc. Susceptibility of planktonic rotifers to a toxic strain of Anabaena flos-aquae John J. Gilbert Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire Abstract Reproduction in Asplanchna girodi, Brachionus calyczjlorus, Keratella cochlearis, and Synchaeta pectinata fed Cryptomonas at 19 C was inhibited by the presence of a strain of Anabaena flos-aquae (IC- 1) producing the neurotoxic alkaloid anatoxin-a. The most susceptible species, B. calycz&!orus, was suppressed at an Anabaena dry mass concentration of 0.5 pg ml-*; the others were suppressed at a concentration of 4 pg ml-. Reproduction of all species at 19 C was inhibited by anatoxin-a; the most sensitive species (S. pectinata) was inhibited at a concentration of 0.2 fig ml-l, and the least sensitive species (B. CalyciJorus), was inhibited at a concentration of 5 pg ml- but not one of 2 pg ml-. In contrast, A. girodi and two clones of B. calyciflorus were not inhibited by filtrates of very dense Anabaena suspensions (1 : 1 and 1 : 3 dilutions of 1 -d-old, 80 pg ml-r suspensions); this showed that the Anabaena did not release extracellular toxin, and thus that it could only inhibit rotifers that ingested it. B. calyczjlorus probably was the most susceptible to the cyanobacterium, even though it was the least sensitive to the soluble toxin, because it ingested the filaments most efficiently. Its relatively low sensitivity to the toxin may reflect an evolutionary response to its greater tendency to ingest toxic cyanobacteria. The effect of a toxic cyanobacterium on the structure of a freshwater zooplankton community should depend on the size and morphology of its colonies. Large colonies should be more readily ingested by daphniids than rotifers, and hence more likely to inhibit competitively dominant daphniids. Small, amorphous colonies or short, thin, nonmucilagecoated filaments should be ingested by, and thus inhibit, some rotifers as well as daphniids. Rotifers are often a major component of freshwater plankton communities, especially when large, competitively superior daphniids are rare or absent (Gilbert 1988a). Thus, any factor that suppresses daphniids more than rotifers should increase the relative importance of rotifers. A well-known example is the presence of visually feeding, zooplanktivorous fish, which feed selectively on large zooplankton (O Brien 1987). Another factor may be blooms of cyanobacteria. Because large cladocerans can eat larger objects than most rotifers (Gilbert 198 5, 1988a), microplanktonic colonies of cy- Acknowledgments I thank Joyce M. Camp for technical assistance, Robert L. Wallace for the Asplanchna girodi from Green Lake, Wayne W. Carmichael for strain IC- 1 of Anabaena flosaquae, Mary E. Claska for maintaining cultures of this cyanobacterium, James Dykes and Stephen A. Wickham for producing the bootstrap program, Steven C. Fradkin for assistance with the use of some statistical software and for determining the relationship between A. flos-aquae dry mass concentration and optical density, and David F. Brakke, Kevin L. Kirk, Peter L. Starkweather, and two anonymous referees for comments on the manuscript. The research was supported by EPA research grant R8 l anobacteria that contain endotoxins or can mechanically interfere with feeding mechanisms should inhibit large daphniids more than rotifers. Unfortunately, few studies have examined interactions between such cyanobacteria and rotifers, and fewer still have compared the susceptibility of rotifers and cladocerans to the cyanobacteria. However, some indirect evidence (see Gilbert 1990) and experimental studies (Gilbert 1990; Gilbert and Durand 1990) support this pattern. The susceptibility of a zooplankton species to a toxic cyanobacterium should depend on 1286 a number of factors. If a toxin is not released extracellularly, the susceptibility of the zooplankton species should be a function of the efficiency at which the cyanobacterium is ingested and its toxin is assimilated and the sensitivity of the tissues to the toxin. If the cyanobacterium also releases toxin(s) extracellularly, then even a zooplankton species un- able to ingest the cyanobacterium could be susceptible to the cyanobacterium. While the question of extracellular release of toxin(s) by cyanobacteria has important implications regarding the susceptibility of zooplankton taxa to cyanobacteria, very little research has been devoted to this subject. Several studies have

2 Rotijk Anabaena interactions 1287 indicated or suggested that certain cyanobacteria produce extracellular toxins (Snell 1980; Ostrofsky et al. 1983; Starkweather and Kellar 1987), but much of the evidence is inconclusive. However, a strain of Anabaena minutissima does release a factor suppressing thoracic limb beat rate in Daphnia carinata (Forsyth et al. 1992). On the other hand, two cyanobacteria very toxic to Daphnia do not release toxins extracellularly-microcystis aeruginosa (Lampert 198 1) and Anabaena afinis (Gilbert 1990). In addition to being much less likely than cladocerans to ingest colonial cyanobacteria, rotifers also may be less sensitive to cyanobacterial toxins. For example, extracts of A. afinis toxic to Daphnia pulex had no effect on three rotifer species (Gilbert 1990). Furthermore, a strain of M. aeruginosa toxic to Daphnia ambigua had no effect on the rotifer Brachionus calyczyorus, although it did on another brachionid, Keratella mixta (Fulton and Pearl 1987). Finally, a strain ofanabaenaflos-aquae thought to contain anatoxin-a was a good food source for B. cazyczjzorus (Starkweather and Kellar 1983). If rotifers generally are more resistant to cyanobacterial toxins than daphniids, then blooms of cyanobacteria would be all the more likely to suppress daphniids more than rotifers and, therefore, could shift the species structure of zooplankton communities in favor of rotifers. The present study investigates the susceptibility of four common planktonic rotifers to a strain of A. flos-aquae which produces the neurotoxic alkaloid anatoxin-a (W. W. Carmichael pers. comm.) and is very toxic to D. pulex (Claska and Gilbert unpubl.). Filaments of this strain are small and likely to be ingested by rotifers as well as cladocerans. To determine the mechanism(s) of any susceptibility of the rotifers to this cyanobacterium, I conducted experiments to assess the effects of filament suspensions, cell-free filtrates of dense filament suspensions, and authentic anatoxin-a on reproduction and survival. The results show that the rotifers are all, but differentially, susceptible to the A. flos-aquae and anatoxina, that the A. jzos-aquae does not release extracellular toxin(s), and that blooms of such toxic A. jlos-aquae in natural systems should suppress both rotifers and cladocerans ingesting the filaments. Methods General procedures - Asplanchna girodi, B. calyciflorus, and Synchaeta pectinata were cultured in filtered (0.45~pm Gelman polysulfone membrane) lake water (Storrs Pond, Hanover, New Hampshire), fed on Cryptomonas erosa var. reflexa (- 1 ng cell-l), and maintained at 19 C in a photoperiod (L/D 16 : 8). KerateZZa cochlearis was cultured in the same way but fed Cryptomonas sp. (-90 pg cell-l). For A. girodi and B. calyczjlorus, the ph of the lake water was adjusted from -7 to 8 with several drops per liter of 0.5 N sodium hydroxide. In two experiments with B. cazyczjzorus, the ph was adjusted to -9 due to a malfunctioning ph meter (experiment 5 with Anabaena suspensions, experiment 2 with filtrates of Anabaena suspensions); however, B. calycijlorus reproduction at ph 9 was about the same as it was at ph 8. All rotifer cultures were clonal. The A. girodi was collected from Green Lake, Wisconsin, by R. L. Wallace. The K. cochlearis and S. pectinata were collected from Star Lake, Norwich, Vermont. The two clones of B. ca- ZyciJlorus were derived from resting eggs purchased from Florida Aqua Farms (Dade City, Florida). Both cryptomonad algae were cultured in modified MBL medium (Stemberger 198 1) at 19 C in a photoperiod (L/D 16 : 8). A. jlosaquae, strain IC- 1 (Cave Lake, Idaho, from W. W. Carmichael), was cultured in ASM-1 medium (Gorham et al. 1964) under similar conditions but with a lower light intensity. This A. flos-aquae produces anatoxin-a and possibly other toxins as well (W. W. Carmichael pers. comm.). Its filaments are 3.8 pm in diameter and arranged in coils with an outside diameter of - 25 pm. The filaments vary greatly in length from single cells -4 pm long, to short, several-celled, curved filaments 7-20 pm long, to long helices pm long. None of the algal cultures was axenic. Cryptomonad suspensions were prepared by removing with a pipet dense patches of motile cells from flasks containing 8-12-d-old cultures, diluting these cells with lake water and determining their concentrations with a hemocytometer at 75 magnifications, and then diluting these suspensions to desired concentrations with lake water. Anabaena suspensions were prepared by removing highly concen-

3 1288 Gilbert trated material by pipet from the surfaces of 7-lo-d-old flask cultures, diluting this material with lake water, determining the biomass concentrations of these suspensions, and then diluting them to desired final concentrations. For determining biomass concentration, a Cahn electrobalance and a Milton Roy Spectronic spectrophotometer were used to produce a regression (r2 = 0.999) relating pg dry mass to optical density at 436 nm. Anabaena suspensions withdrawn from cultures were generally diluted from concentrations of -2 mg ml-l to those of 10 pg ml-l or less. Synthesized anatoxin-a hydrochloride was purchased from Bio-metric Systems, Inc. (lot no ). It was stored at -20 C until use, and then aliquots of known weight were removed from the 10*0.5-mg sample by adding 1 ml of absolute ethanol to the container, withdrawing a known volume of the solution with a micropipet, and then evaporating off the ethanol with nitrogen gas in both the original sample and the aliquot. The dry anatoxin-a from the aliquot was then dissolved in double-distilled water to prepare stock solutions kept at 4 or -20 C. All experiments were conducted at 19 or 22 C (L/D 16 : 8). In experiments with Anabaena suspensions, all vessels with rotifers were placed on a plankton wheel rotating constantly at 1 rpm to prevent heterogeneity in the distribution of the Anabaena. Life-table experiments were preformed with A. girodi, B. ca- ZycijZorus, and S. pectinata. Neonate amictic females were cultured individually for 3 to 7 d, at the end of which time survivorship and total fecundity were determined. A population-growth experiment was performed with K. cochlearis, because the relatively small amount of postnatal growth in this species makes it difficult to distinguish young from parental females, and hence to conduct lifetable experiments. These experiments were conducted in a dimly lit, walk-in incubator at 19 C. In experiments with filtrates of Anabaena suspensions and those with anatoxin-a, vessels were not rotated on a plankton wheel. Lifetable experiments were conducted with A. girodi, B. calyciflorus, and S. pectinata to determine reproductive rates. Neonate amictic females were cultured individually until death. Each day, age-specific survivorship and fecun- dity data were recorded and then used in a bootstrap procedure (1,000 samples) to calculate means and standard errors for the net reproductive rate (R,,) and a bias-corrected intrinsic rate of natural increase (r,) (Meyer et al. 1986). A population-growth experiment was conducted with K. cochlearis. Experiments with filtrates were conducted in a dimly lit, walkin incubator at 19 C; those with anatoxin-a were conducted in a more brightly lit incubator at 22 C. Experiments testing eflects of Anabaena flosaquae on reproduction and survivorship -Cohorts of neonate A. girodi, B. calyczjlorus, and S. pectinata were cultured on C. erosa (2 x 1 O4 cells ml- l, - 20 pg ml- l) with and without A. Jlos-aquae (2, 4, or 10 pg ml-l) for 3, 5, or 7 d at 19 C. Each rotifer was cultured individually in a screwcap vial placed on a plankton wheel rotating continuously at 1 rpm. In experiments l-4, 2-ml glass vials were used. In experiments 5-7, 2.8-ml sterile cryule vials (Wheaton) were used. Every 24 h, survivorship and fecundity were recorded, and living parental females were transferred to fresh treatment conditions. In A. girodi and B. calycijlorus, fecundity was defined as the number of live, female offspring produced per female. In S. pectinata, it was defined as the number of female eggs laid per female, because substantial fractions of the eggs of some females were diapausing and did not hatch for quite a few days. Differences in total fecundity per female in the two treatments were compared with Student s t-tests (2-tailed). Differences in the proportions of living parental females at the end of the experiment were compared with G-tests of independence using Yates correction for continuity. Populations of K. cochlearis were cultured on Cryptomonas sp. (2 X 1 O4 cells ml-l, pg ml-l) with and without A. flos-aquae (4 pg ml-l) at 19 C for 8 d. Six populations, three per treatment, were initiated with 25 individuals of various ages randomly taken from stock cultures. Each population was cultured in a 25- ml, glass, screwcap vial placed on a continuously rotating (1 rpm) plankton wheel. Every 2 d, all individuals in the populations were counted and transferred to fresh treatment conditions. Changes in population size over time in the two treatments were compared with repeated-measures ANOVA.

4 Rotifer Anabaena interactions 1289 With some of the experiments described above, parallel experiments were conducted without rotifers to determine the effect of the A.flos-aquae on the Cryptomonas used as food for the rotifers. Experiments testing for the extracellular release of toxin(s) by Anabaena flos-aquae-the possibility that A. flos-aquae inhibits rotifers by releasing toxin into the medium was tested in three different experiments by culturing A. girodi and B. calyciflorus on C. erosa (2 x 1 O4 cells ml-l) in cell-free filtrates of suspended filaments. In experiment 1 with A. girodi, lake water with A. flos-aquae (120 ml, 80 pug ml- ) and control lake water without the cyanobacterium (120 ml) were incubated at 19 C (L/D 16 : 8) for 1 d without agitation. The A. flosaquae suspension was prepared by diluting an extremely dense suspension removed by pipet from the surfaces of cultures. In this and later experiments (see below), the concentration of the dense suspension varied from to 2.5 mg ml-l. Thus, the proportion of algal growth medium (ASM-1) to lake water in the 80 pg ml-l suspensions varied from - 1 : 19 to 1: 30. After incubation, the 80 pg ml-l Anabaena suspension and the control lake water were each filtered through a separate 76-mm diameter, 0.2~pm cellulose acetate membrane (Schleicher & Schuell) previously washed by passing double-distilled water (100 ml) through it. Experimental and control filtrates were dispensed into sterile, screwcap cryule vials (2 ml per vial) and stored frozen (- 15 C) until use during the next 2 weeks. Each day, sufficient quantities of both filtrates were thawed and diluted (1 : 3) with a suspension of C. erosa in lake water. In experiments 2 and 3 with B. cazyczjzorus, filtrates (200 ml) of lake water with and without A. jlos-aquae were prepared and stored in a similar way, except that the filters used were 0.2-pm nylon membranes (Schleicher & Schuell) and the storage temperature was -80 C. These modifications were used to reduce the possibility that any toxin(s) released into the medium by the cyanobacterium would be either adsorbed onto the filter or degraded during storage. Also, in these experiments, more concentrated filtrates (1 : 1 as well as 1 : 3 dilutions in experiment 2; 1 : 1 dilutions in experiment 3) were used to increase the possibility of detecting toxicity. Cohorts of neonate A. girodi or B. calycijlorus were cultured in the diluted Anabaena suspension and control filtrates. Each rotifer was cultured individually until death in 2 ml (experiment 1) or 1 ml (experiments 2 and 3) of medium in sterile, plastic, 24-well tissue culture plates (Falcon 3047, Becton Dickinson and Co.). Every 24 h, survivorship and fecundity (live, female offspring) were recorded, and living parental females were transferred to fresh treatment conditions. Intrinsic rates of natural increase (r,j and net reproductive rates (R,) were determined with the bootstrap procedure and compared statistically by comparing means and 95% confidence limits. Distributions of such bootstrapped values generally are normally distributed (Efron 1982; James Dykes pers. comm.). Experiments testing e#ect of anatoxin-a concentration on reproduction and lifespan-the susceptibilities of A. girodi, B. calyciflorus, and S. pectinata to anatoxin-a at 22 C were compared by culturing them at toxin concentra- tions where inhibition ranged from negligible or slight to considerable. These concentrations were determined from the results of preliminary experiments. In cohorts of 15-20, rotifers were cultured individually from birth until death in l-ml volumes of lake water with C. erosa (2 x lo4 cells ml-l) and the level of anatoxin-a to be tested. Culture vessels were tissue culture plates (see above) for B. calyciflorus or glass concavity plates in Petri-dish moisture chambers for A. girodi and S. pectinata. Every 12 h (A. girodi, S. pectinata) or 24 h (B. calyciflorus), survivorship and fecundity (live, female offspring in A. girodi and B. ca- Zyciflorus; female eggs in S. pectinata) were recorded; every 24 h, living parental females were transferred to fresh treatment conditions. Net reproductive rates (RO) were determined with the bootstrap procedure and compared with means and 95% confidence limits. Mean life- spans (midpoint method) were compared with ANOVA and Scheffe s F procedure for pairwise comparisons. K. cochlearis populations were cultured on Cryptomonas sp. (2 x lo4 cells ml-l) without and with anatoxin-a (0.5 fig ml-l) at 22 C for 6 d. Eight populations, four per treatment, were initiated with 20 individuals of various ages haphazardly taken from stock cultures. The populations were cultured in plastic Petri dish-

5 1290 Gilbert Table 1. Reproduction and survivorship of the rotifers Asplanchna girodi (A.g.), Brachionus calyciforus (B.C.), and Synchaeta pectinata (S.p.) cultured since birth at 19 C on Cryptomonas erosa (2 x lo4 cells ml-l) with and without Anabaenaflos-aquae. Mean numbers of young (A.g., B.C.) or eggs (S.p.) compared with t-tests. Frequencies of survivors compared with G-tests. Significance of differences: NS (P > 0.05); * (P < 0.025); ** (P < 0.005). Exp Rotifer (and clone no.) A.g. B.c.( 1) B.C.(~) B.C.(~) B.C.(~) s.p. s.p. Young or eggs produced per Duration female during experiment Proportion of of exp. Anabaena concn % reduction cohort surviving (4 CPLX ml-7 Cohort size mean + 1 SD with Anabaena at end of experiment ** ** & ** 92 1.oo** ** ** a ** 95 1.oo** kO z ** 81 1.OONS 1.8k * NS k &2.4** 63 1.oo** k es (35 x 10 mm) containing 5 ml of medium, and every 2 d all individuals in the populations were counted and transferred to fresh treatment conditions. Changes in population size over time in the two treatments were compared with repeated-measures ANOVA. One experiment tested the effect of anatoxin-a on C. erosa. Suspensions of C. erosa (2 X lo4 cells ml-l) were incubated with different concentrations of anatoxin-a at 22 C for 1 d, IOO I 0 Control Day Fig. 1. Effect of Anabaena flos-aquae (4 pg ml- I) on the ability of Keratella cochlearis populations to grow on Cryptomonas sp. (2 x 1 O4 cells ml- ) at 19 C. Values are means + SE of three replicate populations rotated on a plankton wheel. I and then the cell densities of these suspensions were compared. Results A. jzos-aquae significantly suppressed the fecundity and survivorship of all three rotifer species (Table 1). B. calyciflorus was the most sensitive to the presence of the filaments, with the fecundity of clone 2 of this species reduced 8 1% at a concentration of 0.5 pg ml- l. S. pectinata was the least sensitive to the A. jzosaquae filaments. A concentration of 10 pg ml- l had about the same effect on S. pectinata as one of 4 pg ml-l had on A. girodi, and it had somewhat less of an effect on S. pectinata than one of 0.5 pg ml-l had on B. CaZyczjZorus. The population growth of K. cochlearis was significantly suppressed by A. flos-aquae at a concentration of 4 pg ml- l (Fig. 1). A repeatedmeasures ANOVA on the log,,-transformed, population-size data showed no significant treatment effect (F = 3.69 with 1 df, P = 0.13) but a highly significant day x treatment interaction (F = with 4 df, P = ); thus, the presence of A. jlos-aquae changed the trajectory of K. cochlearis population growth. The A. Jlos-aquae had no inhibitory effect on either C. erosa or Cryptomonas sp., as judged by the concentrations of these algae at the end

6 Rotlyer Anabaena interactions 1291 Table 2. Effect of Anabaenaflos-aquae on cell concentration of Cryptomonas at 19 C. In all experiments, initial concentration of Cryptomonas was 2 X lo4 cells ml-l. Number ofreplicates per treatment was four in experiment 3 and three in all other experiments. Significance of differences among means (t-tests): NS (P > 0.05); * (0.02 < P < 0.05). Cryptomonas mean concn at Anabaena end of interval concn Interval fl SD Exp. 64 ml- ) (d) species (cells ml- I x 104) erosa 1.6k0.2Ns 4 1.6kO.l erosa NS 4 1.9kO erosa 1.8&0.1NS 4 1.6kO erosa * kO SP NS 4 1.6kO.2 of l-or 2-d intervals (Table 2). The only significant effect occurred in experiment 4, where the C. erosa concentration was higher in the presence of the cyanobacterium. Under the temperature (19OC) and low-light conditions of these experiments, the Cryptomonas populations in the control treatments showed no growth. The results of the three filtrate experiments (Table 3) show that the inhibitory effect of A. Jlos-aquae on A. girodi and B. calyciflorus cannot be attributed to extracellular toxin(s) re- leased into the environment. The intrinsic rate of natural increase of rotifers in filtrates of Anabaena suspensions was never significantly different from that of rotifers in control filtrates. The net reproductive rate of these rotifers also was similar in both treatments. In experiment 1, R, was significantly lower in the control filtrate than in the filtrate of the Anabaena suspension. The effects of anatoxin-a concentration on the R. and lifespan of each rotifer species are shown in Figs. 2 and 3. For all three rotifers, there was a significant overall effect of toxin concentration on lifespan (Table 4). B. calycijzorus was the least sensitive to the toxin, with both R. and lifespan significantly reduced (P < 0.05) only at a concentration of 5 pg ml-l. S. pectinata was the most sensitive; at 0.5 pg ml-l of the toxin, R. was negligible (0.07) and lifespan was significantly reduced (P < ) to -32% of the control value. A. girodi was less sensitive to anatoxin-a than S. pectinata; at 0.5 hg ml- l, R. was significantly reduced, but to only -50% of the control value, and lifespan was not significantly different from the control value (P = 0.97). In A. girodi, anatoxin-a had little effect on lifespan. At a concentration of 5 pg ml-l, R, was zero but lifespan was still - 65% of the control value. For this reason, the dose of anatoxin-a causing a set reduction in survivorship could not be calculated for A. girodi. Such doses, however, could be calculated for the other two rotifers and further exemplify their different susceptibilities to the toxin. For B. Table 3. Intrinsic rates of natural increase (r,j and net reproductive rates (R,) of Asplanchna girodi and Brachionus calyciflorus (clone 2) cultured from birth to death at 19 C on Cryptomonas erosa (2 x lo4 cells ml- ) in filtrates of Anabaenajlos-aquae suspensions and control lake water. Lake water and cyanobacterial suspensions (80 pg ml-*) were filtered (0.2 pm) after 1 d at 19 C and the filtrates were diluted 1 : 3 and 1 : 1 with lake water. Bootstrapped mean values of r,?, and R, from treatments with control and Anabaena filtrates are compared by determining overlap of 95% confidence limits. Significance of differences between means: NS (P > 0.05); * (P < 0.05). Exp. Rotifer Treatment filtrate 1 A. girodi control Anabaena 2a B. calyciflorus control Anabaena 2b control Anabaena 3 B. calyciflorus control Anabaena Dilution of filtrate Cohort size Mean reproduction rate (+ 1 SE) r,,, d-l 1: (0.01 5)NS 12.79(0.73)* 1: (0.010) 16.41(0.60) 1: (0.01 3)NS 13.15(1.17)NS 1: O(O.0 15) 14.85(0.85) 1: (0.02 l)ns 15.85(1.06)NS 1:l (0.020) 18.12(1.39) 1:l (0.02 l)ns 17.27(1.18)NS 1:l (0.0 17) 14.21(0.89) RO

7 1292 Gilbert 30-1 I] R Lifespan 6 I WI 3 + O dd 0.05.l.2 A O dd 0.05.l Anatoxln-a (pg ml ) 0.05.I Fig. 2. Effect of concentration of anatoxin-a on net Anatoxin-a (pg ml ) reproductive rates (R,) of Asplanchna girodi, Brachionus calyciflorus, and Synchaeta pectinata cultured on Cryp- Fig. 3. Effect of anatoxin-a on lifespans of Asplanchna tomonas erosa (2 x lo4 cells ml-l) at 22 C. Fecundity is girodi, Brachionus calycijlorus, and Synchaeta pectinata offspring produced per female for A. girodi and B. caly- cultured on Cryptomonas erosa (2 x lo4 cells ml-l) at cijlorus, and eggs laid per female for S. pectinata. Values 22 C. Values are means f 95% confidence limits (some >O. 1 are bootstrapped means with 95% confidence limits. obscured by symbol for mean). calyciforus and S. pectinata, the doses (95% confidence limits) giving a survival probability of 0.70 after 2 d were 4.39 ( ) and 0.18 ( ) pg ml-l, respectively (SAS Probit procedure). The population growth of K. cochlearis was significantly suppressed by 0.5 pg ml-l anatoxin-a (Fig. 4). A repeated-measures ANOVA on the log,,-transformed, population-size data showed highly significant treatment and day x treatment effects (F = with 1 df, P = 0.002, and F = with 3 df, P < , respectively). At the highest anatoxin-a concentrations, the tendency for Cryptomonas to divide decreased (Table 5). An ANOVA showed a significant difference among means (F = with 6 df, P < ); conservative pair-wise compari- son tests (Scheffe s) showed that concentrations of 1 pg ml-l and higher significantly reduced final Cryptomonas densities. However, even at the two highest concentrations, Cryptomonas showed no appreciable mortality, maintaining its density near the initial value (2 x lo4 cells ml-l). Under the temperature (22 C) and high-light conditions of these experiments, the C. erosa populations in the control treatment showed appreciable growth. Discussion All four species of rotifers were suppressed by A. flos-aquae filaments (Table 1, Fig. 1). B. calyciflorus was the most susceptible, showing marked inhibition of both survivorship and fecundity at a dry mass concentration of 2 rug ml-l and strong inhibition of fecundity at one

8 Rotifer Anabaena interactions 1293 Table 4. ANOVA table for overall effect of anatoxin-a concentration on lifespans of Asplanchna girodi, Brachionus calycijlorus, and Synchaeta pectinata. Rotifer A. girodi B. calyciflorus A. pectinata df F P <o.ooo <o.ooo Control 100 I of 0.5 pg ml-l. The fecundity of A. girodi and S. pectinata was strongly suppressed at a concentration of 4 pg ml-l. A. flos-aquae had no effect on the cryptomonad algae used as food (Table 2), and so it was probably not affecting the rotifers indirectly through their food source. This strain of A. Jtos-aquae did not excrete detectable amounts of toxin. The reproductive rates of A. girodi and B. calycijlorus were never significantly reduced by filtrates of Anabaena suspensions (Table 3). The experiments designed to investigate the possibility of such excretion greatly amplified the concentration of extracellular toxin that would have been present in the experiments with inhibitory levels of filaments. For example, in experiment 1 with A. girodi, the concentration of any toxin in the 1 : 3 dilution of the filtrate of the l-dold, 80 hg ml-l Anabaena suspension would have been similar to that in a 20 pg ml-l suspension - 5 times the concentration strongly inhibiting this rotifer. Similarly, in experiments 2b and 3 with B. cazyczjzorus, the concentration of any toxin in the 1 : 1 dilution of the filtrate of the Anabaena suspension would have been similar to that in a 40 pg ml-l suspension- 80 times the concentration strongly inhibiting this rotifer. In fact, in experiment 1, the net reproductive rate of A. girodi was sig- nificantly higher in the filtrate of the Anabaena suspension than in the filtrate of the control lake water. Anatoxin-a, known to be produced by this strain of A. flos-aquae, suppressed the survivorship and fecundity of all four rotifers (Figs. 2-4, Table 4). S. pectinata was more sensitive than A. girodi to the toxin, the R0 of the former being significantly inhibited at a concentration of 0.2 pg ml-l and that of the latter only at one of 0.5 pg ml- l. B. calycz@lorus was much less sensitive to the toxin than either of these two rotifers; its R0 was significantly inhibited only at a concentration of 5 pg ml-l, and thus it was -25 and 10 times less sensitive than S. Fig. 4. Effect of anatoxin-a (0.5 pg ml-l) on the ability of Keratella cochlearis populations to grow on Cryptomonus sp. (2 X lo4 cells ml-l) at 22 C. Values are means f SE of four replicate populations. pectinata and A. girodi, respectively. K. cochlearis also was very susceptible to the anatoxin, exhibiting no population growth at 0.5 pg ml- l. However, it is not possible to compare its sensitivity to that of the other rotifers, since lower concentrations were not tested and since comparable life-table data were not obtained. This is the first study on the effect of anatoxin-a on zooplankton. It is unlikely that the anatoxin-a had any appreciable indirect effect on the rotifers through their food source. At concentrations higher than 1 hg ml- l, the toxin did significantly reduce the population growth of C. erosa, but it did not reduce the population density of the alga below the initial density (Table 5). Since the initial density of C. erosa was high (2 x 1 O4 cells ml- )-probably beyond one at Table 5. Effect of l-d exposure to anatoxin-a on cell concentration of Cryptomonas erosa at 22 C. Initial cell concentration was 2 x lo4 cells ml-l. Five replicates per treatment. Vertical lines show groups of means not significantly different from one another (P > 0.05, Scheffe s F-test). Anatoxin-a Ocs ml-7 mean Final Ctyptomonas concn (cells ml-l x 104) SD

9 1294 Gilbert which a further increase would permit an increased numerical response of the rotifers, the failure of C. erosa populations to expand beyond the initial density at concentrations of anatoxin-a > 1 pg ml-l probably would not affect the survivorship and fecundity of the rotifers. The results with the A. flos-aquae filtrates and with the anatoxin-a show that rotifers must ingest the cyanobacterium to be affected by the anatoxin-a (and possibly other toxins) that it produces and to which they are susceptible. These results also shed some light on the mechanisms responsible for the different susceptibilities of the rotifers to the A. flos-aquae. While B. calyciflorus was much less sensitive to soluble anatoxin-a than S. pectinata and A. girodi (Fig. 2), it was much more severely affected by the cyanobacterium (Table 1). This is most likely due to its tendency to ingest the filaments more efficiently and, hence, to receive higher toxin concentrations in its tissues. Regarding feeding mechanism and diet, Brachionus and also KerateZZa are generalists, while both Asplanchna and Synchaeta are specialists (Gilbert 1980; Gilbert and Bogdan 1984; Bogdan and Gilbert 1987). The ability of B. calyciflorus to ingest A. Jlos-aquae efficiently has been shown by Starkweather (198 1) and Fulton (1988). On the other hand, S. pectinata was the most sensitive to the anatoxin-a (Figs. 2 and 3), but the least sensitive to the cyanobacterium (Table 1). The most plausible interpretation for this is that S. pectinata is the least likely to ingest the cyanobacterium and, hence, receives very low concentrations of toxin in its tissues. For these reasons, there may have been little selective pressure for S. pectinata to evolve defenses against the toxin. Future research with a variety of rotifers should determine if there is an inverse relationship between ability to ingest A. flos-aquae and sensitivity to anatoxin-a. Finally, the presence of toxins other than anatoxin-a in A. flos-aquae could confound such a relationship if the relative sensitivities of the rotifer species varied with different toxins. The present study does not support some of the conclusions or suggestions made in earlier studies concerning the effect of A. Jlos-aquae on rotifers or the mechanism(s) by which A. Jlos-aquae may inhibit zooplankton. Snell ( 1980) found that a strain isolated from a pond near Tampa, Florida, inhibited reproduction in A. girodi. He did not observe A. girodi ingesting Anabaena and, therefore, suggested that the cyanobacterium was releasing extracellular toxin. He did not directly test filtrates of filament suspensions for toxicity. In the present study, A. girodi usually did not appear to feed on Anabaena; however, several individuals had stomachs packed with filaments. While A. girodi is very selective and may not initiate feeding responses after contact with the filaments, it may ingest them incidentally while feeding on Cryptomonas. A. girodi captures food items by opening its mouth and rapidly expanding its pharynx to -7% of its body volume (Gilbert and Stemberger 1985). Thus, when ingesting a cryptomonad it may draw in and ingest other items as well. The possibility that A. flos-aquae may release an extracellular toxin was also raised by Ostrofsky et al. (1983). They reported that filtrates of filament suspensions inhibited feeding, survivorship, and reproduction in D. pulex. However, I have reservations about this conclusion. Most importantly, there was no evidence that the strain they used, UTEX 1444, was actually toxic. In fact, several studies have shown that this strain is not toxic to zooplankton. Starkweather (198 1) and Starkweather and Kellar (1983) showed that B. calyciflorus re- produces very well on this strain. Gilbert and Durand (1990) showed that this strain was not toxic to either Daphnia or Keratella; neonate Daphnia galeata mendotae survived and re- produced much better when fed this cyanobacterium (0.5 pg ml-l) than when starved, and the presence of this cyanobacterium increased the population growth rates of K. cochlearis and D. pulex fed on cryptomonads. Furthermore, the UTEX 1444 strain was not toxic in a mammalian bioassay (Carmichael pers. comm. cited by Starkweather and Kellar 1983). The experimental methods of Ostrofsky et al. (1983) also make interpretation of the results difficult. The concentration of filtered filament suspensions was not given, and filtrates of medium without cyanobacteria were not tested as controls. Starkweather and Kellar (1983) showed that B. cazycijzorus reproduced well on a strain of A. jlos-aquae (NRC-44-1) thought to be toxic; thus, they raised the possibility that this rotifer was resistant to its toxin(s). However, although

10 Rotifer Anabaena interactions 1295 this strain had been toxic in the past, there was no evidence that it was toxic to any zooplankton at the time of the study. Since anatoxin-a was produced at one time by this strain (Carmichael et al. 1979), and since the present study has shown that rotifers are sensitive to this toxin, the most parsimonious explanation for the absence of an effect of the strain on B. calyczjlorus is that the strain was no longer toxic. Supporting this conclusion, Starkweather and Kellar (1983) pointed out that the strain was nonlethal in a mammalian bioassay. The finding of the present study that A. flosaquae does not release extracellular toxin is consistent with the results of studies on some other cyanobacteria. Gilbert (1990) found that A. afinis was very toxic to D. pulex but that filtrates of highly inhibitory suspensions had no effect on the daphniid. Similarly, Lampert (198 1) found that filtrates of dense suspensions of M. aeruginosa -up to 100 times more concentrated than necessary to depress the feeding rate of Daphnia pulicaria- had no effect on this daphniid s feeding rate. Three other studies examining the possibility of extracellular release of toxins by this cyanobacterium found little or no evidence for it. Nizan et al. (1986) indicated that supernatants of centrifuged cultures of toxic cells caused no mortality to Daphnia magna. Fulton and Paerl (1987) reported that filtrates of cell suspensions of a toxic strain did not inhibit a susceptible rotifer K mixta) and inhibited a cladoceran (Diaphanosoma brachyurum) no more than filtrates of a good food organism (Chlamydomonas). Starkweather and Kellar (1987) suggested that a strain of M. aeruginosa released a dissolved factor that inhibited reproduction in B. calyciflorus. However, some evidence indicated that this strain had little if any toxicity. Namely, the survival of rotifers fed the cyanobacterium was similar to that of starved rotifers; toxicity is evident when the former is less than the latter (Lampert 1987). Also, the reproduction of B. calyczjlorus fed on Euglena and Microcystis-conditioned medium was not significantly different from that in control medium. Some cyanobacteria, however, do seem to release inhibitory factors or toxins into their environment. Forsyth et al. (1992), working with A. minutissima var. attenuata shown by Burns et al. (1989) to inhibit feeding in D. carinata, found that filtrates of l-d-old suspensions of the field-collected cyanobacterium (15-20 hg ml- ) reduced the filtering rate of this daphniid to -45% of the rate in filtrates of water from another lake without the cyanobacterium. Sivonen (1990), using high performance liquid chromatography to identify hepatotoxin production in Oscillatoria agardhii cultures, found that most toxin was intra- cellular, but that some leaked into the medium, especially in older cultures. Also, Microcystis is known to release endotoxin during decomposition (Berg et al. 1987; Watanabe et al. 1992). Thus, toxic cyanobacteria that normally have to be ingested to affect grazing zooplankton may affect zooplankton species that do not ingest them when they age or lyse and release endotoxins. The results of the present study show that rotifers that ingest a toxic cyanobacterium may be very susceptible to it. The A. flos-aquae filaments were thin (N 3.8 pm) and, especially when short, probably were readily ingested. M. aeruginosa is another cyanobacterium which can be toxic to rotifers that ingest it. Although this species normally occurs primarily as large, amorphous colonies that probably cannot be eaten by most rotifers, it usually becomes uni- cellular in laboratory culture. Its small cells are of a size (3-5 pm diam) that is readily ingestible by generalist, suspension-feeding rotifers, and they are eaten by B. calyczjlorus (Fulton and Paerl 1987). Cell suspensions of toxic strains strongly inhibited K. mixta (Fulton and Paerl 1987) and both K. cochlearis and KerateZZa crassa (Smith and Gilbert unpubl.). Furthermore, Penaloza et al. (1990) showed that toxin extracted from Microcystis-dominated phytoplankton inhibited KerateZZa just as much as it did D. magna. On the other hand, there is evidence that some rotifers may not be sen- sitive to certain cyanobacterial toxins. Extracts of A. afinis that were toxic to D. pulex had no effect on three rotifer species (Gilbert 1990). Also, a strain of M. aeruginosa toxic to some cladocerans and K. mixta was not toxic to B. calycijlorus (Fulton and Paerl 1987). The results of the present study provide further insight into the potential effects of toxic cyanobacteria on the species structure of freshwater zooplankton communities. Because most toxic cyanobacteria probably do not release extracellular toxins, only those zooplankton

11 1296 Gilbert species that ingest the cyanobacteria should be inhibited by their toxins. Therefore, the morphology of a cyanobacterium should play an important role in determining the extent to which the cyanobacterium is ingested by different zooplankton taxa and, hence, the extent to which it affects these taxa. In filamentous forms, critical features determining ingestibility might be filament diameter, filament length, presence of a mucilaginous sheath around the filament, and tendency of filaments to aggregate or entangle into masses. Toxic cyanobacteria occurring as large, amorphous colonies (Microcystis) or as long, aggregated or mucilage-coated filaments should be too large to be ingested by most rotifers but ingestible by some large cladocerans -namely, large daphniids. For example, the straight filaments of a nontoxic strain ofa.jlos-aquae (UTEX 1444) were ingested much more efficiently by Daphnia than by K. cochlearis (Gilbert and Durand 1990), and the straight, mucilage-coated, toxic filaments ofa. afinis were ingested quite efficiently by large daphniids but ingested very inefficiently, if ingested at all, by two species of KerateZZa (Kirk and Gilbert 1992). Therefore, many toxic cyanobacteria, due to the size and shape of their colonies, should be more readily ingested by daphniids than by rotifers, and thus they should inhibit these daphniids more than rotifers. Since large daphniids can competitively suppress rotifers through both exploitative competition for shared food resources and also mechanical interference (Gilbert 1985, 1988a,b; MacIsaac and Gilbert 199 l), the presence of many types of toxic cyanobacteria should differentially inhibit the daphniids and thereby favor the rotifers. If rotifers were also less sensitive to cyanobacterial toxins than the daphniids, then they might benefit even more by the presence of toxic cyanobacteria. However, blooms of toxic cyanobacteria that can be ingested by some rotifers generally should inhibit both these rotifers and cladocerans. The food niches of most rotifers are included within those of cladocerans (Gilbert 1988a), and so any cyanobacterium eaten by a rotifer should also be readily eaten by cladocerans. Thus, blooms of a cyanobacterium such as the toxic A. flos-aquae used in the present study should simultaneously inhibit both the rotifers and cladocerans that eat it. For example, D. pulex is about as sensitive to this strain of A. jlos-aquae as are rotifers; reproduction in D. pulex at 25 C was significantly suppressed by filament suspensions at a concentration of 1 pg ml-l and also by anatoxin-a concentrations of 1 pg ml-l (Claska and Gilbert unpubl.). Relatively little work has been done on interactions between toxic cyanobacteria and herbivorous copepods. What has been done suggests that these copepods should be less affected by the presence of toxic cyanobacteria than cladocerans and perhaps even rotifers. Many copepods are extremely selective feeders and have the potential to avoid ingesting toxic cyanobacteria (Fulton 1988; Vanderploeg et al. 1990; DeMott and Moxter 199 1). In addition, copepods may be able to utilize cyanobacteria that strongly inhibit other zooplankton (Burns et al. 1989). On the other hand, copepods may be sensitive to cyanobacterial toxins. DeMott et al. (199 1) found that Diaptomus birgei was more sensitive than several daphniids to toxins from M. aeruginosa (microcystin-lr) and Nodularia spumigena (nodularin). To understand the effects of toxic cyanobacteria on natural zooplankton communities, we need to conduct considerably more research using a variety of cyanobacterial species and comparing the effects of each species on a variety of rotifers, cladocerans, and copepods. In all studies using culture-collection strains that presumably are toxic to zooplankton, the toxicity of the strain should be firmly established at the time of the study. References BERG, IS., 0. M. SKULBERG, AND R. SKLUBERG Effects of decaying toxic blue-green algae on water quality-a laboratory study. Arch. Hydrobiol. 108: BOGDAN, K. G., AND J. J. GILBERT Quantitative comparison of food niches in some freshwater zooplankton: A multi-tracer-cell approach. Oecologia 72: 33 l-340. BURNS, C. W., AND OTHERS Coexistence and exclusion of zooplankton by Anabaena minutissima var. attenuutu in Lake Rotongaio, New Zealand. Ergeb. Limnol. 32: CARMICHAEL, W. W.,D. F. BEGS, AND M. A. PETERSON Pharmacology of anatoxin-a, produced by the freshwater cyanophyte Anabaena flos-aquae WRC- 44-l. Toxicon 17: DEMOTT, W. R., AND F. MOXTER Foraging on

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