Influence of predator–prey evolutionary history, chemical alarm-cues,and feeding selection on induction of toxin production in a marinedinoflagellate

Christina D. Senft-Batoh,†1 Hans G. Dam,*1 Sandra E. Shumway,1 Gary H. Wikfors,2 Carl D. Schlichting3

1Department of Marine Sciences, University of Connecticut, Groton, Connecticut2National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center,Milford Laboratory, Milford, Connecticut

3Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut

Abstract

The dinoflagellate, Alexandrium fundyense, produces paralytic shellfish toxins and co-occurs with popula-

tions of the copepod, Acartia hudsonica, from Maine, but not New Jersey. The hypothesis that history of co-

occurrence between predator and prey effects the ability of prey to recognize and respond to predators with

increased toxin production was tested for this copepod-alga interaction. When A. fundyense was exposed to

waterborne cues released by copepods from Maine (indirect exposure) that were either starved or fed toxic

cells, cell toxin quota increased by 35% compared to unexposed controls. The induced response was signifi-

cantly less for cells exposed to waterborne cues of copepods from New Jersey, and induction (20%) was only

elicited by this population when fed toxic cells. These results suggest that A. fundyense responded to a kairo-

mone from copepods from Maine, but required a feeding cue from copepods from New Jersey. An increase of

approximately 300% in cell toxin quota, however, occurred when cells were directly exposed to grazing, and

was independent of copepod population. Evolutionary history, therefore, had no apparent effect when induc-

tion was underlain by feeding cues. In assays with a mixture of toxic and nontoxic cells, selection for the lat-

ter was evident, and also independent of copepod population. Selectivity for nontoxic cells, however, could

not account for changes in cell toxin content in the mixture experiments. When A. fundyense was exposed to

extracts of toxic or nontoxic Alexandrium, toxin production increased significantly (23%), suggesting modest

induction by an alga-to-alga alarm signal.

The prevalence and distribution of blooms of toxic algae

has raised concerns of toxin transfer through the food web;

from algae to commercially important finfish and shellfish

species, and subsequently to humans and other apex preda-

tors. Some species and strains of the marine dinoflagellate

genus Alexandrium produce a suite of potent neurotoxins (the

saxitoxins [STX]), which are transferred in such a manner and

cause paralytic shellfish poisoning (PSP; Anderson et al. 2012).

The prevalence of PSP is due to the ubiquitous distribution

of species of Alexandrium in coastal waters world-wide (Lilly

et al. 2007). Although, the mechanisms of initiation, persist-

ence, termination, and toxicity of blooms of Alexandrium are

not fully understood, grazing by zooplankton is recognized as

a factor of bloom regulation (Watras et al. 1985; Colin and

Dam 2007). Ironically, although grazing may initially control

populations of toxic algae, algal survival, and proliferation

may be enhanced when grazing stimulates increased produc-

tion of toxins or chemical deterrents by the threatened spe-

cies of phytoplankton (Wolfe 1996; Jang et al. 2003). Indeed,

copepod-induced production of STX by Alexandrium minutum

has been observed (Selander et al. 2006).

The grazer-induced toxin production by A. minutum is

highly specific to the species of copepod, with some of these

predators eliciting no increased toxin production on the part

of the prey (Bergkvist et al. 2008). The dissimilar responses of

Alexandrium to various species of copepod may be explained

by differences in the history of co-occurrence between the

predators and prey; i.e., prey historically exposed to a preda-

tor, may have evolved to identify and respond to cues

released by that predator (Bergkvist et al. 2008; Sih et al.

2010). Although, this hypothesis has not been tested for

*Correspondence: hans.dam@uconn.edu

†Present address: New York State Department of Environmental Conserva-tion, Long Island City, New York

1

LIMNOLOGYand

OCEANOGRAPHY Limnol. Oceanogr. 00, 2015, 1–21VC 2015 Association for the Sciences of Limnology and Oceanography

doi: 10.1111/lno.10027

marine phytoplankton, examples exist for marine inverte-

brate predator–prey interactions (Freeman and Byers 2006;

Edgell and Neufeld 2008). A potential complication of these

studies is that evolutionary history and phylogenetic effects

on the predator–prey interaction are confounded. This con-

cern is addressed in the present study by working with sepa-

rate populations of a single grazer species.

Cues for inducible defense, such as enhanced toxin pro-

duction, must be specific and effective in alerting prey to

predator presence or activity (Tollrian and Harvell 1999). In

phytoplankton, these cues may be a product of conspecific

cell lysis associated with mechanical damage to the cells and

require no contact with the grazer or its digestive system

[these are cell-to-cell cues also known as “alarm-substances”;

(Dodson et al. 1994)]. Alternatively, the cue may be a metab-

olite produced and released specifically by the grazer, and

require no contact between the predators and prey [also

known as a “kairomone”; (Dodson et al. 1994)]. Finally, the

cue may be feeding-related and released when cells or their

contents have made contact with the feeding or digestive

system of the grazer (Van Donk et al. 2011). Accordingly,

this study was designed to identify the cue(s) (alarm, kairo-

mone, or feeding-related) that induce production of toxins

by the dinoflagellate Alexandrium fundyense.

A hypothesis that has been left untested in previous induc-

tion studies is that selective feeding accounts for a portion of

the observed increase in toxin content of cells exposed

directly to grazers; that is, grazers discriminate against cells

with high toxin content within a prey population (Teegarden

1999; Selander et al. 2006). Because toxin content may vary

widely, even among cells of the same strain of Alexandrium

(Tillmann et al. 2009), grazers may reject the more toxic cells

of a population. Thus, cells remaining at the end of experi-

ments could be those of higher toxin content. Accordingly,

the relative contributions of selective feeding and induced

toxin production to overall toxin content of Alexandrium were

investigated.

The first goal of this study was to test whether or not the

evolutionary history of predator–prey, within a single predator

species, affects toxin induction in phytoplankton. The toxic

dinoflagellate A. fundyense occurs along the northwestern coast

of the Atlantic Ocean from Canadian waters to Long Island

Sound (between Connecticut and New York, latitude 41�). In

laboratory assays, we tested the hypothesis that toxic A. fun-

dyense recognizes and responds to co-occurring populations of

Acartia hudsonica from Maine with increased toxin production,

but not to populations from New Jersey, with which A. fun-

dyense is not known to co-occur (i.e., no blooms of A. fun-

dyense have been reported south of Long Island Sound; Colin

and Dam 2007). To our knowledge, this is the first study to

examine variation of an induced response in phytoplankton

prey to divergent populations of a single predatory species.

Another goal was to test whether induced production of

toxin is stimulated by cues released from the grazer as a kair-

omone, a feeding-related cue, or algal alarm signal. Lastly,

the contribution of feeding-selection to increased toxin con-

tent of A. fundyense exposed to grazers was assessed.

Methods

Collection and culture of copepods

The calanoid copepod A. hudsonica was collected from

Casco Bay, Maine [43�390N, 74�470W; historically co-

occurring with toxic A. fundyense (Colin and Dam 2004,

2007)], and Little Egg Harbor, Tuckerton, New Jersey

(39�630N, 74�330W; no blooms of A. fundyense have been

reported south of Northport Harbor, Long Island Sound,

New York, 40�5401000N 73�2003900W). Triplicate cultures for

each copepod population were maintained as described in

Colin and Dam (2007), with food medium replenished thrice

weekly. Copepods were cultured for at least three genera-

tions (approximately three months) prior to experiments to

eliminate maternal and environmental effects (Falconer and

MacKay 1996). Animals (eggs to adults) in cultures were

transferred monthly to new containers. Prior to assays, adult,

female copepods were acclimatized to experimental condi-

tions (see below) for 24 h and starved during that period to

ensure complete gut evacuation (Dam and Peterson 1988).

Culture of phytoplankton

The dinoflagellates, A. fundyense (toxic strain BF-5, isolated

from Bay of Fundy, Canada) and Alexandrium tamarense [non-

toxic, isolated from Mumford Cove, Groton, Connecticut

(Colin and Dam 2002)], were grown in semicontinuous culture

in F/2 medium without silicate (Guillard 1975). Cultures were

maintained in, and all experiments were conducted in, an envi-

ronmental chamber with fluorescent lighting set to a 12 h :12

h light : dark photoperiod (100 lmol m22 s21 photosyntheti-

cally active radiation) and 18�C. The A. fundyense strain was

used in grazer-enhanced toxin production, feeding-selection,

and algal alarm-cue assays while A. tamarense was used in the

feeding-selection and alarm-cue assays. Aside from production

of paralytic shellfish toxin, A. tamarense and A. fundyense are

nearly identical in shape, size, and carbon and nitrogen content

per cell (Balech 1990), which are properties known to affect

feeding selection in copepods (Vanderploeg 1994).

Assays on direct and indirect induction of toxin

production

Direct and indirect mechanisms of toxin induction were

tested simultaneously using experimental cages similar to

those of Selander et al. (2006). One liter polycarbonate

beakers with bottoms made of 10 lm mesh were nested

within another one liter beaker containing 500 mL of toxic

A. fundyense (300 cells mL21). The mesh isolated these cells

from materials within the cage. Adult female A. hudsonica

(15 individuals) from either Maine or New Jersey were added

to each cage and offered a diet of toxic A. fundyense (300

cells mL21) or were starved (no addition of algal food).

Senft-Batoh et al. Marine dinoflagellate toxin induction

2

Triplicate treatments (n 5 3) of the combinations of cope-

pods and algal food within the cages were: (1) Maine cope-

pods fed toxic algae; (2) Maine copepods starved; (3) New

Jersey copepods fed toxic algae; (4) New Jersey copepods

starved. Control cages (n 5 3) contained 300 cells mL21 of

toxic algae and no copepods.

Assays were run for 72 h, long enough to ensure induc-

tion of toxin production, and incubation conditions were

identical to those of the algal cultures. Cages were lifted

every 12 h to ensure exchange of cues between compart-

ments. At the termination of the assay, cells of A. fundyense

within cages (where applicable) and below cages were col-

lected from treatments and controls for toxin analysis (see

below). A one-way ANOVA coupled with post hoc Holm-

Sidak means analysis (Sokal and Rohlf 2012) was used to

determine significance of differences in cellular toxin con-

tent of A. fundyense grazed by copepods compared to control

cells that had not been exposed to grazers. This same analy-

sis was applied to the algae below the cages to determine

which waterborne cues elicited an increased response relative

to control cells. A 2 3 2 factorial ANOVA was performed for

the indirect induction assay to test significance of the effects

of population (Maine or New Jersey), diet (fed or starved), or

the interaction of these two variables (population 3 diet) on

cell-toxin content of A. fundyense.

Cells within cages (treatments and controls) were enumer-

ated microscopically, before and after incubation, to calculate

copepod ingestion rates (Frost 1972). Differences in ingestion

rate between the populations were assessed by a t-test.

Algal alarm-cue assay

Addition of crushed prey is a common method for testing

induced responses resulting from alarm cues in aquatic envi-

ronments (Chivers and Smith 1998; Schoeppner and Relyea

2009). As a first test to determine if alarm-cues released by A.

fundyense, and a congener (but nontoxic) species, A. tamar-

ense, could induce toxin production in a culture of A. fun-

dyense, extracts of sonicated, conspecific cells, or cells of A.

tamarense (equivalent to 50,000 cells; complete lysis con-

firmed microscopically), were added daily, over a three day

period, to triplicate treatments (n 5 3 for A. fundyense and A.

tamarense extracts) of 500 mL, nutrient-replete (F/2) cultures

of A. fundyense (300 cells mL21). The daily reinoculation of

cue-receiving cultures with sonicated extracts ensured that

potentially labile alarm cues (Selander et al. 2012) were main-

tained throughout the duration of the experiment. Extracts

were not added to control cultures (n 5 3). A one-way

ANOVA, with post hoc Holm-Sidak analysis of means, was

used to test for differences in per-cell toxin content between

treatments and controls at the end of the experiment.

Selective feeding assay

To test the contribution of feeding selectivity to increases

in cell-toxin content during grazing assays, copepods from

Maine and New Jersey were fed a 50 : 50 mixture (150 cells

mL21 each) of toxic A. fundyense and nontoxic A. tamarense.

The toxic strain was labeled with Cell TrackerVR Blue CMAC,

a fluorescent vital stain. A preliminary assay showed that the

label was maintained through cell divisions beyond 72 h (l5 0.2 d21, doubling time 5 2.3 d).

Twenty adult female A. hudsonica (from Maine or New Jer-

sey) were placed in triplicate one liter bottles containing the

50 : 50 diet mixtures in nutrient-replete (F/2) medium. Simi-

lar containers (n 5 3) with no copepods served as controls

for calculating growth rates of each species of Alexandrium.

Cells were maintained in suspension by rotating bottles

slowly, end over end, on a plankton wheel. At the end of

the 72 h assay, subsamples of cells (one milliliter) were pre-

served in 2% formaldehyde (which did not interfere with the

vital stain). Concentration of toxic (stained) cells was deter-

mined using an epifluorescence microscope. Toxic cells were

subtracted from total cell count, made on an inverted micro-

scope, to calculate concentration of nontoxic cells. Ingestion

rates of copepods were calculated by cell disappearance using

the equations of Frost (1972).

The alpha index of selectivity (Chesson 1983) for non-

toxic A. tamarense (an) was calculated as:

an5rn=pn

ðrn=pn1rt=ptÞ(1)

where r is the proportion of food type n (nontoxic A. tamar-

ense) or t (toxic A. fundyense) ingested and p is the proportion

of that corresponding food type offered in the mixture. Pro-

portions based on number of cells ingested rather than car-

bon ingested were used because carbon content of cells was

similar between food types (�2.1 3 1023 [toxic] and �2.0 3

1023 lg C cell21 [nontoxic]). The null hypothesis of no

selection for nontoxic prey (an 5 at 5 0.5; where at is the

alpha index for toxic A. fundyense) was tested against the

alternative (an 6¼ at) by calculating the t statistic,

t5�an20:5ffiffiffiffiffiffiffiffiffiffis2=K

p (2)

where �an is the sample mean and s2 is the sample variance

of the K estimates (K 5 3) of an. If jtj was greater than the

critical point of the t distribution on K 2 1 degrees of free-

dom (df), then the null hypothesis was rejected.

Following incubation, the expected, per-cell toxin content

(toxic 1 nontoxic cells) of grazed mixtures was determined

using measurements of per-cell toxin content of A. fundyense

(10 pgSTXeq cell21) and A. tamarense (0 pgSTXeq cell21)

prior to incubation, assuming that only feeding selection

(no induction of toxin production) had occurred. Actual per-

cell toxin contents of the mixed diets were also measured

following incubation; therefore, after incubation, if toxin

content per cell was significantly greater for the treatments

than what was predicted from feeding selection alone,

induced production of toxins was inferred. The mechanism

Senft-Batoh et al. Marine dinoflagellate toxin induction

3

(enhanced production and feeding selection), grazer popula-

tion (Maine and New Jersey), and the interaction (mecha-

nism 3 population) were analyzed for effects on increased

toxin content using multifactor 2 3 2 ANOVA. Statistical

analyses for this and all prior assays were performed using

SigmaPlot version 11.0 software.

Importantly, the vital stain itself has no effect on selectiv-

ity of copepods in the mixed diet feeding assay. Preliminary

experiments, conducted over 24 h, used a reciprocal design,

in which toxic and nontoxic cells were alternatively labeled

in mixed diet assays for both populations of copepod (Maine

and New Jersey). Ingestion of nontoxic cells was significantly

higher than ingestion of toxic cells irrespective of which algal

species was labeled (Maine: diet [F1,8 5 49.30, p<0.001]; New

Jersey: diet [F1,8 5 49.33, p<0.001]). Labeling of cells, and

the interaction of label and diet, had no effect on selection

by copepods (Maine: label [F1,8 5 0.08, p 5 0.790], label 3

diet [F1,8 5 2.19, p 5 0.177]; New Jersey: label [F1,8 5 4.46, p

5 0.068], label 3 diet [F1,8 5 0.20, p 5 0.668]). These results

are consistent with those of Teegarden (1999).

Toxin analysis

Cells from treatments and controls were collected on a

10 lm mesh and resuspended in filtered seawater in a 50 mL

centrifuge tube. Replicate subsamples (one milliliter) were

taken from each tube, and cells were enumerated micro-

scopically to determine the number of cells present in each

extract (50,000–150,000 cells, depending on the assay). Cells

were centrifuged at 4000 3 g for 20 min. The seawater super-

natant was decanted, and the cell pellet was resuspended in

one milliliter of 0.1 mol L21 acetic acid. Cells were lysed

using a probe sonic dismembrator. Sonication was conducted

with the tube immersed in ice to prevent heating of the sam-

ples. Samples were then centrifuged again at 4000 3 g for

20 min to remove cell debris. The acetic acid supernatant

(extract) was filtered through a 0.45 lm ultracentrifuge filter

cartridge to remove any remaining particles. Samples were

stored at 280�C until analysis.

Concentrations of saxitoxin, neosaxitoxin, and gonyautox-

ins 1 through 4 (GTX 1–4), as well as the sulfamate congeners,

C1 and C2 were measured using high-performance liquid

chromatography with fluorescence detection (Oshima 1995)

after calibration with standards (Certified Reference Materials

Program, NRC Institute for Marine Biosciences, Canada) and

expressed as mass of STX equivalents per cell according to con-

version factors of Oshima (1995). Because ratios of congeners

did not vary as a result of induction, the toxin content of cells

in the unit fmol cell21 may be obtained by multiplying the

content in unit pgSTXeq cell21 by a conversion factor of 2.54.

Results

Direct and indirect induction of toxin production

Toxin content of A. fundyense differed significantly among

treatments of algae directly exposed to grazers and control

cells that had not been exposed to grazers (ANOVA, F2,6 5

480.49, p<0.001; Fig. 1A). Algae directly exposed to either

the Maine or New Jersey populations of copepods, increased

toxin content by >300% (� 80 pgSTXeq cell21) compared to

control cells (20 pgSTXeq cell21; Holm-Sidak, Maine:

p<0.001, New Jersey: p<0.001; Fig. 1A). There was, how-

ever, no difference in direct induction of toxin production

between grazer populations (p 5 0.17; Fig. 1A).

Copepods from Maine consumed toxic cells at a signifi-

cantly higher rate (�50 cells copepod21 h21) than copepods

from New Jersey, (�10 cells copepod21 h21; t-test, t 5 4.528,

df 5 4, p<0.001; Fig. 2).

Toxin content of A. fundyense exposed indirectly to cues

released by copepods differed significantly from control cells

that were not exposed to cues (ANOVA, F4,10 5 18.68,

p<0.001; Fig. 1B). Through indirect mechanisms, copepods

from Maine, either starved or fed toxic A. fundyense, caused

an increase in toxin content of cue-receiving A. fundyense of

35% (up to 27 pgSTXeq cell21; Holm-Sidak, starved:

p<0.001, fed: p<0.001; Fig. 1B) compared to control cells

(20 pgSTXeq cell21). The indirect responses elicited by

starved or fed copepods from Maine did not differ (Holm-

Sidak, p 5 0.97; Fig. 1B). Toxin content of cells that received

cues from grazing copepods from New Jersey, averaged

approximately 25 pgSTXeq cell21; a 20% increase compared

to control cells (Holm-Sidak, p 5 0.012; Fig. 1B). Starved

copepods from New Jersey, however, did not induce toxin

production (Holm-Sidak, p 5 0.37; Fig. 1B). The factorial

ANOVA revealed significant effects of grazer population

(Maine vs. New Jersey; F1,8 5 49.50, p<0.001), diet (fed vs.

starved; F1,8 5 9.35, p 5 0.016), and the interaction of popu-

lation and diet (F1,8 5 9.71, p 5 0.014) on induced toxin

production (Table 1).

Algal alarm cues

Toxin content of A. fundyense receiving cues (extracts)

from lysed conspecific cells or from lysed nontoxic A. tamar-

ense was significantly higher than control cells that were

unexposed to any cues (ANOVA, F2,6 5 7.55, p 5 0.023; Fig.

1C). Toxin content of A. fundyense receiving cues from lysed,

conspecific cells (37 pgSTXeq cell21) increased by 23% com-

pared to control cells (30 pgSTXeq cell21; Holm-Sidak, p 5

0.012; Fig. 1C), whereas an increase of 20% (36 pgSTXeq

cell21; Holm-Sidak, p 5 0.021; Fig. 1C) was observed with

cues from A. tamarense. There was no significant difference

in enhancement of cell-toxin content by lysis products of

toxic or nontoxic cells (Holm-Sidak, p 5 0.65; Fig. 1C).

Selective feeding

Alpha indices indicated that nontoxic A. tamarense (an)

was consumed in higher proportions (an>0.5) than the 50 :

50 proportion in which it was offered, with toxic A. fun-

dyense, by copepods from both Maine (an 5 0.77 (0.06); t 5

4.65, df 5 2, p 5 0.022; Fig. 3) and New Jersey (an 5 0.87

(0.03); t 5 12.427, df 5 2, p<0.003; Fig. 3). Copepods from

Senft-Batoh et al. Marine dinoflagellate toxin induction

4

Maine consumed nontoxic A. tamarense at a rate of 15 cells

copepod21 h21, much higher than the ingestion rate of 5

cells copepod21 h21 of toxic A. fundyense (t 5 4.246, df 5 4,

p 5 0.013; Fig. 3). Likewise, copepods from New Jersey con-

sumed nontoxic cells (18 cells copepod21 h21) at a higher

rate than toxic cells (3 cells copepod21 h21; t 5 2.842, df 5

4, p 5 0.047; Fig. 3). There was no difference in total inges-

tion rate (toxic 1 nontoxic; t 5 0.062, df 5 4, p 5 0.95),

ingestion rate of toxic cells (t 5 1.501, df 5 4, p 5 0.21), or

ingestion rate of nontoxic cells (t 5 0.459, df 5 4, p50.67;

Fig. 3) between copepod populations.

Toxin contents of cells grazed in the mixed-species treat-

ments were much higher than in controls (ANOVA, F2,6 5

55.55, p<0.001) after exposure to copepods from Maine

(92% increase) and New Jersey (134% increase; Table 2). The

overall increase in toxin content, however, did not differ

statistically between copepod populations (t 5 2.654, df 5 4,

p 5 0.057; Table 2). In the food mixtures, selective ingestion

alone accounted for increases in per-cell toxin content of 0.9

pgSTXeq cell21 (18% increase) and 1 pgSTXeq cell21 (20%),

compared to controls (5 pgSTXeq cell21), for Maine and New

Jersey populations, respectively (Table 2). Increase in toxin

content attributable to selective feeding by copepods did not

differ between populations (t 5 0.413, df 5 4, p 5 0.70). In

contrast, increases in cellular toxin content attributable to

induced production were significantly different (t 5 2.92, df

5 4, p<0.043): 3.67 pgSTXeq cell21 corresponding to 73%

enhancement) for copepods from Maine and 5.70 pgSTXeq

cell21 (114%) for copepods from New Jersey (Table 2). The

factorial 2 3 2 ANOVA confirmed that mechanism (feeding-

selection vs. induction) had a significant effect on cell toxin

content (F1,8 5 106.74, p<0.001; Table 3), with the effects

of induced toxin production being stronger than the effects

of feeding selection for both populations (Holm-Sidak,

p<0.001). Population (F1,8 5 8.54, p < 0.019) and the inter-

action of mechanism and population (F1,8 5 7.28, p<0.027)

also had significant effects on cell toxin content (Table 3).

Discussion

This study demonstrates that when copepod grazers, A.

hudsonica, feed on toxic A. fundyense, history of co-

occurrence between the grazer population and the prey has

Fig. 1. Induced toxin production in A. fundyense. (A) Direct induction

(within cage); NJ 1 TOX and ME 1 TOX treatments represent toxincontent (pgSTXeq cell21) of cells directly exposed to grazing by New

Jersey and Maine copepods, respectively. (B) Indirect induction (outsidecage; cue-receiving cells); NJ Starved, ME Starved and NJ 1 TOX, ME 1

TOX treatments represent toxin content of cells exposed to waterborne

cues of starved copepods or copepods grazing on toxic A. fundyense,respectively. (C) Algal-cue induction; NONTOX Extract and TOX Extracttreatments represent induction by cues from lysed cells of nontoxic A.

tamarense and toxic A. fundyense, respectively. Asterisks indicate signifi-cant differences between mean values of groups compared to controls

(that are unexposed to grazing or cues) and also among treatments(ANOVA followed by post hoc Holm-Sidak procedure, p<0.05). A dou-ble asterisk indicates significantly greater mean toxin content of the

treatment compared to other induced treatments indicated by a singleasterisk. Error bars represent 6 1 standard error of the mean (n 5 3).

Senft-Batoh et al. Marine dinoflagellate toxin induction

5

no effect on the degree of enhanced toxin production of the

alga. The results suggest, however, that kairomone cues may

play a role in enhancing toxin production by A. fundyense

when in the presence of historically co-occuring grazers (i.e.,

Maine), but not in the presence of grazers to which the alga

is na€ıve (i.e., New Jersey). For toxin production to be

enhanced in the case of the latter, a feeding-related cue is

necessary. The findings also suggest that algal alarm-cues

may serve as chemical signals for induced toxin production

by A. fundyense. The results of the selection assay imply that

behavioral rejection of highly toxic cells by copepods has a

significant, but relatively small, effect on the observed

increases in per-cell toxin content of grazer-exposed cells.

Direct induction of toxin production

When directly exposed to grazing, induced toxin produc-

tion by A. fundyense was not affected by history of co-

occurrence between the alga and the grazer. There was no

difference in induction when A. fundyense was grazed on

(within cages) by copepods, A. hudsonica, from Maine (with

which the alga co-occurs) or New Jersey (to which the alga is

na€ıve; p 5 0.17; Fig. 1A). The ingestion rate of toxic cells of

copepods from Maine was, however, 400% higher than cope-

pods from New Jersey (Fig. 2). Therefore, the degree of

induction in cells exposed directly to grazers is independent

of grazing rates.

Direct grazing (by either Maine or New Jersey popula-

tions) yielded the greatest increase in toxin production

(�300%) by A. fundyense of any assay (Fig. 1A). The disparity

in toxin production between A. fundyense exposed directly

and indirectly (see Indirect Induction of Toxin Production

section) to grazers was marked, and hypothesized to be the

result of feeding-selection for less-toxic cells by copepods in

the direct-exposure assays. Selectivity, however, accounted

for a smaller percentage of the observed increase in toxin

content of directly grazed A. fundyense than induction (see

Feeding Selection Assay section). The magnitude of direct

induction being greater than indirect induction may stem

from algae being physically closer and perhaps manipulated

by predators when grazed within cages. In other words, mag-

nitude of induced toxin production by A. fundyense may be

related to the immediacy of the threat of predators.

Indirect induction of toxin production

The response of A. fundyense to cues from starved cope-

pods from Maine, but not New Jersey (Fig. 1B), suggests that

this species has evolved signal receptors that are sensitive to

kairomones released by co-occurring copepods (Stacey and

Sorensen 2002). Because A. hudsonica from Maine are toler-

ant of algal STX and can maintain high ingestion rates of

toxic cells (Colin and Dam 2004, 2007; Fig. 2), it would be

evolutionarily beneficial for A. fundyense to recognize cues

emitted by these copepods. Then, prior to attack, cellular

toxin content may be increased to levels that could poten-

tially incapacitate (Teegarden et al. 2008) or more efficiently

deter even tolerant copepods (Teegarden 1999; Selander

et al. 2006; and see Feeding Selectivity section).

Although, cues released by starved copepods from New

Jersey did not increase toxin content of A. fundyense, cues

from the same population of copepods fed toxic cells did

(Fig. 1B). Clearly, there is not only an effect of the grazer

population on indirectly enhanced toxin production but

also an effect of the diet of the grazer (fed toxic algae vs.

starved) and the interaction of diet and population; i.e., the

Fig. 2. Ingestion rates (cells copepod21 h21) of copepods from NewJersey and Maine on toxic A. fundyense (300 cells mL21) in the direct

(within cage) induction assay. An asterisk indicates a significant differ-ence between mean values of the treatments (a 5 0.05). Error bars rep-

resent 6 1 standard error of the mean (n 5 3).

Table 1. Factorial ANOVA results for indirect induction assay.Factors: copepod population (levels: New Jersey and Maine),diet level: fed toxic algae or starved) and the interaction of pop-ulation and diet. The dependent variable is toxin content of A.fundyense. SS(III), type III sum of squares; df, degrees of free-dom; MS, mean sum of squares for ANOVA; F, statistic forANOVA test; p, significance of the ANOVA test.

Source SS(III) df MS F p

Model 225.15 3 75.05 22.85 <0.001

Diet 30.70 1 30.70 9.35 0.016

Population 162.56 1 162.56 49.50 <0.001

Diet�population 31.89 1 31.89 9.71 0.014

Error 26.27 8 3.28

Corrected total 251.42 11

Senft-Batoh et al. Marine dinoflagellate toxin induction

6

response of A. fundyense to each population of copepod

varied depending on whether the animals were fed or

starved (Table 1). Consequently, there seem to be two chem-

ical signals influencing grazer-induced toxin production by

A. fundyense: kairomones released by co-occurring copepods,

and a cue or cues released when algae are physically handled

or digested by copepods (feeding-related cue). Such a

feeding-related cue would also be responsible for induced

toxin production of A. fundyense directly exposed to grazers.

Induced toxin production by algal cues

The contents of mechanically lysed A. fundyense increased

toxin production in conspecific, cue-receiving cells (23%

enhancement Fig. 1C). Thus, copepods feeding on toxic cells

may have caused release of an alarm-cue from grazed cells

[attributable to handling or sloppy feeding (Møller 2007)],

perhaps contributing to the overall induction observed in

the direct induction assays and the indirect induction assay

in which algae had been exposed to waterborne signals

released by actively feeding copepods. The alarm-cue assays,

however, must be interpreted with caution because the

experimental design used has inherent limitations. First, the

concentration of cells used to make the extracts vastly

exceed those in the field, and consequently what copepods

would have consumed. Second, use of lysed cells is equiva-

lent to the release of all cellular contents via sloppy feeding,

and assumes no ingestion of cellular material by grazers.

Therefore, the assays in this study can only suggest that

alarm cues could be triggered on grazing of cells (see Direc-

tions for Future Investigations section).

Importantly, the A. fundyense alarm-cue is not likely a STX

congener. Lysed cellular products of nontoxic A. tamarense (0

pgSTXeq cell21) induced toxin production by the same magni-

tude (20%; Fig. 1C) as closely related toxic A. fundyense. The

ability of A. fundyense to identify and respond to alarm-cues,

termed “the smell of death” (Hay 2009), from damaged con-

specific, and closely related cogeneric cells, indicates that alarm

cues may be used by A. fundyense to respond to predation in

general (not only specifically to threatened conspecifics).

Feeding selectivity

The hypothesis that discrimination by copepods against

toxic cells could affect the overall per-cell toxin content of A.

fundyense exposed to grazers was rejected. The copepods, A.

hudsonica, from Maine and New Jersey preferentially con-

sumed nontoxic A. tamarense when offered as a 50 : 50 mix-

ture with toxic A. fundyense. The percentages of nontoxic cells

consumed as a portion of the total (77% for copepods from

Maine; 87% for copepods from New Jersey) indicated a strong

preference for nontoxic cells (Fig. 3). Such selection, however,

could not explain the observed increase in cell-toxin content

during the experiments. Selection for nontoxic cells by cope-

pods from Maine and New Jersey accounted for 20% (18 : 91)

and 15% (20 : 134) of the observed increase in cell toxin-

content during the selective feeding assays with Maine and

New Jersey copepods, respectively (Table 2).

Active rejection of toxic cells by A. hudsonica confirms

that toxins are an effective grazing deterrent (Teegarden

1999; Selander et al. 2006) and may result in the persistence

Fig. 3. Ingestion rates (cells copepod21 h21) of copepods from Maineand New Jersey on mixed diets of 50% toxic (A. fundyense; dark grey

bars) and 50% nontoxic (A. tamarense; light gray bars) cells. An asteriskindicates significant differences between mean ingestion rates on the two

strains. an is the Chesson index of selectivity. Values >0.5 indicate prefer-ence for nontoxic cells. Standard error values (n 5 3) are in parentheses.

Senft-Batoh et al. Marine dinoflagellate toxin induction

7

of populations of toxic algae (Teegarden 1999; Wolfe 2000;

Schultz and Kiørboe 2009). Furthermore, in another study,

cells of toxic algae that had been indirectly exposed to graz-

ers, and had measurably enhanced toxin content, were

shown to be consumed at significantly lower rates than cells

that were not induced to increase toxin production when

the two prey types were offered as a mixture (Selander et al.

2006). In summary, cells induced to produce more toxin

may enjoy a refuge from grazing.

Possible sources of bias

Three possible sources of bias to the experiments were

considered.

1. Measured cell toxin content at the end of incubation

includes toxins from copepod feces and eggs and results in

erroneously high measurements of induction. Such an arti-

ficial enhancement in measured induction is unlikely as

less than 5% of ingested STX are retained in eggs (our own

unpublished observations) and fecal pellets (Teegarden

et al. 2003). Using the measured per-copepod ingestion

rate (Fig. 2), the concentration of copepods in incubations,

the measured 20 pgSTXeq cell21 (control in Fig. 1), and

STX retention efficiency of 5% for eggs and pellets, total

toxin content in eggs and pellets at the end of the experi-

ment is estimated to be 2 3 104 (New Jersey) and 1.1 3

104 pgSTXeq (Maine). Cell concentrations at the end of

the experiment in the copepod treatments were 635 (New

Jersey) and 544 cells mL21 (Maine). Using the cell toxin

content of 20 pgSTXeq cell21 and experimental volume of

500 mL, the total toxin concentrations were 6.3 3 106

(New Jersey) and 5.4 3 106 pgSTXeq (Maine). Hence, fecal

pellets and eggs would account for only 0.3 (New Jersey)

and 0.2% (Maine) of the expected total cell toxin content

in the feeding-related cue experiments.

2. Ammonium, released as a metabolic byproduct from cope-

pods, enhanced toxin production in A. fundyense (Ferrari

et al. 2010). It is highly unlikely that ammonium had such

an effect on the induction assays. First, all experiments

were conducted in nutrient replete F/2 media (Guillard

1975). The inorganic nitrogen source of F/2 is nitrate

(NO23 ), and it has been shown that ammonium addition to

cultures of Alexandrium spp. will only enhance toxin pro-

duction (compared to nitrate) when phosphate (PO324 ) con-

centrations are below approximately 10 lmol L21 (Lim

et al. 2010). Experimental phosphate concentrations would

have been at least 36 lmol L21 (the concentration of phos-

phate in F/2 media; Guillard 1975); thus, the effect of

ammonium on toxin production rate would be no differ-

ent than that of nitrate in this range (Lim et al. 2010 [see

their Fig. 6]). Second, using the A. hudsonica congener spe-

cies, Acartia tonsa, as a proxy, we estimate that the maxi-

mum concentration of ammonium released into

experimental containers by copepods over the course of

the assays would be 0.625 lmol L21 (based on calculations

using Fig. 8 of Kiørboe et al. 1985). This is an order of mag-

nitude less than concentrations of ammonium reported to

stimulate toxin production (Leong et al. 2004).

More direct experimental evidence refuting a stimulatory

effect of ammonium on cellular toxin content is the

approximately fivefold difference in ingestion rate between

copepod populations in direct grazing assays (Fig. 2). Inges-

tion rates of copepods from Maine were much higher than

those of New Jersey. This would result in a higher excre-

tion rate of ammonium by the copepods from Maine. The

Table 2. Contributions of selective feeding and copepod-induced production to increases in per cell (total toxic and nontoxic cells)toxin content in mixed diet assays. Values are means and standard errors (in parentheses), n 5 3. Italics indicate significant differencesbetween Maine and New Jersey populations. Previous experiments verified that nontoxic cells are not induced to produce toxinwhen exposed to grazers or cues.

Population

Total increase

in toxin

content (%)

Increase in

toxin content

due to selection (%)

Increase in

toxin content

due to selection

(pgSTXeq cell21)

Increase in

toxin content

due to grazer

induction (%)

Increase in toxin

content due to grazer

induction (pgSTXeq cell21)

Maine 91.66 (12.71) 18.24 (0.46) 0.91 (0.02) 73.42 (12.28) 3.67 (0.61)

New Jersey 133.90 (9.58) 19.86 (3.89) 0.99 (0.19) 114.04 (6.54) 5.70 (0.33)

Table 3. ANOVA results for feeding-selection assay. Factors:mechanism (levels: feeding-selection or induced production),copepod population (levels: New Jersey or Maine), and theinteraction of mechanism and population on toxin content ofcells. The dependent variable is toxin content of A. fundyense.SS(III), type III sum of squares; df, degrees of freedom; MS,mean sum of squares for ANOVA; F, statistic for ANOVA test; p,significance of the ANOVA test.

Source SS(III) df MS F p

Model 47.98 3 15.99 41.00 <0.001

Mechanism 41.83 1 41.83 106.74 <0.001

Population 3.35 1 3.35 8.54 0.019

Mechanism�population 2.85 1 2.85 7.28 0.027

Error 3.14 8 0.39

Corrected Total 51.12 11

Senft-Batoh et al. Marine dinoflagellate toxin induction

8

higher ammonium concentration from the Maine treat-

ments, however, did not translate to differences in toxin

content of directly grazed A. fundyense compared to cope-

pods from New Jersey (Fig. 1A). Additionally, nonfeeding

copepods from either population would have similar rates

of ammonium excretion. Toxin content of cells exposed to

waterborne effluent of the nonfeeding copepods, although,

differed between populations, with toxin content being

enhanced for A. fundyense exposed to effluent of copepods

from Maine but not New Jersey.

3. Dissolved organic nitrogen (DON) released from the soni-

cated cellular extracts enhanced toxin production in the

algal-cue induction assay. There is no unequivocal indica-

tion that cell toxin content is higher with DON, com-

pared to nitrate, as a source of nitrogen (Dyhrman and

Anderson 2003; Hattenrath et al. 2010).

Broader implications of induced toxin production

Much like a keystone species, prey toxins can cause cas-

cading effects leading to significant restructuring of aquatic

communities (Pohnert et al. 2007; Zimmer and Ferrer 2007).

Induced toxin production by dinoflagellates could expose

fisheries, shellfisheries, and aquaculture facilities, to a higher

risk for contamination and extended closure as populations

of the toxic species spread geographically and become estab-

lished in new habitats (Bricelj et al. 2005; Matsuyama and

Shumway 2009). In particular, this study suggests that even

grazer species to which a toxic alga is na€ıve may contribute

to perpetuation of blooms of the alga via two mechanisms:

enhanced toxin production when in direct contact with

cells, and avoidance of toxic algal prey when presented with

alternate prey choices in the field.

Directions for future investigations

The possible existence of a kairomone in populations of

A. hudsonica that have a history of exposure to toxic A. fun-

dyense deserves further attention. Studies involving multiple

species of toxic Alexandrium and other grazer groups in addi-

tion to copepods are needed, as well as studies dealing with

dissipation of the kairomone signal, particularly under field-

like conditions. In addition, more experimental designs are

needed to investigate prey alarm-cues triggered by grazers

under realistic field conditions. For example, prey could be

exposed to the dissolved fraction of the water containing

cells and grazers at the end of an experiment. As with the

kairomone studies, alarm-cue tests should be done for several

strains of toxic algae.

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Acknowledgments

We thank David Avery, Hayley Skelton, and Michael Finiguerra for sug-gestions that improved this study, and Claudia Koerting for her assistance

with high-precision liquid chromatography and toxin analyses.Research was supported by grants from the National Oceanographic

and Atmospheric Administration’s Ecology and Oceanography of Harm-ful Algal Blooms program, grant NA06NOS4780249, National ScienceFoundation’s Division of Ocean Sciences grants 0648126 and 1130284,

and Connecticut Sea Grant R/LR-21. Additional support came from theDepartment of Marine Sciences and the Center for Environmental Scien-ces and Engineering at the University of Connecticut, and from award

from the Lerner-Grey Fund of the American Museum of Natural Historyand the Quebec-Labrador Fund, Sounds Conservancy Program.

Submitted 9 May 2014

Revised 12 November 2014

Accepted 12 November 2014

Associate editor: Thomas Kiørboe

Senft-Batoh et al. Marine dinoflagellate toxin induction

11