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Intoxicated copepods: ingesting toxic
phytoplankton leads to risky behaviour
Rachel S. Lasley-Rasher1, Kathryn Nagel2, Aakanksha Angra3
and Jeannette Yen2
Research
Cite this article: Lasley-Rasher RS, Nagel K,
Angra A, Yen J. 2016 Intoxicated copepods:
ingesting toxic phytoplankton leads to risky
behaviour. Proc. R. Soc. B 283: 20160176.
http://dx.doi.org/10.1098/rspb.2016.0176
Received: 26 January 2016
Accepted: 1 April 2016
Subject Areas:
behaviour, ecology, environmental science
Keywords:
harmful algal blooms, Alexandrium fundyense,
copepod behaviour, Temora longicornis
Author for correspondence:
Rachel S. Lasley-Rasher
e-mail: [email protected]
1
School of Marine Sciences, University of Maine, Darling Marine Center, 193 Clarks Cove Road, Walpole,
ME 04573, USA
2
School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, GA 30332, USA
3
Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA
Understanding interactions between harmful algal bloom (HAB) species
and their grazers is essential for determining mechanisms of bloom proliferation and termination. We exposed the common calanoid copepod, Temora
longicornis to the HAB species Alexandrium fundyense and examined effects
on copepod survival, ingestion, egg production and swimming behaviour.
A. fundyense was readily ingested by T. longicornis and significantly altered
copepod swimming behaviour without affecting copepod survival or fitness.
A. fundyense caused T. longicornis to increase their swimming speed, and the
straightness of their path long after the copepods had been removed from
the A. fundyense treatment. Models suggest that these changes could lead to
a 25–56% increase in encounter frequency between copepods and their predators. This work highlights the need to determine how ingesting HAB species
alters grazer behaviour as this can have significant impacts on the fate of
HAB toxins in marine systems.
1. Background
Blooms of toxic phytoplankton, commonly referred to as harmful algal blooms
(HABs), have devastating consequences for coastal economies and can pose a significant threat to human health [1]. As such, there has been a great deal of research
in recent decades targeting mechanisms that control bloom formation, propagation and termination. A key factor responsible for bloom proliferation is the
lack of adequate grazing pressure, reviewed by [2–5]. Therefore, understanding
interactions between grazers and HAB species is of critical importance.
Copepods are important grazers of HABs as they can serve as vectors for toxin
entry into pelagic food webs [6,7]. Therefore, much work has been done to determine the effects of harmful algae on copepod survival and fitness. Behavioural
studies have consisted mostly of measuring copepods’ immediate feeding
response to HAB species, such as cell rejection, avoidance or incapacitation
[8–10]. Fewer studies have documented the effects of harmful algae exposure
on copepod swimming behaviour, but see [11,12]. Owing to the high search
volume rates of marine copepods (approximately 100 l d21 [13]) and the patchy
nature of HABs [14–17], copepods may experience intermittent exposure to
patches containing HAB species followed by HAB-free areas. However, little is
known about the lasting effects of HAB exposure on copepod swimming behaviour. Effects of HABs on copepod behaviour could be important, as swimming
behaviour has a direct impact on copepod dispersal and encounter rates with
predators [18–20], both of which determine the propensity of copepods to
graze HABs and transfer HAB toxins through marine food webs.
Alexandrium fundyense is a neurotoxic dinoflagellate that causes HABs along
northeastern United States and southeastern Canada. These bloom events have
devastating impacts on the economy of these areas owing to shellfish fisheries
closures and outbreaks of paralytic shellfish poisoning (PSP), which cause
severe human health effects [21,22]. Furthermore, the frequency and intensity
of A. fundyense blooms have increased in recent years [22,23]. Grazing interactions
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
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(b) Toxin analysis
To verify the toxicity of our A. fundyense stock culture, two samples
were shipped overnight to Greenwater Laboratories in Palatka, FL.
The first sample was analysed using a competitive enzyme-linked
immunosorbent assay (ELISA) to detect the presence of saxitoxin
(the most potent of the PSP toxin derivatives). However, ELISA
is not reactive with all PSP toxin derivatives and often underestimates the total toxins present. Owing to logistical constraints, a
detailed toxin profile was analysed only for the second sample.
The toxins examined were N-sulfocarbamoyl-11-hydroxysulfate
toxins (C1 and C2), gonyautoxin 1 –4, saxitoxin, neosaxitoxin
and other decarbamoyl derivatives. Toxins were analysed by
high-pressure liquid chromatography (HPLC) with fluorescence
detection following a pre-column oxidation method.
(a) Collection and culture of organisms
(c) Swimming behaviour
Our target grazer species, T. longicornis, is a calanoid copepod
that co-occurs with A. fundyense in the Gulf of Maine. We collected
T. longicornis by boat from the Damariscotta River estuary, Walpole,
ME (438560 N, 698350 W) by obliquely towing a plankton net with a
mesh size of 250 mm at approximately 30 m depth. Upon collection,
animals were immediately transferred into 20 l containers of surface seawater and transported to a temperature-controlled room.
Animals were transferred to 1 l polyethylene bottles within 24 h
of collection. Bottles were packed in an insulated box containing
ice packs and cushioning material to minimize stress and maintain
a cool temperature (approx. 5–108C). Animals were shipped overnight to the Georgia Institute of Technology, Atlanta, GA where
they were diluted in artificial seawater and allowed to acclimate
to their natural temperature over 24 h. Copepods were fed mixed
cultures of Tetraselmis sp., and Isochrysis galbana. After a 24 h acclimation period, adult T. longicornis were sorted from mixed samples
and placed in containers filled with filtered seawater at densities
of fewer than 25 individuals l21.
Alexandrium fundyense was chosen as our ‘HAB’ species
because it is known to contain PSP toxins. The strain we used
(NCMA 1719) was obtained from the National Center for Marine
Algae and Microbiota, Boothbay Harbor, ME. Cell biovolume
(6.61 103 mm3) was calculated treating the cell as a rotational ellipsoid. Cell dimensions 26.6 + 1.9 mm, 21.8 + 0.5 mm
(mean + s.e.) were measured from images using a FlowCAM
(n ¼ 12). Rhodomonas lens (NCMA 739, cell biovolume 3.36 102 mm3 calculated as a rotational ellipsoid from dimensions
13.5 + 1.8 mm, 6.9 + 0.3 mm [mean + s.e.], n ¼ 7) was used as
our non-toxic ‘control’ algae because it is commonly fed to
copepods in culture owing to its nutritional quality [33].
All phytoplankton species were cultured in L1 media, exposed to a
14 : 10 light : dark cycle and maintained at 148C (for A. fundyense)
and 218C (for R. lens). Phytoplankton were kept in exponential
phase during culturing by diluting with media every few days.
Phytoplankton treatments were prepared by conducting triplicate
cell counts (each count consisting of more than 200 cells) of our
stock phytoplankton cultures. We then removed the appropriate
volume with a serological pipette and diluted with 0.7 mm filtered
artificial seawater. The diluted phytoplankton mixture was cooled
to the experimental temperature of 148C if necessary. We inverted
the phytoplankton mixture several times to promote a homogeneous
cell distribution before pouring into experimental vessels.
Carbon : nitrogen ratios for A. fundyense and R. lens were analysed by Micro-Dumas combustion at the University of Georgia,
Athens, GA. Culturing and harvesting conditions were kept constant during all experiments to minimize any physiological
differences within phytoplankton species. All phytoplankton
cultures were in late exponential phase when harvested for
The aim of our behavioural experiment was to determine if the
swimming behaviour of T. longicornis is altered by exposure to
A. fundyense. Fifteen male and 15 female T. longicornis were placed
in a 1 l glass tank containing filtered seawater and fed one of the following treatments: A. fundyense treatment that contained a mixture
of 320 cells ml21 of A. fundyense þ 1120 cells ml21 of R. lens or a control treatment that contained 5600 cells ml21 of R. lens. We
calculated the diet composition using biovolume equivalents to
ensure that copepods were given equivalent total biovolumes of
food in both experimental groups. Copepods were starved for
12 h prior to experiments. We chose 320 cells ml21 for our A. fundyense treatment to ensure that copepods were not food limited.
Although this concentration is quite high for A. fundyense blooms
in the Gulf of Maine, where maximum cell densities are approximately 10 cells ml21 [34], this is within the range of A. fundyense
cell densities measured in other areas [35]. Furthermore, we simultaneously offered T. longicornis an alternative food, R. lens, to
avoid ‘force-feeding’ the copepods A. fundyense.
After exposing copepods to treatments for 2 h, we carefully
transferred them to a tank with filtered seawater amended with
Tetraselmis sp. under non-limiting conditions (ca 103 cells ml21).
Tetraselmis sp. has been fed to copepods during depuration periods
in other A. fundyense grazing experiments [36]. Tanks were covered
with parafilm and placed in a temperature-controlled room where
copepods were allowed to feed for 15 h before being used in behavioural experiments. We repeated this exposure process with five
replicate tanks for both treatments.
We chose a 2 h exposure time to mimic a brief encounter
between a copepod and an A. fundyense patch but allow
enough time for the copepods to fill their guts [37]. The purpose
of a 15 h recovery period was to allow copepods to feed on control algae, so that changes in their behaviour would not be due to
differences in hunger levels (caused by any differences in the
nutritional value of A. fundyense versus R. lens). Fifteen hours
is much longer than the average gut clearance time for marine
copepods (approximately 1 – 2 h [38]), but less than the average
residence time of saxitoxins harboured in copepod guts and
tissues (approximately 33 h [36]).
Following the recovery period, the experimental tanks containing copepods were placed in a Schlieren optical system developed
by Strickler & Hwang [39] and further described by Doall et al. [40].
Observations were conducted in the dark with a near-infrared
laser used to illuminate the copepods. A green laser beam was
directed down the centre of the vessel to attract copepods to the
centre of the tank and reduce effects of vessel walls [40]. Preliminary observations revealed that the light encouraged copepods
to swim in the centre of the tank, maximizing the number of swimming paths that could be analysed. Observations were recorded
2
Proc. R. Soc. B 283: 20160176
2. Materials and methods
experiments. Maximum culture densities were 4.8 107 and
1.3 108 cells l21 for A. fundyense and R. lens, respectively.
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between Alexandrium sp. are variable, reviewed by [3,4]; outcomes of these interactions include copepods behaviourally
rejecting Alexandrium sp. [9,10,24], becoming incapacitated
after feeding [8,25] or showing no apparent adverse effect
[26,27]. Furthermore, Alexandrium sp. can have detrimental
[28] or positive [29,30] effects on egg production rate. The effects
of Alexandrium sp., can vary between copepod species [26],
gender [31], phenotype [29] and historical exposure [25,32].
In this study, we ask the following questions. (i) Does
T. longicornis exhibit altered swimming behaviour in response
to A. fundyense exposure? (ii) Is this behavioural change
explained by nutritional inadequacies or toxicity of the
phytoplankton? (iii) How does this behavioural change affect
copepod encounter rates with predators?
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Table 1. Description of phytoplankton treatments administered during copepod behavioural, survival and reproductive experiments.
description
purpose
Alexandrium fundyense
320 cells ml21 of A. fundyense þ
contains both the HAB species A. fundyense and nutritious species R. lens
21
treatment
high food control
1200 cells ml of Rhodomonas lens
5600 cells ml21 of R. lens
contains equal biomass of total phytoplankton as A. fundyense treatment
low food control
starved
1200 cells ml21 of R. lens
filtered seawater
contains equal biomass of the nutritious phytoplankton, R. lens as A. fundyense treatment
contains no phytoplankton
V¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(xtþ1 xt )2 þ (ytþ1 yt )2 þ (ztþ1 zt )2 p,
where xt, yt, zt and xtþ1, ytþ1, ztþ1 correspond to the copepod’s positions at time t and time t þ 1, respectively, and p is the frame
acquisition rate of our camera (33 frames s21). Instantaneous velocities were averaged over an individual track to obtain a mean
swimming velocity. Swimming speed has important consequences
for T. longicornis distribution and survival, because increased
swimming speed leads to greater dispersal distances, increased
encounter rates with predators and increased conspicuousness
[19,20,41]. We calculated the effect of changes in swimming
velocity on encounter rates between T. longicornis and their
predators (E) using the following equation [18]
E ¼ pK2 (v2pred þ v2prey )0:5 ,
where K is the perceptive distance of the predator and vpred and
vprey are the swimming velocities of the predator and prey, respectively. The relative importance of the prey’s swimming speed
depends on type of predator encountered [19]. For a visual cruising
predator such as a fish larvae, the encounter rate E increases proportionally as prey velocity vprey increases [19]. However, for a
rheotactic predator that relies on hydrodynamic cues from prey,
the predator’s detection distance (K) scales as
Ks
v
prey
v
0:5
,
where (s) and (vprey) are the prey’s size and swimming velocity
and (v) is the sensitivity (threshold speed) that the predator can
detect [19]. Therefore, a faster-swimming organism is more conspicuous to predators. In this case, the predator’s encounter rate
is roughly proportional to v2prey :
Additionally, we calculated the net : gross displacement ratio
(NGDR) for each swimming track. This is a commonly used
metric that varies from 0 to 1 and describes the degree of path tortuosity [42]. An NGDR of 1 represents a perfectly straight path,
whereas an NGDR of 0 describes Brownian motion. Because
net : gross displacement ratios are inherently scale-dependent, we
accounted for this scale-dependency by analysing paths of 8–10 s
in length. High directional persistence of swimming paths indicates
increased encounter rates between individuals [20,43].
(d) Survivorship experiments
To determine the effects of ingesting A. fundyense on copepod
survival, we incubated T. longicornis in 250 ml beakers containing
filtered artificial seawater amended with one of the following
treatments: high food control, A. fundyense treatment, a low
food control and a starved control (table 1). The percentages
were based on biovolume equivalents to account for the different
sizes of the phytoplankton cells.
To control for feeding history, T. longicornis were incubated in
filtered seawater for 12 h prior to experiments. Three male and
three female T. longicornis were randomly assigned to beakers
(n ¼ 10), which were covered with parafilm, placed in a 148C
incubator and exposed to a 14 : 10 h light : dark cycle. For all
experiments, beakers were interspersed with respect to treatment
during incubations. Every 24 – 25 h, copepods were visually
inspected under a dissecting microscope and scored as either
dead or alive. Live copepods were transferred to new beakers
containing fresh phytoplankton and dead copepods were
discarded. Experiments were conducted for 4 days.
(e) Ingestion experiments
We measured copepod ingestion rate in a subset of our beakers
(n ¼ 5) between 24 and 48 h of our survivorship experiment.
Prior to the addition of animals, a 20 ml sample was removed
from experimental beakers and preserved in Lugol’s solution.
The next day, copepods were removed from beakers, and another
20 ml sample was collected and preserved. To determine copepod ingestion rate, the number of cells removed was divided
by the number of surviving copepods in the beaker and
multiplied by the number of hours incubated (24 h).
Visual counts were performed using replicate 300 ml–1 ml
preserved samples, so that a minimum of 500 cells were counted
for each subsample. The rate of copepods ingesting each algal
species was calculated from the formula described by Frost [44]
I¼
(C1 C2 )V
24,
N
where C1 is the cell concentration in the beaker just prior to adding
copepods, C2 is the cell concentration immediately after copepod
removal, V is the cell biovolume and N is the number of live
copepods at the end of the 24 h period. Animals that died during
the incubation were assumed to have died halfway through
the experimental period in calculations. We ran copepod-free controls with mixtures of A. fundyense and R. lens prior to grazing
experiments to measure changes in cell density owing to growth
or settling. We found no significant change in cell density over a
24 h period for either species (A. fundyense, p ¼ 0.96, n ¼ 4;
R. lens, p ¼ 0.68, n ¼ 4, two-tailed t-test). Therefore, we did not
include a growth parameter in our ingestion equation.
(f ) Egg production and hatching success experiments
To determine the effects of A. fundyense on copepod fecundity, we
incubated three male and three female T. longicornis in 250 ml
Proc. R. Soc. B 283: 20160176
onto DVDs and digitized using PRISM video converter 1.82 software and split into clips using SOLVEIGMM video splitter. When
necessary, clips were further processed in Adobe PREMIER PRO
CS5.5 to enhance contrast. Swimming paths were analysed using
LABTRACK software. Our path selection criteria required that copepods were swimming in the centre of the tank for at least 8 s. For
paths longer than 10 s, only the first 10 s were analysed. We then
constructed three-dimensional tracks by matching the common
z-position from superimposed mirror images of individual
copepods.
We calculated instantaneous swimming velocity V (mm s21)
of the animals using the distances between position in the x, y
and z plane over a given time step t.
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treatment
3
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(b)
*
3
2
1
0
Alexandrium
Rhodomonas
4
0.4
copepod net : gross
displacement ratio
4
*
0.3
0.2
0.1
0
Alexandrium
Rhodomonas
beakers containing an ‘egg basket’ (n ¼ 9). Egg baskets consisted of
a 5.5 cm diameter and 10 cm height Plexiglas tube with 160 mm
mesh attached to the bottom and their purpose was to create a
false bottom through which copepod eggs could pass separating
copepods from their eggs and thus preventing egg cannibalism
[45]. To control for previous mating and feeding history, males
and females were separately incubated for 24 h in filtered seawater
prior to experiments. Copepods were individually added to beakers containing one of the following treatments: high food control,
A. fundyense treatment, a low food control and filtered seawater
control (table 1). Beakers were covered with parafilm and placed
in a 148C incubator and exposed to a 14 : 10 h light : dark cycle.
Every 24–25 h, egg baskets containing copepods were moved to
new beakers with fresh phytoplankton. Copepods found lying
on the bottom of the egg basket were visually inspected under
a dissecting microscope. Dead copepods were discarded.
Experiments were conducted for 3 days.
We maintained a 1 : 1 sex ratio within our treatments to control
for any bias in hatching success owing to either fertilization limitation or mate competition. Therefore, if a female died during the
incubation, we removed a male from that replicate. Dead males
were replaced with a new male from a supplemental stock culture
that had been incubated in the appropriate treatment since the
beginning of the experiment.
After moving copepods to new beakers, we gently transferred
eggs to a Petri dish and counted them immediately. A subset of
egg samples were covered with parafilm and placed in a 148C incubator for 48 h (n ¼ 4). After 48 h, we added a few drops of acetic acid
to the Petri dishes to kill and stain the nauplii for enumeration. For
the purpose of analysis, we pooled the total number of eggs and
nauplii produced per female per replicate from 48 h and 72 h time
periods (excluding the 0–24 h time period) to ensure that copepods
had enough time to assimilate ingested phytoplankton [38].
(g) Statistical analyses
The number of trajectories analysed per trial depended on the
number of trajectories that fit our selection criteria (i.e. copepod
swimming near the centre of tank for at least 8 s), which yielded
10 – 15 swimming paths per trial. We averaged individual
swimming speed and NGDR values within trial and consider
experimental tank the unit of replication. Our analysis is based
on a total of 64 trajectories consisting of 19 411 successive animal
positions for the R. lens control and 56 trajectories (16 297
positions) for A. fundyense treatment (n ¼ 5 replicate tanks per
treatment). We used a Welch’s t-test to detect statistical
differences in swimming speed and path tortuosity between
A. fundyense and R. lens treatments.
The total number of surviving copepods and number of
eggs produced per female between phytoplankton treatments
were analysed using an analysis of variance (ANOVA). Per
cent hatching success was calculated from the total number of
nauplii and eggs produced per replicate beaker. Differences in
hatching success across treatments were analysed by a generalized linear model (GLM) with a logit-link function with a
quasi-binomial distribution (to account for overdispersion).
Differences in total ingestion rate between the high food
control and the A. fundyense treatment were compared using a
Welch’s t-test. Differences in the ingestion of R. lens between
the low food control and the A. fundyense treatment were
also compared using a Welch’s t-test. All statistical tests were
conducted using R (v. 2.14.1).
3. Results
After being exposed to phytoplankton treatments for 2 h,
followed by 15 h of consuming non-HAB food, copepods
exposed to the A. fundyense treatment swam faster
than those exposed to the control phytoplankton (figure 1a
t ¼ 2.4, d.f. ¼ 8, p-value , 0.05, Welch’s t-test). Directional persistence was also significantly different (figure 1b;
t ¼ 2.6, d.f. ¼ 8, p-value , 0.05, Welch’s t-test), with copepods exhibiting straighter swimming paths (figure 2)
following A. fundyense exposure.
The average number of survivors did not differ among
phytoplankton treatments (figure 3; F3,36 ¼ 1.42, p ¼ 0.25,
ANOVA). Furthermore, there was no difference between the
amount of total food ingested by copepods in the A. fundyense
treatment versus the high food control (figure 4; t ¼ 0.84,
d.f. ¼ 7, p ¼ 0.43, Welch’s t-test). Additionally, T. longicornis
consumed similar amounts of R. lens in the low food control versus A. fundyense treatment (figure 4; t ¼ 1.3, d.f. ¼ 7,
p ¼ 0.23, Welch’s t-test), indicating that T. longicornis did not
discriminate against or become incapacitated by ingesting
A. fundyense.
There was no difference in copepod egg production
(figure 5a; F3,32 ¼ 0.62, p ¼ 0.61, ANOVA) among the different
food treatments. Hatching success was only significantly
different between the high food treatment and starved
treatment (figure 5b; t ¼ 22.3, d.f. ¼ 12, p , 0.05, GLM,
quasi-binomial distribution). Both samples of our stock cultures contained PSP toxins (as verified by ELISA and HPLC).
As detected by HPLC, our stock culture contained 2.9 pg
total saxitoxin equivalents per cell. The carbon : nitrogen
ratios for R. lens and A. fundyense were 7.16 + 0.44 and
5.04 + 0.17 (mean + s.e., n ¼ 2), respectively.
Proc. R. Soc. B 283: 20160176
Figure 1. Effects of ingesting an Alexandrium fundyense treatment (containing 320 cells ml21 of A. fundyense þ 1200 cells ml21 of Rhodomonas lens) versus a
high food control (containing 5600 cells ml21 of R. lens on the swimming speed (a) and net : gross displacement ratio (b) of T. longicornis (mean + s.e.). Copepods
were incubated in treatments for 2 h and then fed nutritious food for 15 h prior to behavioural recordings. Significant differences were determined using a Welch’s
t-test to compare mean swimming speed, (t ¼ 2.4, d.f. ¼ 7.9, p-value , 0.05) and net : gross displacement ratio (t ¼ 2.6, d.f. ¼ 7.3, p-value , 0.05).
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copepod swimming
speed (mm s–1)
(a)
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(a)
(b)
5
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3
2
1
1
2
position (cm)
3
0
1
2
position (cm)
3
Figure 2. Three-dimensional depictions of 8 – 10 s swimming paths of Temora longicornis after ingesting an Alexandrium fundyense treatment (containing
320 cells ml21 of A. fundyense þ 1200 cells ml21 of Rhodomonas lens) (a) versus a high food control (containing 5600 cells ml21 of R. lens) (b). Plots represent
a subset of the total number of trajectories analysed for visual clarity (n ¼ 40 per treatment). Copepods were incubated in treatments for 2 h and then fed
nutritious food for 15 h prior to behavioural recordings.
phytoplankton ingested
(mm3 copepod–1 d–1)
% survivorship
100
75
high food control
50
Alexandrium treatment
low food control
25
starved
1
Alexandrium
Rhodomonas
1.0 × 109
5.0 × 108
0
high food Alexandrium low food
control
treatment
control
0
0
1.5 × 109
2
day
3
4
Figure 3. Average number of surviving Temora longicornis incubated in an
Alexandrium fundyense treatment (containing 320 cells ml21 of A. fundyense þ
1200 cells ml21 of Rhodomonas lens), a high food control (containing
5600 cells ml21 of R. lens), a low food control (control 1200 cells ml21 of R.
lens) or filtered seawater (starved control). The total number of survivors
at the end of 4 d was compared using an analysis of variance, ANOVA
(F3,36 ¼ 1.42, p ¼ 0.25).
Figure 4. The biomass of phytoplankton ingested by Temora longicornis over a
24 h period when six individuals were incubated in an Alexandrium fundyense
treatment (containing 320 cells ml21 of A. fundyense þ 1200 cells ml21 of
Rhodomonas lens), a high food control (containing 5600 cells ml21 of R.
lens), a low food control (control 1200 cells ml21 of R. lens) or filtered seawater
(starved control). The total amount of food consumed between the high food
control and the A. fundyense treatment was compared using a Welch’s t-test.
The amount of R. lens consumed between the low food control and the A. fundyense treatment was also compared using a Welch’s t-test. There were no
significant differences detected in the total amount of food ingested (t ¼
0.84, d.f. ¼ 7, p ¼ 0.43) or the amount of R. lens ingested (t ¼ 1.3, d.f. ¼
7, p ¼ 0.23).
4. Discussion
To assess the effects of A. fundyense exposure on T. longicornis,
the following factors were examined: copepod swimming behaviour, survival, ingestion rate, as well as egg production
rate and hatching success. Of these factors, the only significant
effect observed was on the copepod’s swimming behaviour.
After being exposed to A. fundyense for 2 h and then allowed
to depurate for 15 h, T. longicornis increased their swimming
velocity and the straightness of their swimming path.
This apparent stimulatory effect of A. fundyense on copepod
swimming behaviour was an unanticipated finding.
The A. fundyense strain used in this experiment contains
saxitoxins that can block sodium channels and interfere with
nerve function, leading to incapacitation, paralysis or death
in vertebrates [7,46] as well as copepods grazers [8,9].
However, T. longicornis swimming significantly faster and
straighter after ingesting A. fundyense does not concur with a
sodium channel-blocking event. It is possible that the population of T. longicornis tested in our experiments were
resistant to A. fundyense toxins owing to their co-occurrence
in the Gulf of Maine. Populations of the copepod Acartia hudsonica from regions exposed to Alexandrium blooms are resistant
to adverse effects [32] and can even exhibit enhanced physiological effects such as increased ingestion rate [25] and
increased egg production rate [47] when fed A. fundyense
versus non-toxic diets. Furthermore, the addition of saxitoxins
to a palatable alga diet have even been shown to stimulate feeding
in amphipod grazers [48]. Therefore, our results add to a growing
body of evidence that suggests saxitoxins and A. fundyense
can have stimulatory effects on grazers in some cases.
Following A. fundyense exposure, T. longicornis increased
its swimming velocity by 25%. The degree to which this
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0
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(b)
6
100
% hatching success
15
10
5
0
80
a
ab
ab
b
60
40
20
0
starved
high Alexandrium low
treatment
food
food
control
control
starved
Figure 5. The number of eggs produced per female (a) and the per cent hatching success (b) of Temora longicornis while incubated in an Alexandrium fundyense
treatment (containing 320 cells ml21 of A. fundyense þ 1200 cells ml21 of Rhodomonas lens), a high food control (containing 5600 cells ml21 of R. lens), a low
food control (control 1200 cells ml21 of R. lens) or filtered seawater (starved control). The total number of eggs per female was analysed using an analysis of
variance, ANOVA (F3,32 ¼ 0.62, p ¼ 0.61). Per cent hatching was analysed using a generalized linear model with a quasi-binomial distribution and logit-link
function (t ¼22.3, d.f.¼ 12, p , 0.05). Different lower case letters indicate differences at the 0.05 alpha level.
increase in speed affects the theoretical encounter rate with
T. longicornis predators depends on the predator’s prey detection mode. Predators that detect copepod prey through visual
(fish larvae) or tactile cues (gelatinous zooplankton) will
experience an increased encounter rate that is roughly proportional to the increase in copepod swimming velocity
[20]. The larger hydrodynamic signal created by an accelerating copepod further enhances encounter rates by extending
the detection distance of rheotactic predators such as mysid
shrimp [20,49]. Therefore, a 25% increase in swimming
speed leads to a 56% increase in the theoretical encounter
rate between copepods and their rheotactic predators [18,19].
Copepods having increased encounter rates with predators
after ingesting HAB species could have important consequences for bloom maintenance and the fate of toxins in food
webs. If copepods that have recently ingested A. fundyense
are selectively removed due to increased predatory encounters,
this could reduce grazing control of HABs. Whether this
increase in encounter rates enhances toxin uptake by copepod
predators depends on both the propensity of T. longicornis to
accumulate PSP toxins and the susceptibility of copepod
predators to those toxins.
Field surveys indicate that PSP toxins are accumulated
by zooplankton in the Gulf of Maine [50–52] and can exceed
acceptable levels of PSP toxins in shellfish for human consumption [53]. T. longicornis is often present in these multispecies field
samples [50,53,54], but, to the best of our knowledge, no one
has ever measured the PSP toxin levels accumulated by
T. longicornis. Laboratory estimates reveal that although copepods can accumulate significant levels of PSP toxins, they
generally accumulate only a small percentage of what is
ingested [6,26,36] and therefore, are not as efficient in toxin
uptake compared with other species such as shellfish. Nonetheless, detectable PSP toxin levels were accumulated in the tissues
of Eurytemora herdmani and Acartia tonsa after only a few hours of
grazing [26] and ingestion of toxin-laden copepods can have
detrimental effects on predators [55].
Copepod predators exhibit different sensitivity levels to PSP
toxins. According to encounter rate models, rheotactic predators such as mysid shrimp will experience the largest increase
in encounter rates with copepods following A. fundyense ingestion [20]. Mysid shrimp can accumulate PSP toxins in their
tissues [56] and suffer reduced survivorship and fecundity following direct exposure to Alexandrium tamarense [57]. However,
it is unclear if indirect exposure through ingesting toxinladen copepods would produce similar effects. Indirect effects
of saxitoxins on fish larvae have been examined through an
A. fundyense–copepod–fish larvae food chain. Results indicate
that ingesting as few as 6–12 toxin-laden copepods is sufficient
to cause mortality in fish larvae [55]. Therefore, fish populations
would likely be detrimentally affected by increased encounters
with toxin-laden copepods. Conversely, gelatinous predators
could potentially benefit from these increased prey encounters
since they possess sodium channels that are insensitive to saxitoxins [58,59]. Therefore, the indirect effects of saxitoxins on
copepod predators is likely species-dependent and further
studies are needed to determine the fate of these toxins in
marine food webs.
It is important to note that encounter rate is not the sole
determinant of predation rate. If ingesting A. fundyense alters
the outcome of these encounters (i.e. by affecting escape behaviour), then this will likely modify the magnitude of these
effects. Furthermore, if predatory encounters occur within an
A. fundyense bloom, copepod predators could suffer direct
effects of toxin exposure that may alter their foraging ability.
Future studies of the direct and indirect effects of A. fundyense
on predation rates and grazer removal are warranted.
In our study, we did not find any effect of A. fundyense
on T. longicornis ingestion (figure 4). Similar to results for
T. longicornis ingesting Alexandrium minutum and A. tamarense
[60]. Our results indicate that T. longicornis do not discriminate
against or become incapacitated by ingesting Alexandrium cells.
Additionally, ingesting A. fundyense did not decrease copepod
survivorship over a 4 day period. A. fundyense toxin concentration was much lower than in other studies [6,25];
therefore, copepod health may be affected by ingesting A. fundyense with higher toxin content. In our study, copepod
mortality was 19–35% during the 4 days experiment, with
the greatest mortality occurring in the starved treatment.
There were no significant differences between any of the treatments. Because there were no differences detected between the
starved and 100% R. lens treatments, we are limited in the conclusions we can make. We cannot conclude whether or not
long-term exposure of A. fundyense could affect T. longicornis
Proc. R. Soc. B 283: 20160176
high Alexandrium low
treatment
food
food
control
control
rspb.royalsocietypublishing.org
eggs female–1 d–1
(a) 20
Downloaded from http://rspb.royalsocietypublishing.org/ on June 16, 2017
harmful phytoplankton. Future work aimed at determining
the outcome of elevated encounters with predators is needed
to fully determine the impact harmful algal species can have
on grazers.
Data accessibility. Data are publicly available at http://dx.doi.org/10.
5061/dryad.1c2j6.
Authors’ contributions. R.S.L.-R. conducted experiments, participated in
Competing interests. We have no competing interests.
Funding. This work was supported by National Science Foundation
grant no. OCE-0728238.
Acknowledgements. We thank J. Kubanek for her generosity in allowing us
to use equipment and laboratory space and for advice on culturing
techniques. We thank A. True for valuable feedback regarding analysis
and M. Heaphy and L. Healy for help with phytoplankton cultures. We
also thank the reviewers for valuable insights and feedback.
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data analysis, participated in the design of the study and drafted the
manuscript; K.N. conducted experiments and participated in data
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participated in the design of the study; coordinated the study; and
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7
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