Limnol. Oceanogr., 56(6), 2011, 000–000
2011, by the American Society of Limnology and Oceanography, Inc.
doi:10.4319/lo.2011.56.6.0000
E
Resting egg production induced by food limitation in the calanoid copepod Acartia
tonsa
Guillaume Drillet,a,b,1,* Benni W. Hansen,b and Thomas Kiørboea
a Danish
Technical University (DTU Aqua), National Institute of Aquatic Resources, Charlottenlund, Denmark
of Environmental, Social and Spatial Change (ENSPAC), Roskilde University, Roskilde, Denmark
b Department
Abstract
;
Three populations of the copepod Acartia tonsa, two from the Baltic Sea and one from the U.S. East Coast,
were compared for resting egg production at conditions of saturating and limiting food availability. All three
populations produced eggs that hatched within 72 h when incubated at 17uC (subitaneous eggs), but the two
Baltic populations in addition produced eggs that hatched at a much slower rate, in the course of a month
(delayed hatching eggs [DHE]). Such eggs were not produced by the U.S. population. The fraction of DHE
increased when food was limiting. Females from a Baltic population that were incubated individually all produced
subitaneous eggs, but about half the females consistently also produced DHE. Cold storage that mimicked boreal
winter conditions synchronized the hatching of DHE after extended storage, indicating that spring hatching of
DHE might seed the water column with nauplii as an adaptation to match the timing of the spring bloom in
boreal marine ecosystems. Low food availability promotes the production of resting eggs in marine copepods.
hatching is not possible even if environmental conditions
are suitable. After termination of the refractory period,
eggs can hatch or remain quiescent if the environmental
conditions are un-suitable.
A third category of eggs was later described (Chen and
Marcus 1997) and is referred to as delayed hatching eggs
(DHE). They do not belong to either of the two established
categories because the hatching is not fast enough to
consider the eggs as subitaneous and the refractory period
is not long enough to categorize the eggs as true diapause
eggs. The DHE category may be controlled through the
same gene expression pathway as for those defined as true
diapause eggs and may simply represent different torpidity
levels, as proposed for freshwater cyclopoid copepods
(Elgmork 1996). These eggs may be considered as eggs
going through oligopause (Alekseev et al. 2007). Diapause,
quiescent, and DHE eggs are all commonly called resting
eggs.
Cues used for diapause induction have been described in
the literature for a wide range of aquatic organisms: for
copepods, mainly abiotic cues (e.g., light and temperature);
rotifers, cues from the biotic environment (e.g., food
quality, quantity, and crowding); and cladocerans, a mix
of both types (Gyllstrom and Hansson 2004). It is less
obvious whether the production of resting eggs is induced
by biotic cues in calanoid copepods. It has been suggested
that induction of resting stages could be density dependent
and due to the accumulation of metabolites in the water
(Ban and Minoda 1994). Production of resting stages under
crowded condition has also been described for other
crustaceans, as, for example, Daphnia lumholtzi (Smith et
al. 2009). However, the link between laboratory studies
examining the population density effects on resting egg
production in marine copepods and the occurrences of high
density swarms in nature has never been properly studied,
even though high densities occur frequently in the natural
environment (Devreker et al. 2008).
Life history strategies may be adapted to unstable
environments in various ways. Under stressful environmental conditions, mobile species can migrate to more
suitable places, but migration in time by going into
dormancy is also a suitable strategy to allow survival
(Hairston and Bohonak 1998). Saving genes over time is
the principle behind dormancy. Dormancy is a widespread
life history adaptation in the plankton, both among
phytoplankton and zooplankton. Phytoplankton, for example, produces resting spores that may survive unproductive seasons in the sediment (Hallegraeff and Bolch
1992). For zooplankton, dormancy is often more prevalent
in freshwater as compared to marine environments, due to
the typically larger fluctuations in limnic habitats. However, some marine crustaceans are known to rest (Alekseev et
al. 2007); and, among marine copepods, diapause has been
described for adult, copepodite, and egg stages (Mauchline
et al. 1998). Diapausing in eggs has been described in close
to 50 species of calanoid copepods (Engel and Hirche 2004)
that all belong to the superfamily of Centropagoidea. Most
if not all of these species are neritic and, thus, occur in
habitats with rather large environmental variability.
Different types of eggs have been described for
copepods, and Grice and Marcus (1981) proposed a
definition separating two categories of eggs. Subitaneous
eggs hatch immediately after spawning under optimal
conditions but are in some species able to arrest development and remain in a quiescent stage if the environmental
conditions are unsuitable. Such eggs will continue embryogenesis and eventually hatch as soon as the environmental
conditions become favorable. Diapause eggs go through a
long, hormonally controlled refractory period during which
* Corresponding author: [email protected]
1 Present address: DHI Water and Environment Pte. Ltd.,
Singapore, Singapore
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Drillet et al.
In this study, we use the copepod Acartia tonsa, a
cosmopolitan euryhaline and eurytherm calanoid that often
dominates the zooplankton community in coastal waters.
The species is known to produce dormant eggs, as either
quiescent, true diapause, or DHE (Zillioux and Gonzalez
1972; Castro-Longoria 2001; Katajisto 2006). We examine
the effect of food availability on the production of resting
eggs and characterize the hatching dynamics of resting eggs
as a function of the duration of their dormancy. Using
laboratory cultures stemming from three populations that
are adapted to different seasonal environments, we further
study population differences in the induction of resting egg
production. Two of these populations (from the Baltic Sea
area) originate from the same mitochondrial clade, while a
population from Florida is genetically distinct (16S
ribosomal RNA and cytochrome oxidase subunit I; Drillet
et al. 2008a).We hypothesize that food limitation induces
production of resting eggs and that the induction differs
among populations adapted to different environments.
Methods
Experimental animals and cultures—Three A. tonsa
cultures originating from Kiel Bright (Germany), Øresund
(Denmark), and Florida were compared in a common
garden experiment for resting egg production. The experimental cultures were initiated with eggs from the source
cultures. Eggs from the source cultures were refrigerated
and sent by express mail (, 2 d) to our laboratory at the
Danish Technical University, where the experimental
cultures were established immediately. The Øresund culture
originated from individuals isolated in 1981 from the
Øresund (56uN; 12uE). The population has been kept at 30
salinity, 17uC, and dim constant light and fed 30,000–
60,000 cell mL21 every 2 d with the haptophyte Rhodomonas salina. The Kiel culture originates from adults isolated
August 2003 from the Kiel Bight in the southwestern Baltic
Sea (54uN; 10uE; Holste and Peck 2005). The culture has
been kept at 18 salinity, 18uC and 13 : 11 light : dark (LD),
and fed daily Rhodomonas sp. at 50,000 cell mL21. The
Florida culture was collected from local waters outside the
Florida State University Coastal and Marine Laboratory
(30uN; 84uW) between 2002 and 2004. The culture was
maintained in the laboratory at salinity 30, 17uC, and
14 : 10 LD, and fed daily a mixed algal diet (Rhodomonas
lens, R. salina, and occasionally Akashiwo sanguinea;
30,000–50,000 cell mL21). Eggs from all cultures were
regularly harvested, rinsed, and stored in test tubes at
1–3uC for later use.
Cultures of the three populations were established in 30liter tanks and run under similar and constant laboratory
conditions for two generations to eliminate parental effects
(17.6uC 6 0.3uC, 34 salinity, 12 : 12 LD, fed in excess with
R. salina). Eggs were then harvested to establish the
experimental cultures. When copepodites appeared, animals from each population were separated into two groups
that were fed different amounts of phytoplankton. The first
group, labeled low food, was offered 2000 cell mL21, and
the high food group was offered 40,000 cell mL21
(subsaturated vs. saturated, Berggreen et al. 1988). Food
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concentrations were adjusted twice daily, and the seawater
in all six tanks was changed weekly. When adults appeared
in the tanks, eggs were harvested to initiate the hatching
experiments.
The density of copepods in the tanks varied between 200
to 700 individuals per liter (mix of copepodites and adults),
and the density was always higher under high food
treatments than under low food treatments.
Hatching experiments—Eggs were harvested when less
than 2 h old by harvesting eggs 1.5 h after a preliminary
harvest and were washed on a sieve. Harvested eggs were
distributed among 10 small Petri dishes (from 47 to 205
eggs per dish, 17.6uC 6 0.3uC, 34 salinity, 12 : 12 LD), and
hatched eggs (nauplii) were regularly counted under a
dissection microscope (Olympus SZ 40) for approximately
72 h (subitaneous eggs). Unhatched eggs were then
transferred to 10 new Petri dishes, while the nauplii were
fixed in 1% final concentration of acid Lugol’s and
counted. The new Petri dishes were inspected daily for
more than a month, and eggs and nauplii were counted and
nauplii removed. Every 2 or 3 d, the eggs were transferred
to new Petri dishes with clean water in order to minimize
the development of fungi or bacteria on the egg shells.
Individual variation—Incubations of Kiel females were
carried out under optimal conditions (saturated food,
Berggreen et al. 1988) to examine whether individual
females were consistently producing the same type of eggs.
Thirty females bearing a spermatophore were incubated
individually under high-food conditions in 600-mL glass
bottles. The bottles were mounted on a plankton wheel (0.5
round per minute) for two days. Each day, the content of
each bottle was emptied through a set of two sieves (180mm and 53-mm mesh size) to separate females from eggs,
and the female was reincubated in a fresh suspension of
algae. Eggs were counted and incubated for 72 h for
hatching and then fixed in Lugol’s. Nauplii and remaining
eggs were then counted to calculate the number of hatched
eggs (subitaneous eggs), decayed (nonviable) eggs, and
nonhatching eggs, which were considered as DHE.
Decaying eggs are easily distinguished from nonhatched
ones on the basis of attached bacteria and a clear, not dark,
egg interior.
Effect of resting duration on hatching success—DHE
from the Kiel population were produced by collecting live
eggs that had not hatched for 5 d. These eggs were stored in
2-mL Eppendorff tubes (500 eggs per tube) in the dark at
1.5uC. Every month, samples of these eggs were transferred
to 17uC, and their hatching success was examined after 48-h
incubation at constant dim light. Constant light is reported
to increase the 48-h hatching success of A. tonsa (Peck et al.
2008).
Results
Egg types and food conditions—Eggs exhibited two types
of hatching patterns: a group of eggs hatched within 72 h
(subitaneous) and another group hatched slowly, in the
Acartia tonsa resting egg induction
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was highest at the low food concentration (Fig. 1). Also,
for the Baltic populations, the 72-h hatching success was
lower for the eggs produced under low food concentration
than under high food concentration (Mann–Whitney rank
sum test, t 5 140,000, n 5 10, p 5 0.009 [Øresund]; t 5
148,000, n 5 10, p 5 0.001 [Kiel]). The Florida population
did not survive the low-food treatment. The Kiel population produced relatively more DHE than the Øresund
population, and the maximum hatching success of DHE
occurred in the Kiel population, whatever the food
treatment. A fraction of the eggs never hatched but
decayed and, hence, were dead.
Fig. 1. Final hatching success after 72 h (subitaneous eggs)
and after 1 month (delayed hatching eggs) in three populations of
Acartia tonsa raised under different food conditions (low: 2000
cell mL21 and high: 40,000 cell mL21 of Rhodomonas salina) in a
common garden experiment.
course of about 1 month (DHE). The two Baltic
populations produced both types of eggs, while the
Floridian population never produced DHE (Figs. 1, 2).
For both of the Baltic populations, the proportion of DHE
Individual variation—There was significant variation
between individuals in their tendency to produce DHE, as
examined for the Kiel population (Fig. 3). Over the 2-d
incubations, all individual females produced subitaneous
eggs, and approximately half the females also produced
DHE. It was the same females that produced DHE on the
first and second day of the incubation: of the 12 females
producing DHE on the second day, 17 had produced DHE
also on the first day.
Effect of cold storage—The 48-h hatching success of coldstored DHE from the Kiel population varied with the
duration of the storage period: it increased during the first
5 months, from 0 to 83% 6 22%, whereupon it decreased until
no more hatching occurred after 12 months (Fig. 4). Samples
incubated for 14 and 22 months also showed no hatching. The
eggs that had been cold-stored for 1 month were allowed to
hatch beyond the 48 h, and, in the course of the subsequent
500 h, 84% 6 5% of these eggs hatched (Fig. 5). Thus, the
Fig. 2. Hatching of delayed hatching eggs over 1.5-month period in two populations of
Acartia tonsa fed at high and low food levels (subitaneous eggs that hatched within 72 h have
been removed).
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Drillet et al.
Fig. 3.
Egg categories produced over 2 consecutive days by 30 individual females of Acartia tonsa from the Kiel population.
effect of cold storage was to reduce the hatching period, from
about 1 month prior to storage, to 10 d after 1 month of
storage, and to only about 48 h after 5 months of storage. In
all cases, the total hatching success was the same, . 80%
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(Fig. 2, Kiel high food treatment, Figs. 4, 5). Eggs that had
been stored for . 12 months never hatched, even after
1 month of incubation, and many had died during storage
(58% after 14 months and 99% after 22 months).
<
Acartia tonsa resting egg induction
Fig. 4. Hatching success of Acartia tonsa DHE eggs at 17uC
and normoxia for 48 h (mean 6 SD) after months of storage in the
dark at 1.5uC. Data after 22 months of storage showed
no hatching.
Discussion
Delayed hatching was proposed for Labidocera scotti
and Pontella meadi by Chen and Marcus (1997) in the Gulf
of Mexico and for A. tonsa in the northern Baltic Sea
(Katajisto 2006) and in our culture (Drillet et al. 2008b).
However, the present study is the first to document DHE
and examine the factors that induce their production.
One difficulty that previous studies had in identifying the
presence of DHE was that egg incubation times for
estimation of hatching rates and success had been based
on models of hatching speed related to temperature
(McLaren et al. 1969; see review by Mauchline et al.
1998). Thus, the incubation time used to estimate hatching
success of A. tonsa eggs, for example, has most often been
limited to approximately 48 h at 17–18uC (Jonasdottir and
Kiørboe 1996; Vargas et al. 2006) and has sometimes been
increased to 5 d when incubated at lower temperature
(Castro-Longoria 2003); and unhatched eggs have been
considered nonviable, while in fact they may have been
DHE. This also implies that, in many studies, the hatching
success of A. tonsa eggs was potentially underestimated and
could have biased conclusions. This is particularly relevant
for studies looking at the effect of phytoplankton quantity,
quality, and toxicity on hatching success (Jonasdottir and
Kiørboe 1996; Vargas et al. 2006).
The production of DHE was increased by low food
availability, suggesting that food quantity in the environment is a cue for the production of DHE, as described for
resting egg production in other aquatic invertebrates
(Gilbert and Schreiber 1998) as well as for summer
diapause induction of nauplii stages of freshwater cyclopoid copepods (Santer and Lampert 1995). Other factors
that we did not examine may similarly induce DHE
production, such as low temperatures, low light, or
copepod density. In another copepod, Eurytemora affinis,
resting egg production is induced via infochemicals in water
in which high population density occurred (Ban and
Minoda 1994). We argue that this was not a major
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Fig. 5. Hatching success as a function of time of delayed
hatching eggs after a month of storage in the dark at 1.5uC (mean
6 SD).
mechanism in our experiments because the density of
copepods was always higher in the high-food treatment
than in the low-food treatment conditions and, thus, would
have countered any effect of food density. However, even
under optimal conditions, a significant fraction of the eggs
in the Baltic population were DHE, suggesting that DHE
production is a risk-spreading strategy.
The eggs that still looked viable after 14 months of
storage, but that did not hatch, survived for another month
at 17uC before decaying. Nonviable eggs of A. tonsa
normally degrade very rapidly at 17uC, suggesting that the
unhatched eggs were still viable but lacked a relevant
environmental cue for hatching. Their hatching may also
have been restricted by the limited presence of ecdysteroids
(i.e., molting hormones), which are believed to play an
important role in diapause formation and exit (Johnson
2003).
The 14 and 22 months of cold storage of DHE in the
dark were lethal for 58% and 99% of the incubated eggs,
respectively, and DHE are thus not produced as a strategy
to survive very long periods in the sediment, as has been
reported for diapause eggs (Marcus et al. 1994; Hairston et
al. 1995). Also, DHE is not a pluri-annual hatching
strategy, as described for plants as a bet-hedging response
to highly fluctuating environments (Philippi 1993).
Individual females were capable of producing subitaneous eggs and DHE simultaneously. This is an example of
a risk-spreading strategy, as similarly described for rotifers
by Gilbert and Schreiber (1998), with the purpose of
ensuring gene spreading in fluctuating environments. The
production of DHE reduces the risk of egg hatching at
unfavorable (low-food) conditions and allows the females
to spread their offspring in the short-term future without
taking the risk associated with deep burial of eggs in the
sediment. The latter is one of the reasons for the decrease of
eggs in an egg bank over time (Caceres and Tessier 2003;
De Stasio 2007). Production of DHE also increases the
spatial spreading potential, particularly in areas where
ocean currents are significant. An extended period of cold
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Drillet et al.
and dark conditions (as hypothetically experienced in the
sediment) increases the synchrony of the hatching when
later facing optimal conditions. Synchronous hatching and
cohort development increase the chances of reaching
maturity simultaneously with conspecifics and therefore
increase the chances of encountering a mate. This may in
particular be important when hatching nauplii colonize the
water column during spring.
The production of DHE gives A. tonsa a great potential
to invade new habitats via egg spreading and ballast water.
In fact, A. tonsa invaded European waters from the United
States and was first described in European water by Rémy
(1927) in Normandy, France. Genetic markers suggest that
the Baltic population of A. tonsa likely originates from a
native population off Rhode Island (Drillet et al. 2008a).
The production of DHE under similar culture conditions
was different among the examined populations. The
Floridian population was not capable of producing DHE
eggs under the tested food availability, but it is known to
survive harsh condition as quiescent eggs for up to a month
(Drillet et al. 2007, 2008a). This population originates from
a different mitochondrial clade than the two other studied
populations and may not exhibit the same life history traits.
A. tonsa is present in Floridian water all year round, and,
though it is possible to find resting eggs of A. tonsa in
seagrass beds, there is no evidence that these are DHE
(Scheef and Marcus 2010). Thus, the A. tonsa Florida
population has apparently either lost or has not evolved the
ability to produce DHE eggs. Because the Florida
population did not survive at the low food concentration,
we cannot entirely rule out the possibility that DHE are
produced at low food availability. The apparent inability to
produce DHE eggs is correlated to a difference in egg size:
the eggs of the Florida populations have a 20% smaller
volume than the eggs of the two Baltic populations (Drillet
et al 2008a). Eggs produced by females from the Floridian
population do not need high reserves to overwinter or rest,
and therefore a female could potentially produce more eggs
as a trade-off. The difference in egg size is suggestive of the
cost of producing eggs that can rest for extended periods.
The two Baltic populations originate from the same
mitochondrial clade (Drillet et al. 2008a) and come from
areas very near to each other with strong ocean currents; we
therefore doubt that they are genetically isolated in nature,
yet they differed somewhat with respect to production of
DHE. The differences may have evolved during the 30 yr of
culturing the Øresund population (. 250 generations), in
which the selective advantage of producing DHE may have
been very limited. Selection pressure has already been shown
to alter the behavioral pattern in the same laboratory
population, in which the individuals have partly lost their
diel rhythm (Tiselius et al. 1995).
Delayed hatching strategy (oligopause) is a very important life history trait that allows the spread of hatching over
a relatively short period of time (a month or two),
increasing the likelihood that some offspring will encounter
good conditions and decreasing the risk of deep burial in
the sediment. Hatching after a delay of a few weeks allows
early nauplii to evolve in an environment in which the
previous generation is likely to have died out, limiting the
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competition for resources. Moreover, synchronous hatching after a cold period (winter) likely increases the chances
of successful mating when reaching adulthood. To our
knowledge, the present observations are the first describing
the effect of food limitation on resting egg production in
marine copepods. These observations support previous
work on other organisms that are known to produce resting
stages under food limitation, strongly suggesting that this
particular cue could be universal for promoting resting egg
production in invertebrates.
Acknowledgments
We are indebted to Linda Holste and Cris Oppert for receiving
eggs from their copepod cultures and the two reviewers for their
pertinent comments. The present project was carried out through a
Ph.D. grant to Guillaume Drillet and was funded by the Eur-Oceans
network of excellence, The Graduate School of Stress Studies
(GESS), and Roskilde University and the Danish Council for
Independent Research Postdoctoral grant 10-094773. TK and BWH
were supported by The Danish Council for Independent Research.
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Received: 08 March 2011
Accepted: 20 July 2011
Amended: 22 July 2011
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Journal: Limnology
Paper: limn-56-06-08
Title: Resting egg production induced by food limitation in the calanoid copepod Acartia tonsa
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