Is postembryonic development in the copepod

Temora stylifera negatively affected by diatom diets?

Ylenia Carotenuto*, Adrianna Ianora, Isabella Buttino,Giovanna Romano, Antonio Miralto

Ecophysiology Laboratory, Stazione Zoologica ‘‘A. Dohrn,’’ Villa Comunale I, 80121 Naples, Italy

Received 30 January 2002; received in revised form 29 May 2002; accepted 11 June 2002

Abstract

Diatoms are major components of the marine microalgae and are generally considered to be the

principal food source for small pelagic crustaceans such as copepods. Recently, some species of this

algal class have been shown to produce abortifacient compounds (aldehydes) that block copepod

embryogenesis, thereby acting as a form of birth control for predatory copepods. To test if diatoms

also have deleterious effects on postembryonic development, several diatom species were used to

rear larval stages of the calanoid copepod Temora stylifera to adulthood. Our results show that T.

stylifera was only able to complete development from hatching to adulthood when reared with the

flagellates Isochrysis galbana and the dinoflagellates Prorocentrum minimum and Oxyrrhis marina.

The daily development and mortality rates observed were in the range of those reported from

previous studies on T. stylifera (0.68–0.82 stage/day and 9.9–12.4%/day, respectively). In contrast,

larvae reared on the diatoms Thalassiosira rotula, Skeletonema costatum and Phaeodactylum

tricornutum were unable to complete development to adulthood and died without passing the

naupliar phase or during the early copepodite stages. Daily mortality rates were higher than for

nondiatom species (20.3–35.5%/day). Inhibitory effects on growth were not correlated to cell size of

the algae. Final survivorship of larvae fed P. minimum and I. galbana significantly improved (70–

80%) when larvae were generated from females preconditioned with P. minimum for 24 h. The same

treatment had no beneficial effect on larvae fed with T. rotula or S. costatum, which died again before

the adult stage. Although larvae completed development in one replicate with T. rotula and final

survivorship improved to 34% (compared to 7% in nauplii from nonconditioned females), this value

was, in any case, lower than with nondiatom diets. No morphological aberrations were found in

larvae fed on diatoms, even though they were unable to complete their life cycle and died for

unknown reasons. By contrast, nauplii produced by females fed the diatom T. rotula for 7 days

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -0981 (02 )00237 -X

* Corresponding author. Present address: Max-Planck-Institut fur Limnologie, D-24306 Plon, Germany.

Tel.: +49-4522-76-3340; fax: +49-4522-76-3310.

E-mail address: ylenia@mpil-ploen.mpg.de (Y. Carotenuto).

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology Ecology

276 (2002) 49–66

showed strong congenital defects such as asymmetrical bodies and reduced number of feeding

appendages. Our results suggest that diatoms, which have already been shown to have deleterious

effects on copepod embryonic development, may also have insidious effects on larval growth of this

copepod species.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Copepods; Diatom–copepod interactions; Larval development; Temora stylifera; Food quality and

fitness; Confocal laser scanning microscopy; Maternal feeding and abnormal development

1. Introduction

Copepods are the most important secondary consumers of the marine environment,

acting as a link between primary producers (phytoplankton) and higher trophic levels (fish,

birds and mammals) (Runge, 1988). Several studies have recently demonstrated that one

of the most important classes of phytoplankton, diatoms, thought to support the secondary

production of pelagic copepods, have deleterious effects on the reproduction of these

consumers. Ianora and Poulet (1993) showed that a monospecific diet of the diatom

Thalassiosira rotula reduced egg-hatching success in the copepod Temora stylifera,

compared to the dinoflagellate Prorocentrum minimum. The effect was not due to anoxia

in the incubation vials (Miralto et al., 1995), nor to bacteria associated with diatom

cultures (Ianora et al., 1996), but to specific diatom embryonic inhibitors. Since then,

several diatom species have been shown to induce low hatching rates and abnormal

naupliar development in several copepod species, representative of marine, estuarine and

freshwater ecosystems (Poulet et al., 1994, 1995; Ianora et al., 1995, 1996; Miralto et al.,

1995; Uye, 1996; Ban et al., 1997; Starr et al., 1999; Lee et al., 1999). The long-chain

aldehydes (C10) responsible for these effects have recently been isolated, and their

antigrowth activity has been demonstrated in several animal models, including copepod

and sea urchin eggs and human adenocarcinoma cells (Miralto et al., 1999). Diatom

aldehydes are produced from eicosanoid (C20) fatty acids seconds after mechanical

damage of cells by zooplankton grazers (Pohnert, 2000). It is believed that such

compounds act as chemical defenses to repel grazers, inducing abortions, birth defects,

poor development and high mortality in unwary predators (Ianora et al., in preparation).

If diatoms have been shown to block copepod embryogenesis, very conflicting results

have been obtained when postembryonic larval stages of copepod species have been reared

on these algae. Some studies have shown that monospecific diatom diets are good food

sources to sustain the development of copepods from hatching to adulthood (Harris and

Paffenhofer, 1976; Paffenhofer and Harris, 1976; Paffenhofer, 1976; Smith and Lane,

1985). However, in other cases, copepods showed a poorer developmental performance

with a diatom diet in terms of final mortality, generation time and adult body size

(Paffenhofer, 1970); or copepods were unable to grow and died at the naupliar or

copepodite stages prior to reaching adulthood (Mullin and Brooks, 1970; Paffenhofer,

1971; Hirche, 1980; Peterson, 1986). The reasons as to why diatoms are ultimately

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6650

insufficient or poor quality foods for copepod development have until now been attributed

to inadequate cell size, age of the culture and/or inadequate mineral/biochemical

composition of the food. Alternatively, there is the possibility that secondary plant

metabolites produced by diatoms, such as the recently described aldehydes, could interfere

not only with embryogenesis of the eggs, but also with postembryonic development of

larval stages.

Not much is known on chemical defense in marine microalgae, but higher plants are

known to produce a variety of secondary compounds as a defensive constituent in the

plant. Such compounds show a wide range of allomonal effects on their victims

including growth reduction and/or developmental arrest, eventually leading to toxicity

and death. For example, the nonprotein amino acids canavanine and canaline found in

the leguminous family are known to elicit severe larval developmental aberrations and

prevent successful larval–pupal ecdysis in the tobacco hornworm and certain moths

(Rosenthal, 1991). Also, the triterpenoid azadinactin is a potent natural insecticide

derived from the neem tree, Azadinachta indica, that when ingested increases larval

mortality and interferes with normal insect growth and development by disrupting the

molting process and inducing pronounced morphological deformities (Addor, 1995).

And natural ecdysones in many higher plants possess powerful molting hormone

activity, targeting the insect endocrine system as a point of attack and causing

developmental and reproductive anomalies when fed to several species of insects

(Harborne, 1988).

There are also several examples on the marine natural products produced by macro-

algae reported to have antigrowth activity on their predators. For example, juvenile

gastropods (Strombus costatus) fed on a control diet and others in which the natural

products halimedatrial (extracted from the sea grasses Halimeda spp.), udoteal (extracted

from Udotea cyathyformis) or caulerpenyne (extracted from Caulerpa taxifolia) had been

added showed the following survivorship over a 2-week period: control diet 100%, udoteal

55%, caulerpenyne 33% and halimedatrial 0% (Hay and Fenical, 1988). The same

compounds have also been shown to be toxic to larval stages of the sea urchin Lytechinus

pictus, and the fishes Pomacentrus coeruleus and Dascyllus auanus (Paul and Fenical,

1986).

Even in the copepod literature, there is some evidence that toxic secondary metabolites

produced by microalgae or cyanobacteria interfere with development. Huntley et al. (1987)

found that Calanus pacificus nauplii showed a low development rate and a high mortality

when fed with the dinoflagellates Gyrodinium resplendens, Amphidinium carterae,

Gonyaulax grindley and Ptychodiscus brevis. The authors suggested that the causes for

loss of neuromuscular control and subsequent death of the nauplii were due to the presence

of secondary metabolites. Also, Kumar and Rao (1998) found that nauplii of the cyclopoid

copepod, Mesocyclops thermocyclopoides, died after 6–9 days in the presence of the toxic

cyanobacteria, Mycrocistis aerogenes, and suggested that this was due to a problem of

poor assimilation and/or toxicity.

The object of this study was, therefore, to better investigate the toxic effects of

diatoms on postembryonic development of the calanoid copepod T. stylifera. The diatom

species selected for our study are among those that have been shown to have adverse

effects on the reproductive success of this copepod species. We compared, in laboratory

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 51

experiments, the effects of various diatom and nondiatom diets on several important

developmental parameters such as development rate and survivorship. The results

obtained are discussed in relation to the recent findings on the toxic effects of diatoms

on copepod embryogenesis.

2. Materials and methods

2.1. Phytoplankton

The diatoms T. rotula, Skeletonema costatum and Phaeodactylum tricornutum were

cultured in 2-l glass jars filled with 0.22-Am filtered sea water enriched with f/2 medium

(Guillard and Ryther, 1962) at 20 jC and on a 12-h dark/12-h light cycle. The autotrophic

flagellates P. minimum and Isochrysis galbana were cultured in K medium (Keller et al.,

1987) under the same experimental conditions as diatoms, while the heterotrophic

dinoflagellate Oxyrrhis marina was cultured in K medium enriched with a culture of

the chlorophyceae Dunaliella tertiolecta. Concentrations and morphological character-

istics of algal species used are reported in Table 1.

2.2. Copepods

Surface zooplankton samples were collected in the Gulf of Naples from October

2000 to June 2001 using a 250-Am net, and were transferred to the laboratory within

1 h. Mature T. stylifera females (N = 15) were sorted under a dissecting microscope and

incubated individually in crystallizing dishes filled with 100 ml of natural 50-Amfiltered seawater (Experiment 1) or with 0.22-Am filtered sea water enriched with P.

minimum (final concentration 5600 cell/ml) (Experiment 2). Both experimental groups

were kept at 20 jC and on a 12-h dark/12-h light cycle. Females were removed after

24 h, and egg production was determined under an inverted microscope. The eggs

produced were incubated again at the same temperature and light conditions, and egg

Table 1

Morphological characteristic and concentrations of diatoms and nondiatoms species used for rearing T. stylifera

Algal species Cell volume (Am3) Carbon content (pg/cell) Cells/ml Ag C/l

Phaeodactylum tricornutum 11 2.3 2.6� 105 600

Skeletonema costatum 196 20.7 3� 104 600

Thalassiosira rotula 2036 121.9 7� 103 850

Isochrysis galbana 65 13 7.7� 104 1000

Prorocentrum minimum 1340 177.1 5� 103 900

Oxyrrhis marina 3619 418.6 2� 103 800

Fig. 1. Development (–.– ) and survival ( –E–) curves of larval stages reared with the nondiatom diets I.

galbana, P. minimum and O. marina. The curves were calculated with a third polynomial function between mean

copepod stage and time and with an exponential function of the percentage of surviving individuals with time.

Mean stages 1–6 correspond to naupliar stages I–VI, 7–11 to copepodite stages I–V and 12 to adult stages.

Symbols represent averages and standard deviations of the replicates.

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6652

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 53

viability was determined 24 h later by counting the number of empty egg membranes

(Ianora and Poulet, 1993). Only when egg viability exceeded 80% of the eggs spawned

were the hatched nauplii used to start rearing experiments.

2.3. Rearing experiments

Two batches of 50 newly hatched nauplius one (NI) were placed in 300-ml glass jars

enriched with one of a series of diatom or nondiatom diets (Experiment 1). Experiments

with no food were also conducted to obtain controls under conditions of starvation. Three

to six replicates were made for each diet. Algal concentrations ranged from 2� 103 to

2.6� 105 cell/ml, corresponding to 600–1000 AgC/l (Table 1).

To assess the effect of maternal diet on neonate fitness, a new set of experiments

(Experiment 2) were started with batches of 50 newly hatched NI produced by females

preconditioned with P. minimum for 24 h. Nauplii thus generated were placed in 300-ml

glass jars filled with 0.22-Am filtered seawater enriched with P. minimum (4500 cell/ml) or

T. rotula (6700 cell/ml) (two replicates), and I. galbana (7.6� 104 cell/ml) or S. costatum

(4.8� 104 cell/ml) (one replicate).

All algae tested were in the exponential growth phase and were provided to nauplii so

as to obtain similar food levels in terms of carbon. Jars were mounted on a rotating wheel

at 0.5 rpm in incubation chamber kept at 20 jC and on a 12-h dark/12-h light cycle.

Individuals were gently collected each day, counted and checked under the microscope to

asses their larval stage, and then transferred to jars with fresh medium. A daily tally was

kept of mean larval stage and percentage of surviving individuals to calculate development

and survival curves for each experiment and, when possible, also the corresponding

development and mortality rates.

2.3.1. Development rate

Development with time was described with a third-order polynomial function. The

development rate of the population corresponded to the slope of the linear regression

calculated on the log10 mean stage against log10 time. These slopes were then used to test

for differences between experiments using covariance analysis (ANCOVA) performed by

‘‘GraphPhad Prism, version 3’’ software. Mean stage of the population was calculated

using the equation reported by Huntley et al. (1987).

2.3.2. Mortality rate

Survival with time was described by a negative exponential function Nt =N0e� Zt,

where N0 is the number of individuals at time = 0, Nt is the number of individuals at

time = t and Z is the instantaneous mortality rate (day� 1) of the population. Slope Z of the

linear regression of lnNt with t was used to test for differences between experiments using

covariance analysis (ANCOVA).

2.4. Confocal laser scanning microscope (CLSM) analysis

T. stylifera nauplii fed with diatom and nondiatom diets were randomly collected from

experimental jars, fixed in 3% glutaraldheyde and stained with ‘DiI’ (dioctadecyl-tetrame-

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6654

thylindocarbocyanine perchlorate; Molecular Probes) using a procedure published else-

where (Carotenuto, 1999). Their morphology was compared to nauplii produced by females

of T. stylifera fed for 7 days on the diatom T. rotula and nauplii obtained from wild females.

Dyed specimens were observed under a CLSM (ZEISS 410) equipped with a He/Ne laser

(543 nm k) at 40� magnification and with a 1.2-N.A. water immersion objective; images

were collected for each single focal plane, scanning the whole preparation. Zeiss software

was used to create three-dimensional (3-D) images of the specimens.

3. Results

3.1. Experiment 1

In all replicates, larval stages of T. stylifera reached adulthood when fed with P.

minimum, I. galbana and O. marina (nondiatoms) diets. The generation time from

Fig. 2. Development (–.– ) and survival (–E–) curves of larval stages fed with the diatom diets T. rotula, S.

costatum and P. tricornutum, and also under conditions of starvation. For T. rotula, separate curves were fitted for

the experiment in which larvae completed development (1) and experiments in which they did not (2). The curves

were calculated as reported in Fig. 1.

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 55

hatching to the adult stage ranged between 19 and 28 days, whereas percentage of

survival on the last day of the experiment was between 1% and 34.5%. The calculated

development and survival curves for these diets were very similar (Fig. 1). Develop-

ment was not isochronal and it increased almost linearly with time throughout the

naupliar and early copepodite stages, but became slower during the last copepodite and

until the adult stages. Survival curves showed a typical exponential trend with a very

sharp decrease in the number of surviving individuals during the naupliar and

copepodite stages, after which numbers remained almost constant with time until the

end of the experiment.

In contrast, larval stages of T. stylifera that were fed upon T. rotula, S. costatum and P.

tricornutum (diatoms) diets never reached adulthood except in one replicate with T. rotula

(22 days and 7% survival) (Fig. 2). Larvae generally survived only for 4–9 days and never

passed the naupliar stages with any of the diatom species tested. However, with T. rotula,

they survived for 12–19 days, reaching the early copepodite stages (CII). Although

development and survival with time followed the same polynomial and exponential model

as with nondiatom species, the shapes of the corresponding curves were very different

(except for larvae grown on T. rotula). They showed, in fact, a very flattened devel-

opmental curve and a more constant diminution in survival rates with time. Under

starvation conditions, the number of surviving larvae decreased linearly in only 3 days

and larvae molted only to the second nauplius (NII) stage (Fig. 2). That, as observed from

gut contents, is the first feeding stage.

Development rates, corresponding to the slopes of linear regressions relating log10

mean stage against log10 time for each diatom and nondiatom species, and with no food,

were significantly different (ANCOVA, F = 15.228, p < 0.0001), and are reported in Table

2. Development rate for nondiatom species ranged between 0.68 and 0.82 stage/day

compared to 0.58 and 0.73 stage/day for diatom species and 0.52 stage/day under

conditions of starvation.

In each rearing condition, except under conditions of starvation, survival was

exponential and the slope of the linear regression of ln(N) against time corresponded to

the mortality rate of the population. Mortality rates for larvae fed with each diatom and

Table 2

Parameters of linear regression of development, relating log10(mean copepod stage) to log10(time) for nondiatom

and diatom species

Algal species Slope (stage/day) S.D. Intercept r2

Isochrysis galbana 0.8228 0.015 0.013 0.98

Prorocentrum minimum 0.6800 0.009 0.123 0.98

Oxyrrhis marina 0.6771 0.013 0.138 0.97

Thalassiosira rotula (1) 0.7341 0.017 0.080 0.99

Thalassiosira rotula (2) 0.7193 0.017 0.048 0.97

Phaeodactylum tricornutum 0.5818 0.038 0.058 0.85

Skeletonema costatum 0.6118 0.027 0.057 0.96

No food 0.5177 0.084 0.049 0.79

Slopes correspond to development rates of the larval populations.

Analysis of covariance (ANCOVA) demonstrated that the slopes were significantly different (df = 7, F = 15.228,

p< 0.0001).

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6656

nondiatom species, were significantly different (ANCOVA, F = 9.010, p < 0.0001), and are

reported in Table 3. Larvae reared with nondiatom species showed a daily mortality

ranging from of 9.9% to 12.0%, compared to 20.3% to 35.5% for diatom species. In the

one case in which larvae could grow on T. rotula, the daily mortality was 10.3%.

With no food, survival decreased linearly with time; hence, mortality rate was

calculated as a linear regression between the percentages of individuals that survived

each day with respect to time. The slope of this line corresponded to the mortality rate of

the population (slope =� 27.12F 2.43; intercept = 128.7; r2 = 0.90).

Fig. 3. Correlation between development (stage/day) (.) and mortality (day 1) (o) rates of larval stages reared

with diatom and nondiatom diets, and cell volume of the algae. No correlation was found between these

parameters (r = 0.269, p> 0.05, and r =� 0.718, p>0.05, respectively).

Table 3

Parameters of linear regression of mortality, relating ln(N) to time for nondiatom and diatom species

Algal species Slope (stage/day) S.D. Intercept r2

Isochrysis galbana � 0.1240 0.023 3.934 0.30

Prorocentrum minimum � 0.0988 0.008 4.513 0.57

Oxyrrhis marina � 0.1205 0.005 4.568 0.85

Thalassiosira rotula (1) � 0.1031 0.008 4.005 0.88

Thalassiosira rotula (2) � 0.2030 0.016 4.540 0.79

Phaeodactylum tricornutum � 0.3553 0.038 5.120 0.68

Skeletonema costatum � 0.2365 0.040 4.762 0.59

Slopes correspond to mortality rates of populations.

Analysis of covariance (ANCOVA) demonstrated that the slopes were significantly different (df= 6, F = 9.010,

p< 0.0001).

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 57

A correlation was also made between development and mortality rates of the larval

stages reared with diatom and nondiatom species and the log10 of cell volume, which is a

measure of cell size (Fig. 3). No relationship was found between size of the algae and

developmental (Pearson r = 0.269, p>0.05) or mortality rates (Pearson r =� 0.718, p>0.05).

Hence, the differences within rates were not linked to morphology of the algal food.

3.2. Experiment 2

All nauplii reared with P. minimum and I. galbana reached adulthood after 22–23 and

19 days, respectively, with a very high final survivorship (80–70% and 80%) that was

much higher than that obtained in Experiment 1.

Fig. 4. Development and survival curves of larval stages generated from P. minimum preconditioned females and

then reared with nondiatom diets I. galbana and P. minimum, or with the diatom diets T. rotula and S. costatum.

For T. rotula, separate curves were fitted for the experiment in which larvae completed development (1), and

experiments in which they did not (2). The developmental curves were calculated as reported in Fig. 1, while all

the mortality curves were obtained as connecting lines through the points.

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6658

By contrast, when nauplii were fed with the diatoms T. rotula and S. costatum, again

they completed their development with T. rotula in only one replicate. Development time

was very similar to that obtained with nondiatoms (19 days), but was worse in terms of

final survival (34%). In another replicate with T. rotula, where development did not

proceed to adulthood, and also with the other diatom S. costatum, larvae died after 14

days, reaching the CII stages, or after 7 days, not passing the NIV stage (Fig. 4).

The trend for the development curves for all diets were very similar to those of

Experiment 1, proceeding almost linearly through time until the later copepodite stages.

Development rates for larvae fed with all diets are reported in Table 4, with values ranging

between 0.64 stage/day for S. costatum and 0.84 stage/day for I. galbana. These rates are

significantly different from one another (ANCOVA, F = 6.41, p < 0.0001).

The shapes of the survival curves were, in contrast, very different from the previous

experiments (Experiment 1). Since survival did not decrease with time following an

exponential trend, it was not possible to calculate the mortality rates as for Experiment 1.

Hence, to compare different diets in which larvae completed development, we performed a

repeated-measures ANOVA on the number of individuals surviving each day during time.

Significant differences were recorded between P. minimum, I. galbana and T. rotula

(ANOVA, F = 36.31, p < 0.0001), except for P. minimum and I. galbana (Tukey’s Post-hoc

test, p>0.05). Survival at the end of the experiment with T. rotula was 34% compared to

70–80% with either P. minimum or I. galbana.

3.3. CLSM analysis

3-D confocal images of naupliar stage NI generated from wild females and with the

control diet were normal (Fig. 5A). The cephalosome carried three pairs of well-shaped

symmetrical mouth appendages with normal segmentation and setation, and the metasome

was normal with the characteristic caudal spines and setae. Also, the nauplii generated by

mothers fed T. rotula for 24 h were normal as opposed to those obtained from females fed

T. rotula for 7 days. Abnormal nauplii showed strong congenital defects consisting in

asymmetrical bodies, which were at times laterally flattened, and which had a reduced

number of mouth appendages (Fig. 5B). Nauplii obtained from mothers fed P. minimum

Table 4

Parameters of linear regression of development, relating log10(mean copepod stage) to log10(time) for nondiatom

and diatom species

Algal species Slope (stage/day) S.D. Intercept r2

Prorocentrum minimum 0.7514 0.015 0.027 0.98

Thalassiosira rotula (1) 0.8022 0.014 0.0473 0.99

Thalassiosira rotula (2) 0.7632 0.018 0.0421 0.99

Isochrysis galbana 0.8447 0.024 0.0265 0.98

Skeletonema costatum 0.6451 0.053 0.059 0.96

Slopes correspond to development rates of populations.

Analysis of covariance (ANCOVA) demonstrated that the slopes were significantly different (df= 4, F = 122.0,

p< 0.0001).

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 59

for 24 h and successively switched to T. rotula also did not show deformities during

development, as for the NIII stage in Fig. 5C.

4. Discussion

Larval stages of T. stylifera were unable to develop from hatching to the adult stage

when fed on monocultures of the diatoms T. rotula, S. costatum and P. tricornutum, and

could only complete development in the presence of the flagellate I. galbana and the

dinoflagellates O. marina and P. minimum. Generation times of 19–22 days with the

flagellate were close to the 20 days reported by Abou Debs (1979) for T. stylifera reared

with the Haptophycea Hymenomonas elongata, but were longer than the 15 days obtained

by Yassen (1981) with a mixture of Cricosphaera elongata, Monochrysis lutheri and P.

tricornutum. This latter author reported a daily mortality rate of 6.7%, which is close to the

mortality rates obtained in our experiments with the nondiatom diets (9.9% and 12%). This

Fig. 5. 3-D reconstruction of confocal images of (A) well-formed naupliar stage NI from wild females with high

initial hatching success (z 80%), used to start rearing experiments with each diet. (B) Abnormal naupliar stage NI

hatched from females fed T. rotula for 7 days. (C) Normal naupliar stage NIII reared with the diatom T. rotula,

which was unable to complete development to adulthood. Scale bars = 55 Am.

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6660

implies that monocultures of flagellates or dinoflagellates are a suitable food to sustain the

complete development of larval stages of T. stylifera.

As for diatoms, in only one replicate with T. rotula did larvae reach the adult stage and

complete their development. Developmental parameters (generation time and survival) in

this case were in the same range as those for larvae fed with nondiatom species (22 days

and 7%). All other diatoms could not sustain larval development at all, and most animals

died before passing the naupliar phase or, as for other replicates with T. rotula, soon after

the early copepodite stages. Development and mortality rates of larvae fed with diatoms

were worse than those of larvae reared with nondiatoms (Figs. 1 and 2).

The adverse effects of diatom diets on larval development of T. stylifera were also

apparent when females were preconditioned with a good-quality diet (P. minimum) to

reduce the negative effects of maternal diet and past feeding history of females prior to

capture. In Experiment 2, larvae reared with nondiatoms (P. minimum and I. galbana)

showed higher survivorship compared to Experiment 1 (75.5% vs. 13% and 80%

vs.13.7%, respectively). These values are in the range or even better than those reported

in the literature for other copepod species (Mauchline, 1998 and references therein). By

contrast, larvae fed with diatoms completed their development in only one case with T.

rotula and, although survivorship was higher than in Experiment 1 for the same diet (34%

compared to 7%), it was nonetheless lower than with the control diets. A faster develop-

ment was observed in larvae that completed development with T. rotula (curve 1, Fig. 4),

compared to P. minimum. Analogous results were obtained with C. helgolandicus nauplii

fed on S. costatum and P. minimum (Ianora et al., in preparation), but the causes for

enhanced growth albeit high mortality rates are unknown.

Diatoms have always been considered as a suitable source of energy for zooplankton,

and in particular copepods, to sustain secondary production in terms of reproduction and/

or growth. Harris and Paffenhofer (1976) were able to grow larval stages of T. longicornis

on the diatom T. rotula, with a generation time of 24 days and a final mortality of 37.5%.

Similar results (29 days and 25.3%) were obtained for Pseudocalanus elongatus

(Paffenhofer and Harris, 1976). Various other copepod species have also been successfully

grown with diatom foods, such as C. helgolandicus on Lauderia borealis with a final

mortality less than 10% (Paffenhofer, 1976), and Centropages typicus on T. weissflogii in

more than 40 days (Smith and Lane, 1985). In the latter case, however, the authors did not

estimate mortality because of the possible damage of individuals during transfer.

On the other hand, there is also some evidence that diatoms are not an optimal source of

food to grow copepods, since they lengthen the generation time and increase mortality

rates. Paffenhofer (1970), for example, found that larval stages of C. helgolandicus grew

slower (36 and 24 days) and had a higher mortality (33.9% and 13.5%) with the diatoms S.

costatum and L. borealis, respectively, compared with the dinoflagellate Gymnodinium

splendens (18 days and 2.3%) given at the same food concentration. Moreover, females of

Calanus grown on diatoms were smaller than those obtained with dinoflagellates, which

were of the same size range as females collected at sea. The author concluded that these

results were not due to differences in food quality, but to the higher ingestion of

dinoflagellates compared to diatoms. More recently, Koski et al. (1998) demonstrated

that the copepod P. elongatus could grow on the diatom T. weissflogii with a very low

daily mortality rate (3.8%/day) but development and growth rates were slower compared

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 61

to the flagellate Rhodomonas spp. and the dinoflagellate G. simplex. The authors

concluded that the reason for the scarce growth success was not due to an absence of

ingestion by P. elongatus, or to the toxicity of the food species, but probably to its

digestibility or mineral/biochemical composition. Recently, Rey et al. (2001) reported that

C. helgolandicus nauplii showed higher development rates when fed with I. galbana and

R. baltica, compared to nauplii reared with T. weissflogii. However, the authors did not

explain as to why developmental performance was poorer with the diatom food. Ianora et

al. (in preparation) also found that C. helgolandicus could grow on the diatom S. costatum,

even though mortality rates were higher than with the nondiatom P. minimum.

Our results show that except for two replicates with T. rotula, larvae of T. stylifera were

unable to grow on diatoms at all, as also reported by Mullin and Brooks (1970), who found

that nauplii of C. helgolandicus were unable to grow with Ditylum brightwelli. Also,

Hirche (1980) found that all nauplii of the copepod Calanoides carinatus fed on P.

tricornutum died after 8–10 days, soon after passing the naupliar phase. In contrast, larvae

grew on the dinoflagellate G. splendens, with a development time of 20 days and a final

survival of 13%. Also, Peterson (1986) found that larval stages of C. marshallae grew on a

mixture of T. weissflogii and I. galbana in only one experiment, with a final survival of

19% and a generation time of 36 days because specimens of other replicates did not pass

beyond the fifth copepodite stages.

No clear explanation was given in these studies as to why larvae could not be reared on

diatoms, although usually the negative effect of food was assumed to be due to its

inadequate nutritional content. In most cases, the term quality included different aspects of

the food, such as taxonomy (Koski et al., 1998), morphological parameters (Berggreen et

al., 1988), mineral composition (Kiørboe, 1989) and/or content of some specific bio-

chemical compounds such as amino acids (Cowie and Hedges, 1996), polyunsaturated

fatty acids (Jonasdottir, 1994) and sugars and vitamins (Brown et al., 1997). Hence, the

inability of a diet to allow the development of copepod species has been associated with

size of the algae (Mullin and Brooks, 1970; Hirche, 1980), age of the culture (Paffenhofer,

1971), and inadequate mineral/biochemical composition of the phytoplankton (Huntley et

al., 1987; Koski et al., 1998). Our results, however, show that differences in developmental

performances of T. stylifera were not due to cell size of the species used as food (Fig. 3), or

to age of the culture since these were always in the exponential growth phase. It also seems

improbable that noxious metabolites were accumulated in the culture medium of larvae fed

diatoms because cultures were substituted with fresh filtered seawater and algae daily.

We cannot exclude the possibility that diatoms were nutritionally inferior food, because

no data were collected on their chemical composition, or that the larvae with respect to

nondiatoms less efficiently assimilated these cells. In all cases, larvae ingested diatom cells

as observed from microscopic analysis of gut fullness, and from the high number of faecal

pellets produced during the rearing experiments. Brown and Jeffery (1995) examined the

chemical composition of six different diatom species, including S. costatum, and found

that all diatoms were rich in high-quality proteins (31–38% dry weight) and lipids (18–

20% dry weights). These data are in the range of 30–60% and 10–20%, respectively,

reported by Brown et al. (1989) for diets that have been successfully used as food for early

prawn larvae and, in general, induce satisfactory growth for crustaceans. Diatoms and

flagellates are also generally considered as good-quality food because of their high level of

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–6662

fatty acids (Brett and Muller-Navarra, 1997), which are considered essential for zoo-

plankton larval growth (Muller-Navarra, 1995; Gulati and Demott, 1997).

However, notwithstanding their high nutritional content, numerous recent studies

have shown that diatoms have deleterious effects on copepod reproduction. When

ingested in large quantities, diatoms block normal embryogenesis and eggs fail to

develop to hatching (Turner et al., 2001 and references therein). We here suggest that

diatoms may also have insidious effects on larval postembryonic growth. We cannot say

with certainty whether these negative effects on growth were due to inhibition by

diatom aldehydes, or to poor efficiency in capturing, ingesting or digesting diatoms by

larvae. However, from our results still in progress, we have found that C. helgolandicus

fed on P. minimum cultures with the addition of diatom aldehyde (decadienal) were

unable to complete development and grew only to the sixth naupliar stages after 3

weeks, indicating that these molecules have negative effects on larval development of

copepods (Ianora et al., in preparation).

Although no morphological aberrations were found in larvae fed on diatoms

observed at the confocal microscope, such individuals died soon after birth, or did

not develop beyond the naupliar stage. Nauplii reared on diatoms were structurally

similar to larval stages reared on nondiatom food, and did not show any of the birth

defects of nauplii generated when females were fed on a diatom diet for several days

(Fig. 5). Previous studies have already shown that deformed nauplii are produced by

females that are fed dense diatoms cultures (Poulet et al., 1995; Uye, 1996; Starr et al.,

1999; Ianora et al., in preparation). And similar anomalies have also been found in eggs

spawned by wild C. helgolandicus from the English Channel during the spring-diatom

bloom (Laabir et al., 1995). Ban et al. (2000) found that the proportion of deformed

nauplii produced by wild females of P. neumanii was negatively correlated with egg

hatching success, but the authors did not find any correlation between hatching success

and diatom abundance at sea. However, when females were fed in the laboratory with a

nondiatom diet (Pavlova spp.), the percentage of deformed nauplii decreased with time

to zero.

Our results suggest that the production of deformed nauplii seems to be related only to

maternal consumption of diatoms and transfer of toxins that induce teratogenesis in

developing embryos. By contrast, when nauplii are spawned from females having high

initial hatching rates (i.e. in our experiments hatching success was always z 80%), diatom

metabolites do not seem to induce morphological aberrations during growth even though

they still induce slower development rates and higher naupliar mortality, leading to

extinction of the population before reaching adulthood. Regardless of the method of the

loss or the impact, therefore, the production of these secondary plant metabolites, probably

produced by the plant as a part of a defensive mechanism against predation, has

successfully eliminated potential predators.

We conclude that diatoms not only induce insidious abortive effects during embryo-

genesis, but also antigrowth effects on later larval stages. Even when maternal effects on

neonates are minimal (as inferred from the high initial egg viability values in Experiment

1), or reduced (due to preconditioning as in the Experiment 2), juveniles of certain

copepods species may not develop on a diatom food, or survivorship will at times be lower

than with a nondiatom diet. The latter experiments indicate that the nutritional condition of

Y. Carotenuto et al. / J. Exp. Mar. Biol. Ecol. 276 (2002) 49–66 63

mothers is fundamental for the well being of their offspring. It is likely that the different

results induced by the same diatom T. rotula (indicated as curves 1 and 2, Figs. 2 and 4)

were due to past-history feeding of the females that promoted the production of healthy or

less healthy individuals with higher or lower chances of survivorship independently from

the food that the nauplii will receive during growth.

The fact that some diatoms (T. rotula) induced less deleterious effects on development

of T. stylifera than others (S. costatum and P. tricornutum) may denote differences in

toxicity among diatom species. Copepod species may also differ in their capacity to

detoxify such compounds. For example, with the diatom T. rotula, hatching success was

reduced to 0% after 15 days (Turner et al., 2001) but only to 65% in C. helgolandicus

(Chaudron et al., 1996). This would explain why some diatoms promote good growth in

some copepods but not others, and why not all diatoms induce the same negative effects.

The study of chemical defense in diatoms is still in its infancy and much remains to be

explored on diatom–copepod interaction and the role of secondary plant metabolites in

shaping predatory copepod populations. In the long run, such studies will shed light on the

mechanisms underlying energy transfer through the marine food chain, from primary

producers to higher trophic levels.

Acknowledgements

We sincerely thank Dr. F. Esposito for his technical assistance and for preparation and

maintenance of phytoplankton cultures. Ylenia Carotenuto acknowledges the financial

support from the Italian Murst University Programs and the ‘‘Stazione Zoologica’’ of

Naples for completion of her PhD. [RW]

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