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Effects of different monoalgal diets on egg production, hatching success andapoptosis induction in a Mediterranean population of the calanoid copepodAcartia tonsa (Dana)

Jianshe Zhang, Changwen Wu, David Pellegrini, Giovanna Romano,Francesco Esposito, Adrianna Ianora, Isabella Buttino

PII: S0044-8486(13)00097-5DOI: doi: 10.1016/j.aquaculture.2013.02.032Reference: AQUA 630569

To appear in: Aquaculture

Received date: 5 September 2012Revised date: 20 December 2012Accepted date: 26 February 2013

Please cite this article as: Zhang, Jianshe, Wu, Changwen, Pellegrini, David, Romano,Giovanna, Esposito, Francesco, Ianora, Adrianna, Buttino, Isabella, Effects of differ-ent monoalgal diets on egg production, hatching success and apoptosis induction ina Mediterranean population of the calanoid copepod Acartia tonsa (Dana), Aquaculture(2013), doi: 10.1016/j.aquaculture.2013.02.032

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Effects of different monoalgal diets on egg production, hatching success

and apoptosis induction in a Mediterranean population of the calanoid

copepod Acartia tonsa (Dana)

Jianshe Zhang1,2

, Changwen Wu1, David Pellegrini

2, Giovanna Romano

3,

Francesco Esposito3, Adrianna Ianora

3 and Isabella Buttino

2,3,4 *

1) College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan, PRC

2) ISPRA Istituto Superiore per la Ricerca e Protezione Ambientale STS-Livorno- Piazzale dei Marmi

Terminal Crociere – 57128 Livorno (Italy)

3) Stazione Zoologica Anton Dohrn – Napoli- Villa Comunale 80121 Napoli

4) CRIAcq-Interdepartmental Research Center for Hydrobiological Resources Management and for

Aquaculture. University of Naples “Federico II”.

* Corresponding author

e-mail:Isabella.buttino @isprambiente.it

Abstract

The influence of six monoalgal diets was tested on the reproductive success of

the copepod Acartia tonsa over a 15-day period in order to define the most

favourable diet for the optimization of this copepod species to be used in

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aquaculture and in ecotoxicology applications. The cryptophytes Rhinomonas

reticulata and Rhodomonas baltica induced highest egg production rates (mean

= 24.4 eggs female-1

day-1

and 21.9 eggs female-1

day-1

) and hatching success

(mean = 76% and 86.1%) over the 15-day period, respectively. Lowest egg

production rates were recorded with both diatoms Phaeodactylum tricornutum

(mean = 8.8 eggs female-1

day-1

) and Skeletonema marinoi (mean = 8.4 eggs

female-1

day-1

). These two diatoms also had detrimental effects on hatching

success (mean = 44.1% and 46.5%, respectively) and adult survival. No adults

survived for longer than 13 days with a diet of P. tricornutum. Moreover,

nauplii produced by females that had fed on diatoms for >10 d, were positively

stained for the apoptotic fluorescent marker TUNEL, indicating imminent

death. The prasinophyta Tetraselmis suecica induced low fecundity (mean =

13.4 eggs female-1

day-1

) and hatching success (mean= 62%), and after 15 days

only 37.5% of the adults survived. Isochrysis galbana induced low egg

production per female (< 10 eggs female-1

day-1

) after 15 days.

Keywords: copepods, phytoplankton, egg production, hatching success, fecal

pellet production

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1. Introduction

In recent years there has been a growing interest in the massive cultivation of

marine copepods to be used for different purposes, for example, in aquaculture,

as live feed for fish larvae (Drillet et al.; 2006a, Buttino et al., 2012; Olivotto et

al., 2008 and 2009), or in ecotoxicology studies, as animal models to predict

the impact of chemicals on marine zooplankton physiology (Buttino et al.,

2011; Carotenuto et al., 2012; Fang et al., 2006; Gorbi et al. 2012;).

Cultivation of copepods at high densities is difficult, especially for temperate

species with longer developmental times compared to tropical and subtropical

copepods (Payne and Rippingale, 2001; Støttrup 2000). Therefore, massive

copepod production remains a bottleneck for their extensive use in aquaculture

or for other laboratory applications.

Rearing techniques have focused on how to improve copepod fitness and

increase their productivity, especially in indoor systems (Buttino et al., 2012;

Støttrup 2000) since copepod production is mainly dependent on the quality

and quantity of food supplied (Buttino et al., 2011; Ceballos and Ianora 2003;

Kleppel et al., 1998; Zheng et al., 2011). Recently Drillet and coauthors (2011a)

showed that a microbial preparation in addition to an algal food, can enhance the

performance of A. tonsa in terms of egg production and hatching success.

Copepods are at the base of the marine food web, linking primary producers

with higher trophic levels and, hence, the nutritional quality of phytoplankton

will reflect the quality of fish larvae growing on a food-based copepod diet

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(McKinnon et al., 2003; Olivotto et al., 2008). Acartia tonsa is one of the most

investigated calanoid species, occurring in a wide range of geographic areas

from temperate to subtropical waters (Paffenhöfer and Stearns, 1988). In the

Mediterranean Sea, this eurythermic and euryhaline species was introduced in

the 1980’s (Farabegoli et al., 1989; Gaudy and Viñas, 1985), becoming the

dominant species in Northern Adriatic lagoons (Comaschi et al., 2000; Sei et

al., 1996) and recently found in confined waters in Southern Italy (Belmonte

and Potenza, 2001).

Acartia tonsa species may have a future in aquaculture because is a

cosmopolitan species and "easy to grow" compared to other calanoid copepods,

have a full life cycle in the water column (see Støttrup 2006 and Drillet et al.,

2011b for reviews) and their eggs can be stored longer in

temperature-controlled conditions (Drillet et al. 2006b; 2008). Moreover, this

species has widely been proposed as an animal model in ecotoxicology

standardized assays (Gorbi et al., 2012; ISO 1999; Widdows 1993).

Many authors have tested a variety of phytoplankton cultures, in terms of cell

density or strain quality, on Acartia tonsa production in different experimental

rearing conditions: from massive cultivation in large volumes, to small

aquariums in laboratory-based cultures (Amin et al., 2011; Colin and Dam,

2002; Leandro et al., 2006, Marcus and Wilcox, 2007). Recently, Drillet and

co-authors (2008, 2011c) found that populations of A. tonsa coming from

different geographic areas, showed distinct mitochondrial clades and life

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history traits, such as generation times and productivity, which differed for

each clade even if the same food conditions were supplied. Therefore, the

relationship between feeding and productivity of A. tonsa must be

re-considered in the light of these findings and the origin of different

populations must be taken into account before any generalizations can be made

on the productivity of this copepod species.

In our laboratory, the copepod A. tonsa collected in the lagoon area in the

Northern Adriatic Sea (Comacchio Valley) has been reared as an animal model

for ecotoxicological studies and for future applications in aquaculture as live

feed for fish larvae, since 4 years. In the present study we test which of 6

monoalgal diets was the best food, in terms of egg production and egg viability,

for the Mediterranean strain of A. tonsa for 15 days, and compared our results

with those obtained by other authors for different populations of this copepod

species. The aim of the present study is to define the most favourable

monoalgal diet for the optimization of A. tonsa to be used both in

ecotoxicology applications, as an animal model to test the toxicity of different

pollutants, and to be proposed as first-feed for fish larvae in aquaculture

farming, as a supplement to classical Artemia and rotifer diets.

Of the algae tested, it is well documented that two diatoms (Skeletonema

marinoi and Phaeodactylum tricornutum) produce toxic oxylipins which

induce apoptosis in other copepod species (reviewed by Ianora and Miralto,

2010). Because there is no information about the effect of these diatom diets on

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A. tonsa reproduction, the aim of this study was also to test these algae on this

copepod species. The other phytoplankton species tested (Isochrysis galbana,

Rhinomonas reticulata, Rhodomonas baltica, Tetraselmis suecica) are not

known to produce toxic metabolites and are generally used as mixed diets for

copepod feed (Buttino et al., 2009, 2012; Carotenuto et al., 2012; Mauchline,

1998).

2. Materials and methods

2.1 Algal cultures

Two diatom species Phaeodactylum tricornutum (FE1 corresponding to RCC

69) (PHAE) and Skeletonema marinoi (Sarno et al., 2005) (CCMP 2092, SKE),

two cryptophytes Rhodomonas baltica (Zimmermann) (FE 202, RHO) and

Rhinomonas reticulata (FE 208 corresponding to CCAP 995/2, RHINO), the

prymnesiophyte Isochrysis galbana (FE 207 corresponding to CCMP 1323,

ISO) and the prasinophyte Tetraselmis suecica (Butcher) (FE 205

corresponding to CCMP 906, TETRA) were used as food for adult Acartia

tonsa (Dana) copepods. These algae were cultured in a temperature-controlled

room, using 500 ml flasks filled with 0.22μm filtered seawater (FSW) with a

salinity of 35‰ for the two diatoms PHAE and SKE and 30‰ for the others.

Filtered seawater was previously treated for 24h with HClO (0.04% v:v), and

then with sodium thiosulphate 12.5% (v:v) for another 24h. Sea water was

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aerated for 24h (Lavens and Sorgeloos, 1996) to remove chloride residues. All

algae, coming from cultures at the Stazione Zoologica Anton Dohrn in Naples,

were cultured using F/2 medium (Guillard, 1975), with silicate for the diatom

cultures and without silicate for the other algae. The algae were cultured on a

14 h light:10 h dark cycle.

Algal cultures were supplied to copepods during their exponential growth

phases at the following concentrations corresponding to 500μg C L-1

: ISO

3.8x104 cells mL

-1 (~65µm

3 volume); RHINO 0.90x10

4 cells mL

-1 (~321 µm

3);

RHO 0.4x104 cells mL

-1 (~942 µm

3); TETRA 1.1x10

4 cells mL

-1 (~298µm

3);

SKE 2.5x104 cells mL

-1 (~196µm

3 volume); PHAE: 7.35x10

4 cells m

-L

-1

(~11µm3 volume). Carbon contents were converted from cell volumes

according to the formula reported by Strathmann (1967).

Copepod rearing experiments

A. tonsa used for this study were reared, for more than 70 generations, at the

ISPRA laboratory in Livorno (Italy). Adults, originally obtained from cultures

coming from the University of Parma (courtesy of Gorbi G. and Sei S.), were

reared in a 50L aquarium containing 20L of 0.22 μm filtered seawater

(Millipore 90 mm holder YY3009000) at 30‰ salinity and fed with a mixed

algal diet of ISO, RHINO and RHO at a final concentration > 300 μg C L-1

per

day. The aquarium was maintained at 20°C and a 14h L: 10h D photoperiod.

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2.2 Egg production, fecal pellets and hatching success

After the in vitro population reached the adult stage, healthy mature females

and males, with intact appendages and that were actively swimming, were

selected from the tank and 8 couples were incubated pair wise in 100 ml

crystallizing dishes containing 50 ml 0.22 µm for each of the 6 test algal diets

Containers were maintained on a very slow agitator to favour algal suspension.

After 24h, copepods were transferred to new crystallizing dishes containing

fresh media and eggs and fecal pellets were counted under an inverted

microscope (x10, 20 magnification). Egg hatching success was determined 48h

after egg laying, by adding 0.2 ml Lugol solution and counting the number of

nauplii that had settled on the bottom of the containers. Nauplii were

considered abnormal when the body or appendages were asymmetrical such as

previously described for Calanus helgolandicus (Poulet et al., 1995). Egg

production experiments were run for 15 days to test if the diet significantly

modified copepod reproductive responses. Experiments were run in duplicates

for each algal diet (N=96).

2.3 Fluorescence labeling and image acquisition

Apoptosis in nauplii was verified using TdT-mediated dUTP nick end labeling

(TUNEL) (Roche Diagnostics). Acartia tonsa nauplii, obtained from females

fed for 10-15 days on each of the monoalgal diets, were fixed overnight in 4%

paraformaldehyde and 0.2M NaCl in PBS, pH 7.4, rinsed in PBS, and frozen in

liquid nitrogen to fracture the carapace. Samples, incubated for 20 h in 0.3 unit

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mL-1

chitinase enzyme (Sigma-Aldrich) at 25°C to permeabilize the chitin,

were then treated according to the TUNEL manufacturer’s instructions.

To verify if SKE and PHAE induced apoptosis in newly hatched nauplii, we

incubated nauplii with TUNEL and with Hoechst to visualize all nuclei.

Acartia tonsa nauplii were incubated with 10 µg mL-1

of the vital fluorescent

dye Hoechst 33342 (Sigma-Aldrich) for 20 minutes at room temperature to

stain nucleic acids. Nauplii were then observed with the confocal

laser-scanning microscope Zeiss LSM-510, using a 488 nm wavelength laser,

to visualize TUNEL-positive areas (green), with a 543 nm wavelength laser, to

visualize chlorophyll autofluorescence (red), and with 405 nm wavelength laser

to observe nuclei stained with Hoechst (violet) (Buttino et al., 2004, 2011).

Acartia tonsa adults fed TETRA for 15 days were observed with a Zeiss

Primovert Monitor inverted.

2.4 Data analysis

One-way ANOVA analysis of variance and Bonferroni’s multiple comparison

tests were used to analyze significant differences among treatments. All

statistical analyses were conducted using GraphPad Prisma Program. Daily

data in the graphs are presented as means ± standard errors (s.e.), means

calculated over the whole experimental period are reported in each graph ±

standard deviation (s.d.).

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3. Results

The number of eggs, fecal pellets (fp) and % egg hatching success for

Acartia tonsa copepods fed six monoalgal cultures are reported in Figures 1

and 2. Each diet induced variable daily egg production rates during the 15-d

incubation period with lowest mean values of 8.4 eggs f-1

obtained with the

diatom SKE, to a maximum of 24.4 eggs f-1

recorded with the cryptophte

RHINO.

With a diet of ISO, egg production increased from 11.7 to a maximum of

19.4 eggs f-1

after 4 days and remained at these levels until day 9. After 10 days

egg production declined to reach a minimum of 5.0 eggs f-1

on day 13 (Fig. 1A).

Mean egg production per female during the whole experimental period was

12.7 ± 5 (s.d.). Fecal pellet production followed a similar trend with a mean

value of 95.2 ± 24.4 (s.d.) fp couple-1

day-1

recorded during the whole

experimental period. Egg hatching success was high during the first week of

feeding but dropped to less than 50% after 12 days, with a mean value of

78.2% (Fig. 1B).

A RHINO diet induced a rapid increase in egg production after one week,

with a maximum of > 30 eggs f-1

, followed by a slight decline after 11 days

with a production of almost 20 eggs f-1

day-1

(Fig. 1C). Mean egg production

24.21 ± 8.57 eggs f-1

day-1

(s.d.), calculated over the whole period, was the

highest recorded for all of the tested diets.

Fecal pellet production was very high with a mean of 138 fp couple-1

day-1

.

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The % egg hatching success slowly declined during the experimental period

from greater than 85% to 60% after 15 days. On average, egg hatching success

calculated over the whole period was 76%.

RHO diet induced a similar egg production rate as for RHINO with almost

21.9 ± 5.9 eggs f-1

day-1

(s.d.) and the highest egg hatching success (86.1%)

recorded for the entire period (Fig. 1 E,F).

When couples were fed with TETRA fecundity remained low (< 20 eggs

f-1

day-1

) and was strongly reduced after the first week. A minimum egg

production rate (less than 5 eggs f-1

) was recorded at the end of the experiment

and, on average, egg production was only 13.4 eggs per female during the

whole experimental period (Fig. 2A). In contrast, fp production was the highest

recorded of the six diets, with a mean of 142 fp couple-1

day-1

. Egg hatching

success declined very quickly, from almost 100% on the first day to less than

60% after 10 days, and to only 20% after 14 days (Fig. 2B). On average egg

hatching success calculated for the whole experimental period, was 62.2%.

With the diatom diets PHAE and SKE, egg production was the lowest recorded

(almost 8 eggs f-1

d-1

). In particular with PHAE egg production was strongly

reduced after 5 days from almost 15 eggs f-1

d-1

to less than 4 eggs after 8 days

(Fig. 2C). Fecal pellet production declined sharply during the experimental

period, from an initial value of almost 200 fp couple-1

recorded the first day to

about 50 fp on day 11, suggesting that couples did not feed as at the beginning

of the experiment. Also egg hatching success declined very rapidly, and on day

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9 only 20% of the eggs hatched (Fig. 2D). No apparent abnormal nauplii were

observed during the experiment, but those that survived were sluggish and

showed limited swimming capabilities. With this diet only one couple survived

after 12 days and the experiment was therefore terminated on day 12.

With the other diatom SKE, egg production remained low throughout the

experimental period, with a minimum of 3.8 eggs f-1

recorded after 14 days

(Fig. 2E). By contrast, fp production was very high at the beginning, with more

than 150 fp couple-1

on the first 3 days. It then became very unstable: dropping

to about 100 fp couple-1

for the following 4 days but increasing again on day 9

to 200 fp couple-1

. Production declined again during the second week. The %

egg hatching success declined in the first week, from 80% to less than 40% on

day 7. During the second week egg hatching success was more than 50% but

then declined again to less than 40% after three days (Fig. 2F).

Figure 3 shows the % of female survival with the different diets. PHAE and

TETRA diets induced high adult mortality in females after 10 and 13 days,

respectively.

Nauplii, produced by females fed ISO were used as controls, due to the high %

egg viability recorded during the entire experimental period (Fig. 1B). Figure 5

shows fluorescent nauplii stained with both dyes and observed with

fluorescence microscopy. Fig. 5A is a normal nauplius produced by a female

fed ISO for 10 days; the only visible fluorescence is the violet, due to Hoechst,

and red autofluorescence due to chlorophyll inside the gut. A similar

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fluorescent staining was observed for nauplii produced by A. tonsa females fed

TETRA for 10 days (data not reported), indicating that such nauplii were not

undergoing any apoptotic processes. Figures 5 B and C show nauplii produced

by females fed SKE for 10 and 12 days, respectively. Green fluorescence

signals are well visible in the legs and along the body; red fluorescence due to

chlorophyll is not visible. After 12 days many hatched nauplii appeared

strongly deformed with a relevant portion of tissues that were positively

stained with TUNEL (Fig. 5C). These nauplii were characterized by slow

movements and did not survive long after observation.

Nauplii produced by females fed PHAE appeared normal but showed strong

positivity to TUNEL in the legs and inner body (Fig. 5D) indicating that

apoptotic processes were under way.

4. Discussion

The copepod species A. tonsa has been proposed in aquaculture and as

animal model in bioassay protocols to text marine pollutants (Drillet et al.,

2006a, 2011b; Gorbi et al., 2012). Therefore, it is important to found better

conditions to rear this copepod with reduced efforts in terms of costs and time.

Moreover, it has recently been reported that different copepod strains, living in

different geographic areas, could have different physiology and life history

traits (Drillet et al., 2008, 2011c; Lauritano et al., 2012).

Our results on the productivity of A. tonsa copepods, collected in a

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Mediterranean area and reared in labotory since 4 years, indicated that diatoms

are deleterious for their fecundity and naupliar viability, while the best

monoalgal diets are the cryptophytes RHINO and RHO. Statistical comparison

among A. tonsa fed six different monoalgal diets suggests three different

groups showed no significant differences in terms of productivity: the first one

included two cryptophytes, with the highest egg production and egg hatching

success, the second group of ISO and TETRA with reduced egg production and

relatively high egg hatching, and the diatoms PHAE and SKE, as a third group,

with very low fecundity and hatching success and with also a reduction in

the % of adult survival (Table 1). However, significant differences occurred

among these three groups. With the two cryptophytes RHO and RHINO, egg

production was the highest recorded, increasing 5-fold after the first 10 days

with RHINO and 4-fold at the end of the experiment. RHO also induced the

highest egg hatching success during the entire experimental period, followed

by ISO and RHINO. The excellence of a RHO diet has also been reported by

other authors for different copepod species (Buttino et al., 2009; Dahl et al.,

2009; Ismar et al., 2008) providing high survival rates and fast naupliar

development at concentrations ranging from 150 to 300 µg C L-1

. Egg

production per female per day recorded for our Mediterranean A. tonsa

population, did not reach the production rates recorded by Holste and Peck

(2006) for the Baltic population (almost 50 eggs per female per day). However,

this production was recorded supplying 5 times the concentration used in our

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experiments suggesting that these two diets alone can support extremely high

production rates also for Mediterranean population of A. tonsa, when given at

saturating food concentrations. By contrast, ISO alone is not a good diet as also

reported by other authors for other copepod species (Buttino et al., 2011;

Carotenuto et al., 2002, Knuckey et al., 2005; Ismar et al., 2008). Our results

indicate that the % of egg hatching success declined during the second week

suggesting that this diet cannot support the cultivation of A. tonsa for periods

longer than one week. We also recorded a reduction in the % of adult survival

after 12 days; to our knowledge, a similar adult mortality has never been

reported before for A. tonsa fed ISO. Ismar et al. (2008) reported that naupliar

survivorship was high with a diet of ISO at concentrations of 150 µg C L-1

, but

that this diet did not support their development until reproduction. However,

Carotenuto et al. (2002) found that the copepod Temora stylifera was able to

complete development from hatching to adulthood when reared with this

flagellate. Our results indicate that nauplii produced by females fed ISO

developed normally and that no more than 10% were positively stained for

apoptotic fluorescent marker TUNEL, however we did not follow development

until adulthood. The reduction in egg hatching success after 15 days of feeding

on ISO may be due to the poor quality of this alga which is known to contain

low levels of eicosapentaenoic acid (EPA) (Muller-Fuega et al., 2003,

Tremblay et al., 2007).

Interestingly, also TETRA, commonly used to rear A. tonsa for

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ecotoxicological studies in combination with ISO and RHINO (APHA 1989;

Gorbi et al., 2012) did not support high egg production rates in the

Mediterranean population of A. tonsa, for a period longer than 2 weeks. Colin

and Dam (2002) considered Tetraselmis spp. as a control monoculture in their

3-day feeding experiments; the authors reported that this alga stimulated

ingestion and egg production after three days. Our results agree in part with

their findings: we observed significantly higher fp production rates with this

diet compared to PHAE, RHO and ISO. If we consider only three days of

feeding, TETRA seems to enhance egg production with values doubling after

two days of feeding. However, if we consider longer incubation periods, mean

egg production rates decline with time with a strong reduction in A. tonsa

productivity. In consideration of our results, this algae can be avoided, even if

used as supplemented food with other algae RHINO and ISO, with high

advantages in terms of time and resources employed. In fact, reduction of the

mixture food will influence economic costs.

Moreover, in our study adult survivorship was strongly reduced after 13

days. Even if TETRA did not significantly reduce adult survival when the

whole experimental period was considered, % mortality increased from 25 to

more than 60% during the last 12-15 days (Fig. 3) confirming that this diet

cannot sustain the rearing of A. tonsa for period longer than 10 days. Females

and males appeared covered by filamentous structures in the mouth regions

that slowed down their swimming movements. Probably, their appendages and

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filtration apparatus were clogged by such structures and animals were unable to

feed. This is the first time that a similar effect has been described in copepods

after ingestion of TETRA. Induction of apoptosis was not evident in nauplii

produced by females fed with this culture (< 10%). Hence we suggest that

reduced egg production and high adult mortality after 12 days could be due to a

mechanical impediment in feeding due to filamentous structures which we

were unable to identify. On the other hand, we cannot explain why there was

also a reduction in egg hatching success. It is also known that less food

availability can trigger delayed hatching eggs (resting eggs) (Drillet et al.,

2011c) and we cannot exclude the possibility that unhatched embryos could be

entered in dormancy, even if this type of embryos have never been reported for

the Mediterranean A. tonsa copepod. It is well known that subitaneous eggs of

A tonsa can enter into quiescence when challenged with temperature decline, or

when salinity and oxygen change (Holmstrup et al., 2006; Drillet et al., 2006b,

Hansen et al., 2010), or by food limitation (Drillet et al., 2011c). However,

non-viable eggs are clearly distinguished from resting eggs due to their shape

and opacity, and generally degenerate a few hours after spawning (also our

observations). Knuckey et al., (2005) reported that Acartia sinjiensis

copepodites feeding on Tetraselmis spp. did not develop beyond copepodite

stage C1 and appeared strongly deformed showing the absence of an eyespot

and/or reduction in size. In our experiments completely developed nauplii were

visible within many of the unhatched eggs indicating that development had

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proceeded normally until an advanced stage but was then blocked due to

unknown reasons. We conclude that this alga is a poor food item for A. tonsa

reproduction and development but it remains unclear as to why TETRA is such

a poor diet. In a study by Dhal et al. (2009), who compared fatty acid content

in different algae used to feed the harpacticoid copepod Nitocra spinipes,

TETRA was reported as having very low % of HUFA (1.7) with respect to ISO

and Rhodomonas salina, this latter species having a 20 times higher highly

unsaturated fatty acids (HUFA) concentration compared to TETRA. The

nutritional contents of the six algae used in our experiments are well known in

literature and the classical nutritional parameters considered essential for the

correct development of copepods are reported in Table 2. Diatoms are very

high proportions of EPA and DHA and very low linolenic and linoleic acid

content, while cryptophytes have very high proportions of EPA and DHA as

well as linolenic acid. Patil and coauthors (2007) reported that the highest

amount of EPA was found in P. tricornutum (28.4 mg g–1

), followed by T.

suecica (4.8 mg g–1

) and R. baltica (4.4 mg g–1

), while DHA was abundant only

in I. galbana (15.8 mg g–1

).

Finally, diatoms, as expected, showed strong effects on the productivity of A.

tonsa (Table 1), as also described for other marine organisms (reviewed by

Caldwell, 2009; Ianora and Miralto, 2010). Two different effects were observed

with PHAE and SKE; the first diatom induced a slight increase in egg

production rates after the first week with a simultaneous reduction in fp

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production and egg hatching success. A dramatic, significant decline in egg

production, fp production, hatching success and adult survivorship were

observed the second week. In particular, after 13 days no adults that had fed

PHAE had survived. Even though PHAE lacks the volatile aldehydes

decadienal and decatrienal, Pohnert et al. (2000; 2002) reported the presence of

the aldheydic acids 9-oxo-(5Z,7E)-nonadienoic acid (9-ONDE) and

12-oxo-(5Z,8Z,10E)-dodecatrienoic acid (12-ODTE) which affected sea urchin

development (Adolph et al., 2003). Dahl et al., 2009 reported that the

development of the harpacticoid copepod N. spinipes was affected when

animals were fed P. tricornutum, with only 6.9% juvenile survival after 7 days,

and a total arrest in development from NI to CI stage. Even in combination

with another algae, Dunaniella tertiolecta, development of N. spinipes was

inhibited further confirming that poor survival and development was due to the

presence of toxins rather than to poor food quality. On the contrary, SKE did

not affect adult survival but strongly reduced egg production and egg hatching

success after three days of feeding and until the end of the experiment. This is

in agreement with our previous findings obtained for other copepod species

(e.g. Ianora et al., 2004). Fecal pellet production remained high with mean

values similar to TETRA and RHINO, suggesting that this alga was actively

fed upon. It is interesting to note that nauplii produced by females fed SKE and

PHAE were strongly positively marked for apoptosis. However, only those

produced by females fed SKE were strongly deformed (Fig. 5C). Such

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deformities increased the longer the females ingested this food, suggesting that

possible toxins present in both diatoms could have different targets on copepod

development. Apoptosis in nauplii was accounted for more than 60% in nauplii

produced by females fed PHAE and SKE, after day 4 and 8, respectively.

In conclusion, this study reveals the complexity in cultivating planktonic

copepods such as Acartia tonsa in controlled systems using monoalgal diets,

and confirms the toxicity of two diatoms on A. tonsa reproduction. Choosing

the right feed item will benefit the ambition of mass cultivation of calanoids for

aquaculture and bioassays purposes, also for temperate species.

Acknowledgments:

This work was partially supported by the Danish National Strategic Research Council -

IMPAQ - grant no. 10-093522 to IB, by Zhejiang Ocean University-Zhoushan- PR-CHINA

and by ISPRA-STS Ecotoxicology and Plankton Biology Lab. -Livorno-Italy. The authors

thank the Inter-University Consortium for Marine Biology–CIBM in Livorno for logistic

accommodation and Prof. Barone Maria Carmela, director of the Inter-departmental Center

for Hydrobiological Resource Management and Aquaculture - CRIAcq of University of

Naples “Federico II” for the support in hosting Prof. Zhang in Italy.

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Figure legend

Figure 1. Acartia tonsa daily egg production per female, fecal pellet production per female

and male couples and % of egg hatching success with monoalgal diets: Isochrysis galbana

(A, B), Rhinomonas reticulata (C,D) and Rhodomonas baltica (E, F).

Figure 2. Acartia tonsa daily egg production per female, fecal pellet production per female

and male couples and % of egg hatching success with monoalgal diets: Tetraselmis suecica

(A, B), Phaeodactylum tricornutum (C, D) and Skeletonema marinoi (E, F).

Figure 3. Acartia tonsa daily % adult survival with monoalgal diets: Isochrysis galbana,

Rhinomonas reticulata, Rhodomonas baltica, Tetraselmis suecica, Phaeodactylum

tricornutum and Skeletonema marinoi.

Figure 4. Acartia tonsa females fed Tetraselmis suecica for 13 days and observed with a

Zeiss Primovert Monitor microscope. Arrows indicate the filamentous structures attached to

the mouth appendages. (A) dorsal view; (B) lateral view. (400x).

Figure 5. Acartia tonsa nauplii stained with Hoechst (violet) and Tunel (green), produced by

adult females fed different monoalgal diets, and observed with the confocal laser scanning

microscope Zeiss LSM 510. Each image is a three-dimensional reconstruction of different

z-stacks acquired along the whole body. (A) Nauplii produced by females Isochrysis

galbana fed 10 days, hoechst marks all body tissues; the red spot inside the gut is

chlorophyll. (B,C) Nauplii produced by females fed Skeletonema marinoi 10 and 12 days,

respectively. Green fluorescence of the legs indicates apopotitc cells. B is slightly deformed

whereas C is strongly abnormal due to longer maternal feeding on S. marinoi. (D) Nauplii

produced by females fed Phaeodactylum tricornutum for 9 days; green fluorescence in the

legs and buccal region indicates apoptotic cells.

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Table 1. Comparison between mean fecundity, fecal pellet and hatching success for A. tonsa

fed six different diets.

Different capital letters indicate differences statistically significant (df=5, p > 0.001).

Fecundity

(eggs per female per day)

F =22.57

Fecal pellets

(per couple per day)

F =7.597

% hatching success

F =16. 05

% adult

Survival

F =8.305

RHINO

24.4±7.0A

138.0±15.8AB

76.0±9.4A

98.33±4.39 A

RHO 21.9±5.9A 108.6±15.80

C 86.11±4.36

A 95.00±6.33

A

PHAEO 8.8±4.6C 121.4±35.7

B 44.1±25.6

C 60.83±39.77

B

SKE 8.40±3.2C 136.8±31.0

AB 46.5±16.0

C 98.33±4.39

A

ISO 12.78±5.0B 95.2±24.40

C 78.2±13.4

A 92.5±12.32

A

TETRA 13.4±5.4B 142.3±31.0

A 62.2±21.7

B 88.30±19.75

A

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Table 2. Comparison of proteins and fatty acids content in the six algal species (SFA=

saturated fatty acids; MUFA= Monounsaturated fatty acids; PUFA= Polyunsaturated fatty

acids).

Nutritional

content

I. galbana

Cryptophiceae

T. suecica

S. costatum

P. tricornutum

Protein

(% dry weght)

23-29 20-31 25 30

SFA

(% total fatty acid)

30.3 14.4 -22.6 21.7-24.7 32.0-33.0 35.2

MUFA

(% total fatty acid)

11.3 6-11 12.9-16.5 27.7-29.4 34.9

PUFA

(% total fatty acid)

33-56.4 75.2-65.8 27.2-52.5 36.8-34-8 17-27

EPA 20:5(n-3)

(mg/g dry weight)

0.9 2-4.4 4.3 -4.8 28.4

DHA 22:6(n-3)

(mg/g dry weight)

15.8 12- 18 0.1 -0.2 0.2-0.74

References Albentosa et al., 1996; FAO 1996; Li et al., 2006; Patil et al., 2007; Renaud et al., 1999

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0 2 4 6 8 10 12 14 160

20

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days

% e

gg

hatc

hed

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20

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days

% e

gg

hatc

hed

ISO

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10

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0

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gs f

em

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fecal p

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ou

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-1

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10

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0

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days

% e

gg

hatc

hed

RHINO

m= 76.0 9.4

m= 78.2 13.4

RHO

m= 86.1 4.3

m= 12.78 5.0

m= 95.2 24.4

eggs

fecal pellets

fecal pellets

m= 24.4 7.0

m= 138.0 15.8

m= 21.9 5.9

m= 108.6 15.8

eggs

A B

C D

E F

eggs

fecal pellets

Fig 1

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0 2 4 6 8 10 12 14 160

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20

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0

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10

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0

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TETRA

0 2 4 6 8 10 12 14 160

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% e

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hatc

hed

m= 8.4 3.2

m= 136.8 31

eggs

fecal pellets

m= 8.8 4.6

m= 121.4 35.7

eggs

fecal pellets

m= 13.4 5.4

m= 142.3 31

eggs

fecal pellets

m= 46.5 16

m= 44.1 25.6

m= 62.2 21.7

A B

C D

E F

Fig. 2

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Highlights for the paper entitled: Effects of different monoalgal diets on egg

production, hatching success and apoptosis induction in a Mediterranean

population of Acartia tonsa

By Zhang and coauthors.

The influence of six monoalgal diets was tested on the reproductive

success of the copepod Acartia tonsa;

The cryptophytes Rhinomonas reticulata and Rhodomonas baltica

induced highest egg production rates;

Egg production rates, hatching success, and adult survival were

reduced with both diatoms Phaeodactylum tricornutum and

Skeletonema marinoi; which also induced apopotosis in newly hatched

nauplii;

Tetraselmis suecica induced low fecundity and hatching success and

after 15 days only 37.5% of the adults survived with this food, but does

not induced apopotosis.