Journal of Experimental Marine Biology and Ecology 352 (2007) 187 199 www.elsevier.

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Collapse of Calanus chilensis reproduction in a marine environment with high diatom concentration
S.A. Poulet a,, R. Escribano b , P. Hidalgo b , A. Cueff a , T. Wichard c , V. Aguilera b , C.A. Vargas b , G. Pohnert d
b

Station Biologique de Roscoff, CNRS, INSU, UPMC Paris VI, UMR 7150-Unit Mer et Sant, Roscoff 29682, France Center of Oceanography for the Eastern South Pacific (COPAS), Universidad de Concepcion, P.O. Box 160 C, Concepcion, Chile c Max Planck Institute for Chemical Ecology, Hans-Knll-Str. 8; D-07745 Jena, Germany Ecole Polytechnique Fdrale de Lausanne (EPFL) Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland Received 17 July 2007; accepted 18 July 2007

Abstract Variations of egg production rate (EPR), hatching success (HS), production of abnormal larvae (AL) and histology of gonads have been investigated with Calanus chilensis females sampled weekly, from late November to December 2004, at a station located in the coastal zone off Dichato (Chile), at time diatom concentration in phytoplankton bloom was high. Weekly EPR estimate in nature did not change significantly during this period. It remained close to normal values (2540 eggs/female/day), whereas HS was constantly low and high proportions of AL were observed. In parallel, bioassays revealed that EPR was strongly depressed by artificially enriched diets, corresponding to natural diatom assemblages (NDA) occurring in the field, while abnormal HS and AL values could not be improved. Ingestion of diatoms by females was estimated by faecal pellet production rates and SEM examination of diatom remains in pellet samples. Low HS and the high amounts of abnormal larvae were not reversible when females were offered a favourable food, the dinoflagellate P. minimum (PM). Minor cell degradations were observed in gonads of females fed NDA diets. In comparison with other environments, present results show that impairment of Calanoid copepod reproductive factors can occur at both high and low diatom concentrations, depending on maternal diets and diatom species in blooms. 2007 Elsevier B.V. All rights reserved.
Keywords: Calanus; Copepods; Diatoms; Reproduction

1. Introduction Egg production rate (EPR), egg hatching success (HS) and production of abnormal larvae (AL) are the three main factors used to describe reproduction and recruitment success of marine copepods. The reproduc Corresponding author. E-mail address: poulet@sb-roscoff.fr (S.A. Poulet). 0022-0981/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2007.07.019

tive response determines the demography and copepod population dynamics and is strongly influenced by the maternal food. Up to now maternal diets can be characterised by food quality parameters and their content of potential adverse chemical compounds. In bioassays conduced under laboratory conditions, several authors (Ban et al., 1997; see reviews by Ianora et al., 2003; Paffenhfer et al., 2005) found, with combinations of different copepods fed high diatom concentrations

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(103104 cells/ml), that certain single diatom diets can arrest one, two or all factors. But others (e.g. Colin and Dam, 2002) have not observed such effects in the laboratory. Field observations and bioassays mimicking natural phytoplankton diets have shown that either EPR, or HS and/or AL values, monitored during the breeding season of several species of Calanoid copepods, were impaired during diatoms blooms occurring in the Adriatic Sea (Miralto et al., 1999; Ianora et al., 2004), in the English Channel (Poulet et al., 1995, 2006, in press; Wichard et al., 2007, submitted for publication), in Norvegian Fjords (Ask et al., 2006; Koski, 2007), in the North and South Pacific Ocean (Halsband-Lenk et al., 2005; Vargas et al., 2006). But another global field survey revealed no significant deleterious effect of diatom high concentration on copepod egg hatching success (Irigoien et al., 2002). We have recently established a model that links EPR, HS and the amount of AL to the ingestion of different diets, which can either have positive effects, impair vitellogenesis by interfering with oocyte maturation resulting in low EPR (in the following referred to inhibitory mechanism 1), or interfere with the embryonic development resulting in low HS and high proportions of AL (inhibitory mechanism 2) (Poulet et al., 2007). Past results have shown that there is a high variability of copepod responses to diatom diets. This is not surprising if we consider that nearly all results are based on tests of different copepod and diatom species. Obviously a high variance exists in inhibitory properties of food algae (see e.g. Wichard et al., 2005), as well as in the susceptibility of copepods (Ianora et al., 2003). These results apparently contradict other groups of laboratory and field observations that showed no significant deleterious effect of diatoms on copepod egg production and hatching success (Colin and Dam, 2002; Irigoien et al., 2002). In this context, our main objective was to clarify these two opposite points of view related to the diatom effects observed particularly in diatom-rich environments, such as those surveyed by Irigoien et al. (2002) and temporarily occurring in upwelling environments. To do so, the reproductive response of Calanus chilensis was evaluated during the summer phytoplankton bloom 2004 in Chile in a nutrient rich upwelling coastal zone, characterised by high diatom concentration. This study was complementary of a seasonal survey conduced with small-size copepods (Acartia tonsa, Paracalanus parvus and Centropages brachiatus: see Vargas et al., 2006). C. chilensis is a common large-size copepod occurring in the Southern Pacific Ocean, along the coastal zone, occupying the same ecological niche as C. helgolandicus.

The ecology, feeding and growth patterns of C. chilensis in the Chilean coastal areas have been described earlier (Escribano et al., 1997, 1998; Escribano and McLaren, 1999; Torres and Escribano, 2003). However, less is known about the reproductive responses of this species in the field. C. chilensis supposedly reproduces continuously year round (Escribano and McLaren, 1999), although reproduction seems more intense between August and December during the Austral spring (Peterson et al., 1988; Gonzlez et al., 1989; Escribano and Rodriguez, 1994; Escribano, 1998), coinciding with successions of phytoplankton blooms dominated by very high diatom concentration (103104 cells/ml). In the Dichato area, where this study was conduced, chlorophyll a values ranged between 5 and 25 g/l (Vargas et al., 2006), which are in the same range as the diatom richest regions monitored by Irigoien et al. (2002) and about 25 times higher than in the Roscoff coastal waters (Sournia and Birrien, 1995; Laabir et al., 1998; Poulet et al., 2006) and comparable to diatom blooms previously investigated in Dabob Bay (Horner et al., 2006). This contribution is part of a series of experiments performed in the coastal waters off Dichato (Chile) and was aimed to get an improved understanding of the very variable reproductive success of Calanoids in the field. Our major goal was to revisit Irigoien et al. (2002) global observation and show that their conclusion does not apply to every diatom-rich environments, specially those where chemical factors are identified in copepod maternal diets (supposedly responsible for the reproductive failure : e.g. diatom toxicity related to aldehyde or other oxylipin production: Pohnert et al., 2002; Pohnert, 2005, or diatoms with low DHA/EPA ratios b 2 defining food deficiency threshold in copepod food: Arendt et al., 2005; Poulet et al., 2007). 2. Materials and methods 2.1. Estimates of reproductive success in nature Field estimates of EPR, HS and AL were carried out during spring bloom, from the 29th November 2004 to the 04th January 2005. The same methods, as for C. helgolandicus (Laabir et al., 1998; Poulet et al., 2006, 2007), were used in the experiments with C. chilensis. Copepod specimens were collected several times a week offshore Dichato, Chile (36 5 S; 73 20 W) in the South Pacific Ocean, by towing a 200 m mesh plankton net obliquely from 20 to 0 m. Samples were transported within 2 h to the laboratory, where adult, sexually mature females (20 in total) for each experiment were sorted and incubated individually in dishes

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containing 100 ml of 0.22 m filtered sea water during 24 h in order to estimate initial EPR, HS and AL, corresponding to food-field conditions (see day1: Laabir et al., 1995a; Poulet et al., 2006, 2007). All incubations were performed at 12 1 C. The length of incubation for hatching success measurements was 24 to 64 h, longer than the temperature-dependent egg hatching time (e.g. around 19 h at 12 C). Batch of eggs (530 per sample) corresponding to female daily egg clutches were incubated in open, separated incubators containing 35 ml natural filtered seawater each. Abnormal larvae (nauplius stage N1) observed the next day were characterised by deformed, unsymmetrical appendages and/or abnormal swimming pattern (see Fig. 2B, C, D). Differences of EPR and HS between the initial values on Day 1 and those on the following days with enriched natural diatom assemblages (NDA diets) were tested with the non-parametric Wilcoxon signed-rank test. 2.2. Diatom isolation and cultivation Five single diatom species (TR: Thalassiosira rotula, SJ: Skeletonema japonicus, CD: Chaetoceros dydimus, C sp.: Chaetoceros sp. and N sp.: Nitzschia sp.) were successfully isolated from Dichato phytoplankton samples from October to November 2004 and cultured in filtered seawater enriched with K-medium at 12 C with a 12:12 light:dark cycle. Isolation, purification and culture of these five diatom species were achieved according to standard methods (Guillard and Ryther, 1962; Keller et al., 1987) and identified according to Tomas (1997). At least five of the isolated diatom strains (TR, SJ, CM, C sp. and N sp.) are involved in the yearto-year spring blooms observed in the Dichato coastal waters. 2.3. Experiments with diatom-enriched diets Samples of mixed species in natural phytoplankton assemblages (N 11 m) (NDA1, 2, 3, 4) were collected at four different occasions during the field survey at the same station as the copepod females and used to test their effects on EPR, HS and AL (see Figs. 1 and 3). Sub-surface sea water samples (25 m depth) were gently filtered by gravity through a Sartorius filtering funnel, supporting a 11 m mesh Nitex sieve (Millipore, 45 mm diameter). Pre-filtration with a larger mesh sieve (350 m), normally used to remove large particles and zooplankton (Poulet et al., 2006) was not utilised, due to the size of diatom chains often 200 m, which could have been removed from diets. Samples corresponding to 200 ml sea water were collected on the 11 m mesh

Fig. 1. Calanus chilensis. Variations of the weekly means of egg production rate, hatching rate and proportion of abnormal larvae produced by females incubated in filtered sea water, reflecting the reproductive responses in the field. Observations during summer bloom were conduced from November 30th to December 29th 2004. Arrows in the top panel give dates and start of feeding incubations with NDA diets (see Fig. 2). Error bars are standard deviations. Sample size was N = 20 females maximum at each sampling date. : no values in relation to zero hatching rate. Same symbol as in Figs. 3 and 5.

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Table 1 Concentration and proportion of diatoms, abundance of non-diatom organisms and mean values of chlorophyll a, POC and PON in phytoplankton samples measured in untreated sea water samples collected off Dichato before preparation of NDA diets Sampling date 09/11/2004 01/12/2004 02/12/2004 06/12/2004 09/12/2004 11/12/2004 15/12/2004 16/12/2004 20/12/2004 28/12/2004 Diatom species Proportion (%) Total diatoms (cells/ml) ? 7.5 103 15 103 0.32 103 0.93 103 ? ? 0.22 103 8.11 0.74 103 TR 40 SJ 35 Total non-diatoms (cells/ml) ? ? ? 6.2 34.8 ? ? 10.62 6.93 8.3 C sp. 1 21 Chlorophyll a Inshore (g/l) 5.83 23.19 ? 9.9 14.2 10.78 ? ? ? ? CD 12 N sp. 1 2

: Date of sampling at sea and start of assays with NDA diets are same as in Figs. 1 and 3.

and re-suspended in incubators containing 100 ml filtered sea water (Millipore, 0.22 m). Thus, the final diatom concentrations of NDA diets in each incubator was approximately 2 times higher than the initial abundance in nature (Table 1). Filtered sea water and diet were renewed every day in each incubator. Untreated sea water samples were preserved with Lugol's solution to allow identification of the diatom species in the NDA diets and to estimate cell numbers in the incubators (Table 1). This approach allowed to increasing artificially food biomass above field level, in order to boost copepod reproductive responses. New sea water stocks were collected twice to three times a week offshore Dichato at the same station as females and were used to renew NDA diets every day during the entire incubation periods. Particles in these sea water samples, kept in 50 l transparent plastic reservoirs in the same incubation room as the females, were resuspended by hands twice a day. In order to obtain a representative spectrum of diatom species occurring successively during the spring blooms, samples for NDA1, 2, 3 and 4 diets were collected from the end of November to late December, respectively (see date of experiments in Fig. 1 and legend of Table 1). Microscopic observations of NDA diets indicated that they were dominated by chain-forming diatoms mixed with other microorganisms, belonging to unidentified autotrophic and nanoflagellates and some dinoflagellates such as Protoperidinium and Gymnodinium species (Vargas et al., 2006). We assumed that these NDA diets

resembled natural phytoplankton assemblages, which the copepod females should have encountered in the field before capture. At the end of incubation with NDA4 diet and sea water (day 5: Fig. 5), the dinoflagellate Prorocentrum minimum (PM: same strain as used in Poulet et al., 2006; Wichard et al., submitted for publication) was tested as a favourable diet at concentrations corresponding to 104 cells ml 1 in the incubators. The growth condition of this alga was the same as the other diatom isolates. As shown previously with C. helgolandicus (Poulet et al., 2006, 2007), this non-diatom diet was used to evaluate the reversible reproductive capacity of C. chilensis, when EPR, HS and AL had collapsed, following initial reproductive responses to ingestion of NDA or field diets. The proportions of dominant diatom species and date of bioassays with NDA1-4 diets are given in Tables 13 and Fig. 1. Each bioassay was conduced once with a different cohort at day 1, each with 20 carefully selected females, with undamaged antenna, swimming legs and furca and well mature genital segment. Bioassays conduced with PM, were performed with the same female cohorts, initially fed during 45 days with NDA4 or field diets (Table 2). It was repeated a second time with another cohort (results not shown). Female mortality during the assays was 15%. 2.4. Faecal pellet analysis During assays with NDA and PM diets, ingestion of algal cells by single females was estimated indirectly, through daily counts of faecal pellet production in each
Table 2 Calanus chilensis faecal pellets production Date Diet 02/12/2004 NDA1 Mean Day 1 2 3 4 5 6 7 8 9 10 93 106 58 68 S.D. 46 39 31 26 06/12/2004 NDA2 Mean 61 82 68 42 S.D. 20 37 24 21 16/12/2004 NDA3 Mean 87 121 170 167 211 S.D. 35 23 35 22 31 20/12/2004 NDA4-PM Mean 48 37 44 61 54 68 S.D.

18 26 26 32 22 32

Values are means of faecal pellet production standard deviation measured daily during assays with NDA1, 2 and 3 diets, and with PM diet following pre-incubation with NDA4 diet (: not measured).

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Table 3 Production of polyunsaturated aldehydes (PUA: fmol/cell) by the most abundant single diatom species blooming in the Dichato coastal waters and by mixed diatom assemblages in NDA3, 4 diets collected during the survey (see date of sampling in Fig. 1 and Table 2) Strain Total PUA fmol/cell S.D. 0.10 0.02 1.41 0.22 0 0 0 3.12 0.48 11.12 2.52 Category of PUA Heptadienal % 56 23 Octadienal % 30 77 Octatrienal % 14 0 Decadienal % 0 0 Decatrienal % 0 0

SJ TR CD C sp. N sp. Mixed diatom diet NDA3 NDA4

67 45

15 19

18 35

0 0

0 0

Five category of toxic PUA were analysed.

incubator (Table 2). The methods used for faecal pellet examination have been described earlier by Laabir et al. (1995b). A scanning electronic miscrocopy (SEM) method was applied with selected samples, collected from the 14 to 19th of December 2004 and corresponding to NDA3 diet assays (Fig. 3; Poulet et al., 2006). 2.5. Phytoplankton biomass Untreated sub-surface sea water samples were collected at the offshore station along with copepod females and NDA samples used for the incubation tests. One part of the sample (150 ml) was preserved with Lugol's solution to determine and to evaluate the proportions and concentrations of diatoms. The other part of the sample was used to determine the chlorophyll a concentrations by filtering 3 replicate samples (100 200 ml) of sea water onto GF/F filters and frozen ( 30 C). Subsequently the samples were analysed using a Turner Design fluorometer, according to Yentsch and Menzel (1963) and the concentration of chlorophyll a was calculated through Lorenzen's (1966) equation. Complementary information on phytoplankton biomass for the area during this period was available from the time series study off Concepcin carried out by the COPAS Oceanographic Center (www.copas.cl). 2.6. PUA analysis in phytoplankton At two occasions during the survey phytoplankton samples were collected for determination of diatomderived polyunsaturated aldehydes (PUA) in phytoplankton. Each sample was pre-sieved on a 11 m Nitex mesh and the retained phytoplankton was split in three sub-samples of equal volume, corresponding to NDA3 and NDA4. The PUA were trapped and preserved at Dichato following a method described by Wichard et al.

(2004) and sent to Jena (Germany) for determination of potential PUA production in phytoplankton. Five single diatom species (TR: T. rotula, SJ: S. japonicus, CD: C. dydimus, C sp.: Chaetoceros sp., N sp.: Nitzschia sp.), first isolated at Dichato, were further cultured at Roscoff and posted to Jena for evaluation of PUA production. These complementary chemical analyses allowed determining if NDA diets used in bioassays and the major, single diatom components of the phytoplankton bloom were PUAproducers. Sample volumes collected for PUA analysis with NDA3 and 4 were 10 l and 15 l, respectively. Cell

Fig. 2. Calanus chilensis. A: pictures of microscope photos of normal eggs (1) and of abnormal eggs (2 to 3). B: photos of a normal nauplius larva, 56 h old. CD: photos of abnormal larva, same age as B. Scale: 100 m.

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Fig. 3. Calanus chilensis. Variations with time of (EPR), (HS) and (AL) reflecting the negative effects of NDA diets on the reproductive responses of females incubated 46 days under laboratory conditions. Sampling dates for NDA14 are given in Fig. 1. Arrows give estimated field values at day 1. : no value.

density in diatom cultures sampled at the stationary phase ranged between 104 and 1.2 106 cells/ml. 2.7. Histology preparation and observation of gonads Since reproductive response depends also on the maturation of the gonads, five females per sample were sacrificed and fixed for histological examination of semi-thin sections of gonads on the days 1 and 5 during bioassays with NDA3 and 4 diets. At Dichato, female samples were incubated 48 h with fixing-solution (1% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer in seawater 20%, pH 7.2) and stored in a rinsing solution (cacodylate buffer 0.2 M in seawater 20% and sucrose 0.45 M, pH 7.2) until arrival to Roscoff, where they were dehydrated using standard ethanol series (RPE, Carlo Erba) and subsequently examined under a light microscope (Olympus BX61) (Lacoste et al., 2001; Poulet et al., 2006). Longitudinal semi-thin sections of one to three females per sample were examined. Pictures were taken at the same magnification ( 200) using a digital Spot RT cooled CDD camera. 3. Results Phytoplankton biomass of near-surface waters in terms of total chlorophyll a remained high during the study at the inshore and offshore stations (Table 1). Although measurements were not achieved during the last part of the survey, chlorophyll values were probably of the same order of magnitude as the first half of December judging from the total diatom cell concentration, except on the 20th. The mean in situ EPR values varied between 1828 eggs/female/day. These values were relatively high in comparison with optimum rates estimated in others co-generic Calanoid species

(Mauchline, 1998). In contrast, HS values, expressed as % of EPR, were almost completely depressed, except the first week (Fig. 1). Moreover, the majority of hatched larvae were morphologically abnormal as shown by mean AL values always close or equal to 100% (Figs. 1, 2). These larvae did not survive after first nauplius development stage at weeks 1, 2, 3, 4 and 5. During the first half period of observation, EPR decreased and slightly increased during the second half, although these variations were not significantly different (T test, n = 5, p = 0.05). In order to understand the impact of diatom-enriched diets on EPR, HS and AL during upwelling driven diatom blooms, four different batches of females, collected at weeks 1 to 4 for field estimates, were further incubated with NDA14 diets during incubation periods 6 days. We expected that this food-enrichment protocol could improve the reproductive responses simply by decreasing a potential food deficiency in diatom diets. EPR, HS and AL values measured daily are given in Fig. 3. Mean values at day 1 corresponded

Fig. 4. SEM photos of faecal pellets produced by copepods during bioassays with NDA14 diets. A: frustule remains of TR (Thalassiosira rotula). B: frustule remains of SJ (Skeletonema japonicus).

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to in situ estimates, same as in Fig. 1. Doubling the diatom concentrations in diets had a significant (nonparametric Wilcoxon signed-rank test, p b 0.01) higher adverse effect on EPR and HS than diets with natural diatom concentrations. From day 1 to 6, the EPR values decreased significantly below 10 eggs/female/day. HS was strongly affected by NDA diets, although only differences with NDA1 diet were significant between day 1 and the following days (non-parametric Wilcoxon signed-rank test, p b 0.01) and almost completely depressed with the other NDA diets most of the time. Once again hatched larvae were scarce and morphologically abnormal, ressembling those shown in Fig. 2B, C, D. Obviously NDA diets could not improve nor sustain EPR and HS rates at optimum values (normally around 30 eggs/female/day and 80% in Calanus spp.). Daily faecal pellet production was estimated with NDA1, 2 and 3 diets. High values shown in Table 2 suggested that these diets were ingested by copepods. Photographs of the faecal pellets taken by SEM reveal that the majority of ingested diatoms belong to the bloom forming species (e.g. TR and SJ: Fig. 4, Table 1). Two independent assays were conducted with two different cohorts of 20 females, which had been fed either with NDA4 diet during 4 days (Fig. 1) , or following a 24 h incubation period in filtered sea water (data not shown). In Fig. 5 EPR values decreased significantly between days 1 and 2 (non-parametric Wilcoxon signed-rank test, p b 0.01). Each group was further fed the same PM diet for 10 and 6 days, respectively. At the end of the PM feeding regime, EPR had partially recovered from the negative NDA4 diet effect (Fig. 5). However, mean EPR values were still below but not significantly different from values observed in the field 10 days before (non-parametric Wilcoxon signed-rank test, p b 0.01; day 1: Fig. 1). In contrast, neither HS nor AL values could return to in situ rates nor be improved with PM diet. Results in Fig. 5 illustrated the negative and irreversible effects of dense diatom diets on HS and AL. Same result was obtained in different bioassays using females pre-conditioned in filtered sea water 24 h (day 1) before addition of PM diet renewed during 6 days. This test confirmed the negative influence of the past-feeding history (e.g. natural diets consumed in the field before day 1) on both HS and production of morphologically abnormal larvae (AL). Egg development and nauplius larvae (development stage N1) were monitored daily during each bioassay under a light microscope. Malformed, non-hatched eggs (Fig. 2A: 2 3 4) were compared to unobtrusive eggs (Fig. 2). Those eggs were qualified as pseudo-normal, because they did not tend to hatch, or gave birth to

Fig. 5. Calanus chilensis. Bioassay showing the reproductive responses of females fed successively in the field (arrow: in situ), NDA 4 diet and PM (control). Deleterious effects of NDA 4 were not modified by PM diet. : no values.

morphologically abnormal larvae. Because of the different types of eggs, it turns out that eggs had to be classified according to the size, shape and colour of their blastomers. Egg-type 1: Pseudo-normal eggs had equal size, pale-brown blastomers; egg-type 2: numerous small blastomers with irregular sizes, egg-type 3: dark, homogeneous matrix and egg-type 4: enormous blastomers associated with smaller ones. Spines were present on all egg membrane, except egg-type 3. Majority of

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egg-type 1 could hatch, but most of them gave birth to abnormal nauplii (Figs. 2C, D, and 3). Proportions of egg-types were not monitored. Morphology of normal nauplius larva shown in Fig. 2B were characterised by symmetrical body and appendages. By contrast, abnormal larvae shown in Fig. 2C and D presented several morphological symptoms, characterised by deformed, non-symmetrical body and appendages. The same types

of egg and larval morphological anomalies were observed in all field and bioassay samples (Fig. 2E, F). Pictures of longitudinal sections in gonads and oviducts of females, belonging to egg-types A (characterised by pseudo-normal egg production rates, very low hatching success, high production of abnormal larvae) and B (characterised by low egg production rates and extremely low hatching success) are shown in Fig. 6.

Fig. 6. Calanus chilensis. Cytological examination of gonads in females fed NDA3 and 4 diets in the laboratory (see Figs. 1 and 3). A: semi-thin longitudinal section in a female with normal egg production rate (EPR) and abnormally low hatching rate (HS) and high abnormal larvae production (AL). B: similar section in another female, in which egg production was arrested. OO: oogonia, OS13: oocyte development stages. Cell anomalies go (vitellus granules and cell organelles) and v(unidentified vesicles), shown in the samples A and B, are focused in the pictures CE. CDE: semi-thin sections of female oviducts. C: OS3 in females characterised by high EPR-low HS-high AL. Homogeneous distribution of vitellus granules and organelles (e.g. mitochondria) in normal oocyte cytoplasm. D: OS3 in a sterile female (EPR value was zero at time of sampling). Heterogeneous distribution of granules and organelles (go), with a tendency to aggregate around nucleus in abnormal cytoplasm, characterised anomaly no. 1. E: Abundance of small vesicles (v), observed in between cell membranes of oocytes and follicular cells, characterised anomaly no. 2. n: nucleus. AB scale: 200. CDE scale: 800.

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Normal oogonies (OO) and oocyte development stages OS1, OS2, OS3 were always observed in these samples (for definition of OO and OS: see Niehoff, 1998, 2003). At time of sampling, around 9 h AM, oldest oocyte development OS4 stages were scarce, because the majority had been spawned earlier in the morning. Minor cell anomalies in OS3 were occurring in females fed NDA and PM diets (see go and v in Fig 6A, B, C, D, E). Micro-structures, scattered in the cytoplasm (go: vitellus granules and cell organelles) and vesicles (v: unidentified vesicles) sandwiched between oocyte and follicular cell membranes, were observed in many OS3. In spawning females (Fig. 6A), pseudo-normal OS3 had a uniform colour because go were homogeneously scattered in the cytoplasm (Fig. 6A, C). In nonspawning, or low-spawning females (Fig. 6B) colour of oocyte cytoplasm was not uniformed, due to the heterogeneous distribution and concentration of go, aggregated around the nucleus (defining anomaly no. 1: Fig. 6B, D), while v seemed to be more frequent (defining anomaly no. 2: Fig. 6B, E). Since polyunsaturated aldehydes are supposed to affect the copepod reproductive response, chemical analysis of the Chilean phytoplankton samples and isolated diatom species were conducted. At two occasions the PUA production of phytoplankton samples (NDA 3 and 4) were investigated (Table 3). Because PUA production is diatom species- and strain-dependent (Pohnert et al., 2002; Wichard et al., 2005), complementary analyses of single diatom species in cultures were achieved to determine which of the major diatoms occurring during the phytoplankton bloom were PUA producers. The two dominant, blooming species SJ and TR were PUA producers, whereas the three investigated less abundant species, CD, C sp. and N sp 1 did not release PUA. PUA production by isolated and unialgal cultured diatoms was around one order of magnitude lower than samples of mixed diatom assemblages. Whereas PUAcomposition was similar in all samples, the change of PUA-proportion indicates the variable pool of those polyunsaturated fatty acids transformed into PUA (Table 3, Wichard et al., 2007). 4. Discussion Results in Figs. 1, 2, and 3 showed that EPR, HS and AL were impaired in C. chilensis during upwelling driven summer dense diatom blooms in the field, or by semi-artificial NDA diets, as shown before with C. helgolandicus fed diatoms at much lower concentration (Poulet et al., 2006, 2007; Wichard et al., submitted for publication).

These species occupy similar ecological niches in the Southern and Northern Hemispheres, respectively. It means that different diatoms occurring in areas located at the antipodes can exert severe impacts on the reproduction. In all investigated systems (Adriatic sea, Dabob bay: North Pacific, coastal waters off Rroscoff: English Channel, upwelling system: South Pacific, Norvegian fjords: Ianora et al., 2004; Halsband-Lenk et al., 2005; Poulet et al., 2006; Vargas et al., 2006; Koski, 2007, respectively) a diatom driven reduction of reproductive success can be observed. Observations of the reproductive responses of C. chilensis females in the Dichato coastal waters (Chile) were achieved, following the same protocols as with C. helgolandicus in the Roscoff coastal waters (Laabir et al., 1995a,b; Poulet et al., 2006, 2007) and thus, allowing safe comparison of results between the two copepod species and regions. Results in Fig. 1 indicate that mean EPR values for C. chilensis varied between 2030 eggs/female/day during the summer bloom, resembling normal specific values in Calanus sp. (around 33 eggs/female/day: Peterson et al., 1988; Mauchline, 1998). HS remained abnormally low in the field with mean value 50%, while AL was close to 100% (Fig. 1). Exceptionally long periods of reproductive breakdown have been already observed with C. helgolandicus (Poulet et al., 2006; Wichard et al., submitted for publication). Egg production rates decreased significantly in comparison to day 1, when C. chilensis females were offered diatoms in NDA diets twice their concentrations in nature (Table 1). EPR values, low HS values and high larval morphological anomalies (AL) were not reversible when females were fed PM diet during 6 or 10 days (Fig. 5). In contrast, C. helgolandicus females always returned to normal EPR, HS and AL values when fed PM diet. We first assumed that irreversibility was due both to highest diatom concentrations and to longest exposure of C. chilensis to extremely abundant deleterious diatoms in nature, prior to bioassays in the laboratory. We already have mentioned such a phenomenon in C. helgolandicus females exposed to NDA diets, when feeding periods were 7 days (Poulet et al., 2006, 2007). The cumulative effects of both diatom concentration and duration of female exposure have also been already documented for EPR and HS (Chaudron et al., 1996). Alternatively, different detoxification mechanisms and different diatom toxicity levels, as well as deficiency of specific nutrients in diets could be the causes of such irreversibility. Apparently, both field and laboratory observations are coherent among these two co-generic copepod species. Histological examination of gonads provides valuable information about the reason why reproductive factors varied between different seasons, or areas, when

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females feed on diatom-rich diets. With C. chilensis, minor cell anomalies observed in gonads coincided with normal/high EPR, low HS and high AL values (Figs. 6B and C; 3). This pattern was already observed with C. helgolandicus (corresponding to inhibitory mechanism (2): normal/high EPR, low HS and/or high AL, defined by Poulet et al., 2007). At Roscoff, it was related to ingestion of Navicula sp., Nitzschia sp., Skeletonema costatum and to a minor extend to T. rotula (b 40% HS anomalies). As reported earlier by Ban et al. (1997), another type of inhibition was identified corresponding to inhibitory mechanism (1), defined by Poulet et al. (2007) and characterised by the presence of severe cell anomalies in oocytes matching low EPR, high and/or low HSAL values. At Roscoff, this inhibitory pattern was influenced by several single diatom species in diets, in particular Chaetoceros calcitrans, Guinardia delicatula, G. striata, Rhizosolenia setigera, Thalassiosira pseudonana, Stephanopyxis turris, Odontella regia. It was shown that the level of cell degradations in oocytes and reproductive breakdown were diatom species dependent (Poulet et al., 2007). In C. chilensis, the reason why only minor oocyte anomalies and strong inhibition in HS were observed may be due to three causes: 1. absence of cell degradations in OS3 coincides with high and normal EPR values (Figs. 1, 3 and 6; Niehoff, 2003; Poulet et al., 2007); 2. concentration of diatoms at Dichato was 25 times higher than Roscoff, thus providing high food supply (Tables 1 and 3; Poulet et al., 2006), and 3. TR and SJ remains were extremely abundant in faecal pellets, suggesting that they were heavily fed upon by copepods (Fig. 4). TR and SJ were PUA producers and favoured high egg production, while the other species, CD, C sp. and the less abundant N sp. were not (Table 3; Wichard et al., 2005). It has been recently demonstrated that diatomPUA producers do not impair EPR (Poulet et al., 2006, 2007; Wichard et al., submitted for publication), whereas they can partially or strongly depress HS and/or increase AL (Pohnert et al., 2002; Ianora et al., 2004; Poulet et al., 2007; Wichard et al., submitted for publication), even though no significant correlations could be found in the field between PUAs production, EPR, HS and AL at Roscoff (Wichard et al., submitted for publication). However, several TR strains known as strong PUA producers are capable to induce either very low or medium hatching failure in C. helgolandicus (b 40%: Pohnert et al., 2002, Wichard et al., 2005, submitted for

publication, Poulet et al., 2007). These results support three conclusions. First, other toxic oxylipins, metabolised along the PUA production pathways might be involved in these inhibitory mechanisms. Second, food deficiency in several diatoms might be related to DHA/ EPA ratios (b 2) below values requested to sustain normal copepod reproduction (Arendt et al., 2005; Poulet et al., 2007). Third, production of PUA is fuelled with PUFAs acting as precursors, the concentration of which decreases with time and thus induces indirect fatty acid deficiency in diet, as shown by Wichard et al. (2007). Therefore, we assumed that TR and SJ Chilean strains were affecting only HS and AL in C. chilensis, because they resemble TR and SK activities in C. helgolandicus (T. rotula and S. costatum strains assayed at Roscoff known as PUA producers, which did not impair EPR: Wichard et al., 2005; Ianora et al., 2004; Ask et al., 2006; Poulet et al., 2006), (Figs. 1 and 3). These results suggest that inhibitory mechanism (2) was also prevailing at Dichato at time of sampling, because TR and SJ were the most abundant diatoms and heavily ingested by C. chilensis females (Fig. 1, Tables 1, 2, Fig. 4). This conclusion was supported by complementary results obtained by Vargas et al. (2006). These authors observed the same inhibitory mechanism (2), due to highly nutritious diatoms occurring in the field and fed upon by A. tonsa, P. parvus and C. brachiatus during diatom springsummer blooms. Since their field survey and assays lasted a complete year, these authors could also notice that inhibitory mechanism (2) was replaced by another reproductive inhibitory pattern (lower EPR, normal high HS and low AL values), when diatom diets were seasonally replaced by non-diatom preys comprising mainly nanoflagellates, ciliates and dinoflagellates. Vargas et al. (2006) showed that this pattern, apparently resembling inhibitory mechanism (1), was due to low biomass of non-toxic preys, thus inducing a typical food limitation linked to the relative decrease of PUFA and HUFA per cell known to support high EPR (Verity and Paffenhfer, 1996; Paffenhfer et al., 2005). This third reproductive pattern was typically linked to a food shortage and nutrient deficiency. As such, it can be defined as a passive inhibitory mechanism (3). Succession of reproductive inhibitory patterns (2) and (3) occurred during the summerfall and winterspring transitions; when low biomass, non-toxic, non-diatom preys were progressively replacing high biomass of diatoms prevailing during springsummer. Results with C. chilensis further showed that inhibitory mechanism (1) was not directly involved in the Chilean coastal waters at time of sampling (Fig. 1). However, inhibitory

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mechanism (1) was probably latent in the field. It could be expressed in the laboratory and was superimposed to inhibitory mechanism (2), when C. chilensis was fed very dense NDA diets (Fig. 3: see incubation time 4 days). Expression of mechanism (1) was assumed to be due to the artificial increase of CD and C sp., relative to TR and SJ. These two Chaetoceros species which did not produce PUA were abundant in the Dichato coastal waters (Table 3) and twice as much in NDA diets. They were sharing same inhibitory pattern as another cogeneric species C. calcitrans, a non-PUA producer, which can express inhibitory mechanism (1): see Poulet et al. (2006), Wichard et al. (2005). Oithona nana, another common small-size copepod, which coexisted in the Dichato coastal waters, was not affected by deleterious diatoms (unpublished data), because Oithona sp. usually selects different food resources like detritus and faecal pellets (Gonzlez and Smetacek, 1994), or live preys belonging to the microbial-food web (nanoflagellates b 10 m: Vargas and Gonzlez, 2004). In contrast, reproduction of four co-occurring Calanoid copepods was deeply impaired by diatoms, which were selected and ingested by those (Fig. 4; Vargas et al., 2006). It may be the reason why Irigoien et al. (2002) did not find any inhibitory patterns with Metridia sp. or Pleuromamma sp., because these copepods are carnivorous. The same reasoning applies to C. pacificus, which can avoid deleterious Thalassiosira sp. (Leising et al., 2005; Halsband-Lenk et al., 2005). Similarly, Calanoides acutus, Rhincalanus gigas, Calanus finmarchicus and C. marshallae could be much less influenced by diatoms than C. helgolandicus or C. chilensis, may be because they could be post-diaposing and thus, might be metabolically relying on their lipid reserves for spawning at time of sampling (Hagen and Auel, 2001; Kosobokova and Hirche, 2001; Niehoff, 2004). Extrapolating to copepods results obtained with Daphnia sp. (Carotenuto et al., 2005), the reason could be a better detoxification mechanism in these four species. Accumulating evidences on the deleterious influence of diatom-rich diets fed upon by both C. helgolandicus and C. chilensis plead for the expression of inhibitory mechanisms (1) and (2) by diatoms in nature. These mechanisms are not directly related to the concentration of phytoplankton expressed by the number of cells, chlorophyll a, POC and PON, neither by PUFA nor HUFA deficiency in diets (Laabir et al., 1998; Lacoste et al., 2001; Poulet et al., 2006, 2007; Vargas et al., 2006; Wichard et al., 2007, submitted for publication). Moreover, these inhibitory mechanisms were not correlated to PUAs production with C. helgolandicus at

Roscoff (Wichard et al., submitted for publication). Recent results with Eurytemora affinis (Ask et al., 2006), A. tonsa, P. parvus and C. brachiatus (Vargas et al., 2006), C. helgolandicus (Ianora et al., 2004; Poulet et al., 2006, 2007; Wichard et al., submitted for publication), C. pacificus (Halsband-Lenk et al., 2005) and C. chilensis (Tables 1 and 2, Figs. 15) support the idea that reproductive failure in several Calanoid copepods is primarily linked to the ingestion of specific deleterious diatoms. The chemical compounds responsible for the deleterious variability have to be further investigated. In conclusion, various phytoplankton blooms occur in different ecosystems with similar diatom genus composition but different species offering distinct chemical properties. Thus, positive or negative activities on the reproductive responses can be observed following post-ingestion of diatoms by copepod females. When different inhibitory mechanisms are involved, they can be understood by histology of female gonads, classification of egg-inhibition and morphological aspect of larvae. Therefore, conclusion raised by Irigoien et al. (2002) does not apply to every marine ecosystems, because chemical properties and biological activities expressed by diatoms are globally variable. Acknowledgements This work has been partly funded by a CONICYTCNRS exchange programme and by the French Biodiversity programme, by Max Planck Institute and by the COPAS FONDAP Center. Thanks are due to Dr. Carmen Morales for permission to use her chlorophyll data, to Dr. Marc Blondel for sharing his Olympus microscope and to Dr. Adrianna Zingone for identification of diatoms (SJ). [SS] References
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