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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

d

Station Biologique de Roscoff, CNRS, INSU, UPMC Paris VI, UMR 7150-Unité Mer et Santé, 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-Knöll-Str. 8; D-07745 Jena, Germany Ecole Polytechnique Fédérale de Lausanne (EPFL) Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland Received 17 July 2007; accepted 18 July 2007

a

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 (25–40 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; Paffenhöfer et al., 2005) found, with combinations of different copepods fed high diatom concentrations

chilensis. 1998). chilensis supposedly reproduces continuously year round (Escribano and McLaren.188 S. or HS and/or AL values. Poulet et al. 2007). HS and the amount of AL to the ingestion of different diets. (2002) and temporarily occurring in upwelling environments. impair vitellogenesis by interfering with oocyte maturation resulting in low EPR (in the following referred to inhibitory mechanism 1). 2006). helgolandicus (Laabir et al. 2003). Poulet et al. Poulet et al. Colin and Dam. chilensis is a common large-size copepod occurring in the Southern Pacific Ocean. But another global field survey revealed no significant deleterious effect of diatom high concentration on copepod egg hatching success (Irigoien et al. But others (e...g. Samples were transported within 2 h to the laboratory. such as those surveyed by Irigoien et al. 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. (2002) and about 2–5 times higher than in the Roscoff coastal waters (Sournia and Birrien. 2005. sexually mature females (20 in total) for each experiment were sorted and incubated individually in dishes . our main objective was to clarify these two opposite points of view related to the diatom effects observed particularly in diatom-rich environments.. We have recently established a model that links EPR. 1997. HS and AL were carried out during spring bloom. Poulet et al. 2005). González et al.. 2005. 1994. The same methods. In the Dichato area. 1995.g.. Paracalanus parvus and Centropages brachiatus: see Vargas et al. 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. 1998.. Our major goal was to revisit Irigoien et al. chlorophyll a values ranged between 5 and 25 μg/l (Vargas et al.. Materials and methods 2. Copepod specimens were collected several times a week offshore Dichato. Irigoien et al. helgolandicus. 73° 20′ W) in the South Pacific Ocean. along the coastal zone.. 2004). monitored during the breeding season of several species of Calanoid copepods. or diatoms with low DHA/EPA ratios b 2 defining food deficiency threshold in copepod food: Arendt et al. two or all factors. 2006) and comparable to diatom blooms previously investigated in Dabob Bay (Horner et al. chilensis in the Chilean coastal areas have been described earlier (Escribano et al. 1999). as for C.1. 1998. 1999. diatom toxicity related to aldehyde or other oxylipin production: Pohnert et al. 1998. 2002). feeding and growth patterns of C.. 1989. characterised by high diatom concentration. where adult. Chile (36° 5′ S. 2003). coinciding with successions of phytoplankton blooms dominated by very high diatom concentration (103–104 cells/ml)... from the 29th November 2004 to the 04th January 2005. 2002) have not observed such effects in the laboratory. which can either have positive effects. less is known about the reproductive responses of this species in the field. Estimates of reproductive success in nature Field estimates of EPR. 2. 2007). were impaired during diatoms blooms occurring in the Adriatic Sea (Miralto et al.. C. Wichard et al. Field observations and bioassays mimicking natural phytoplankton diets have shown that either EPR. However. in the English Channel (Poulet et al. although reproduction seems more intense between August and December during the Austral spring (Peterson et al. 2007). 2002. 2002. 2006. C. This study was complementary of a seasonal survey conduced with small-size copepods (Acartia tonsa. in Norvegian Fjords (Ask et al. Past results have shown that there is a high variability of copepod responses to diatom diets.. as well as in the susceptibility of copepods (Ianora et al. that certain single diatom diets can arrest one. 2006).. occupying the same ecological niche as C. Vargas et al. Pohnert. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 (103–104 cells/ml). the reproductive response of Calanus chilensis was evaluated during the summer phytoplankton bloom 2004 in Chile in a nutrient rich upwelling coastal zone.. 2007. where this study was conduced. (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. Escribano.. In this context. 1999. submitted for publication). 2005.. in the North and South Pacific Ocean (Halsband-Lenk et al. Escribano and McLaren.g. Laabir et al. This is not surprising if we consider that nearly all results are based on tests of different copepod and diatom species.. 1988.. Torres and Escribano.. which are in the same range as the diatom richest regions monitored by Irigoien et al.. by towing a 200 μm mesh plankton net obliquely from 20 to 0 m. 2006). or interfere with the embryonic development resulting in low HS and high proportions of AL (inhibitory mechanism 2) (Poulet et al. in press.. Ianora et al. 2006. The ecology.A. To do so. 2006).. Koski. Obviously a high variance exists in inhibitory properties of food algae (see e. Escribano and Rodriguez. 2007).. Wichard et al. were used in the experiments with C.. 2006. 1995. 2002).

. 1987) and identified according to Tomas (1997). D).: Nitzschia sp. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 189 containing 100 ml of 0. All incubations were performed at 12 ± 1 °C.) are involved in the yearto-year spring blooms observed in the Dichato coastal waters.: Chaetoceros sp. C sp. Batch of eggs (5–30 per sample) corresponding to female daily egg clutches were incubated in open. 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. 45 mm diameter). Variations of the weekly means of egg production rate.. 2006) was not utilised. reflecting the reproductive responses in the field. Isolation. Pre-filtration with a larger mesh sieve (350 μm). corresponding to food-field conditions (see day1: Laabir et al.3. longer than the temperature-dependent egg hatching time (e. Diatom isolation and cultivation Five single diatom species (TR: Thalassiosira rotula.) 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.A. SJ: Skeletonema japonicus. CM. 3. due to the size of diatom chains often ≥ 200 μm. HS and AL (see Figs. 2B. 2. 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. and N sp. Abnormal larvae (nauplius stage N1) observed the next day were characterised by deformed.g. Experiments with diatom-enriched diets Samples of mixed species in natural phytoplankton assemblages (N 11 μm) (NDA1. 2. 3 and 5. Keller et al. normally used to remove large particles and zooplankton (Poulet et al. 2006. Observations during summer bloom were conduced from November 30th to December 29th 2004. separated incubators containing 3–5 ml natural filtered seawater each. 1962. C sp. ⁎: no values in relation to zero hatching rate. purification and culture of these five diatom species were achieved according to standard methods (Guillard and Ryther. The length of incubation for hatching success measurements was 24 to 64 h. Calanus chilensis. 2. 2007). Arrows in the top panel give dates and start of feeding incubations with NDA diets (see Fig. C. Same symbol as in Figs. 2).2. Sample size was N = 20 females maximum at each sampling date.. . Samples corresponding to 200 ml sea water were collected on the 11 μm mesh Fig. CD: Chaetoceros dydimus. SJ. HS and AL. 1995a. hatching rate and proportion of abnormal larvae produced by females incubated in filtered sea water. Error bars are standard deviations. supporting a 11 μm mesh Nitex sieve (Millipore. 1 and 3). and N sp. 1. Sub-surface sea water samples (2–5 m depth) were gently filtered by gravity through a Sartorius filtering funnel..S. At least five of the isolated diatom strains (TR. Poulet et al. around 19 h at 12 °C). unsymmetrical appendages and/or abnormal swimming pattern (see Fig.22 μm filtered sea water during 24 h in order to estimate initial EPR. which could have been removed from diets. Poulet et al.

35 23 35 22 31 20/12/2004 NDA4-PM Mean – – – – – 48 37 44 61 54 68 S. 2.3 C sp.93 8..D. Female mortality during the assays was ≤ 15%. the final diatom concentrations of NDA diets in each incubator was approximately 2 times higher than the initial abundance in nature (Table 1).. The proportions of dominant diatom species and date of bioassays with NDA1-4 diets are given in Tables 1–3 and Fig.5 × 103 15 × 103 0. Particles in these sea water samples.D. ingestion of algal cells by single females was estimated indirectly. 0. Microscopic observations of NDA diets indicated that they were dominated by chain-forming diatoms mixed with other microorganisms. abundance of non-diatom organisms and mean values of chlorophyll a. in order to boost copepod reproductive responses.74 × 103 TR 40 SJ 35 Total non-diatoms (cells/ml) ? ? ? 6.. 3 and 4 diets were collected from the end of November to late December. 2006). which the copepod females should have encountered in the field before capture. 2006.D. Thus. samples for NDA1. this non-diatom diet was used to evaluate the reversible reproductive capacity of C. 1 2 ⁎: Date of sampling at sea and start of assays with NDA diets are same as in Figs. 1 21 Chlorophyll a Inshore (μg/l) 5.2 34. As shown previously with C.D.A.190 S. Poulet et al. and with PM diet following pre-incubation with NDA4 diet (–: not measured). Each bioassay was conduced once with a different cohort at day 1. 2006. Filtered sea water and diet were renewed every day in each incubator.22 μm).2 10.93 × 103 ? ? 0. 18 26 26 32 22 32 Values are means of faecal pellet production ± standard deviation measured daily during assays with NDA1.11 0. It was repeated a second time with another cohort (results not shown).83 23. 2 and 3 diets. Bioassays conduced with PM. 1 and 3. were resuspended by hands twice a day. 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. Faecal pellet analysis During assays with NDA and PM diets.9 14. 1 and legend of Table 1).78 ? ? ? ? CD 12 N sp. 20 37 24 21 16/12/2004 NDA3 Mean 87 121 170 167 211 S. chilensis. 46 39 31 26 06/12/2004 NDA2 Mean 61 82 68 42 S. helgolandicus (Poulet et al. were performed with the same female cohorts. submitted for publication) was tested as a favourable diet at concentrations corresponding to 104 cells ml− 1 in the incubators. This approach allowed to increasing artificially food biomass above field level. following initial reproductive responses to ingestion of NDA or field diets. We assumed that these NDA diets resembled natural phytoplankton assemblages..4. 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. kept in 50 l transparent plastic reservoirs in the same incubation room as the females.22 × 103 8. 2007). 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). 2. each with 20 carefully selected females. with undamaged antenna. . / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 Table 1 Concentration and proportion of diatoms.32 × 103 0. and re-suspended in incubators containing 100 ml filtered sea water (Millipore.19 ? 9. initially fed during 4–5 days with NDA4 or field diets (Table 2). 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. At the end of incubation with NDA4 diet and sea water (day 5: Fig.62 6. HS and AL had collapsed.8 ? ? 10. Wichard et al. In order to obtain a representative spectrum of diatom species occurring successively during the spring blooms. 5). swimming legs and furca and well mature genital segment. The growth condition of this alga was the same as the other diatom isolates. the dinoflagellate Prorocentrum minimum (PM: same strain as used in Poulet et al. respectively (see date of experiments in Fig. belonging to unidentified autotrophic and nanoflagellates and some dinoflagellates such as Protoperidinium and Gymnodinium species (Vargas et al. when EPR. 1.

Poulet et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 191 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.cl). japonicus. C–D: photos of abnormal larva. dydimus. were further cultured at Roscoff and posted to Jena for evaluation of PUA production. 2. 4 diets collected during the survey (see date of sampling in Fig. The PUA were trapped and preserved at Dichato following a method described by Wichard et al.12 ± 2. Mixed diatom diet NDA3 NDA4 67 45 15 19 18 35 0 0 0 0 Five category of toxic PUA were analysed. 2. 2. N sp.D.48 11. 1 and Table 2) Strain Total PUA fmol/cell ± S. CD: C.. B: photos of a normal nauplius larva.A.02 1. (1995b). 0.: Nitzschia sp. single diatom components of the phytoplankton bloom were PUAproducers. A: pictures of microscope photos of normal eggs (1) and of abnormal eggs (2 to 3). These complementary chemical analyses allowed determining if NDA diets used in bioassays and the major. PUA analysis in phytoplankton At two occasions during the survey phytoplankton samples were collected for determination of diatomderived polyunsaturated aldehydes (PUA) in phytoplankton.52 Category of PUA Heptadienal % 56 23 Octadienal % 30 77 Octatrienal % 14 0 Decadienal % 0 0 Decatrienal % 0 0 SJ TR CD C sp. . Scale: 100 μm.: Chaetoceros sp. Calanus chilensis.). 3. first isolated at Dichato. collected from the 14 to 19th of December 2004 and corresponding to NDA3 diet assays (Fig. N sp.6. The methods used for faecal pellet examination have been described earlier by Laabir et al. according to Yentsch and Menzel (1963) and the concentration of chlorophyll a was calculated through Lorenzen's (1966) equation.5. 2006). Poulet et al.22 0 0 0 3. Five single diatom species (TR: T. Each sample was pre-sieved on a 11 μm Nitex mesh and the retained phytoplankton was split in three sub-samples of equal volume. Complementary information on phytoplankton biomass for the area during this period was available from the time series study off Concepción carried out by the COPAS Oceanographic Center (www. One part of the sample (150 ml) was preserved with Lugol's solution to determine and to evaluate the proportions and concentrations of diatoms.10 ± 0. 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). Cell Fig.41 ± 0.12 ± 0. rotula.copas. 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. Subsequently the samples were analysed using a Turner Design fluorometer. respectively.S. SJ: S. (2004) and sent to Jena (Germany) for determination of potential PUA production in phytoplankton. incubator (Table 2). C sp. A scanning electronic miscrocopy (SEM) method was applied with selected samples. Sample volumes collected for PUA analysis with NDA3 and 4 were 10 l and 15 l. 5–6 h old. same age as B. corresponding to NDA3 and NDA4..

2 M sodium cacodylate buffer in seawater 20%. Moreover. were further incubated with NDA1–4 diets during incubation periods ≤ 6 days. Histology preparation and observation of gonads Since reproductive response depends also on the maturation of the gonads. Mean values at day 1 corresponded Fig. except the first week (Fig. 4. 2001. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 Fig. 3. 2). SEM photos of faecal pellets produced by copepods during bioassays with NDA1–4 diets. 1. Longitudinal semi-thin sections of one to three females per sample were examined. Arrows give estimated field values at day 1.5% glutaraldehyde in 0. These larvae did not survive after first nauplius development stage at weeks 1. female samples were incubated 48 h with fixing-solution (1% paraformaldehyde and 2.7. pH 7. A: frustule remains of TR (Thalassiosira rotula). . Sampling dates for NDA1–4 are given in Fig. HS and AL during upwelling driven diatom blooms. p = 0. Carlo Erba) and subsequently examined under a light microscope (Olympus BX61) (Lacoste et al. ⁎: no value. 1).192 S. B: frustule remains of SJ (Skeletonema japonicus). where they were dehydrated using standard ethanol series (RPE. pH 7. collected at weeks 1 to 4 for field estimates.2 × 106 cells/ml. four different batches of females. chlorophyll values were probably of the same order of magnitude as the first half of December judging from the total diatom cell concentration. 3.. 3. expressed as % of EPR.2) and stored in a rinsing solution (cacodylate buffer 0. HS and AL values measured daily are given in Fig. although these variations were not significantly different (T test. 1. Although measurements were not achieved during the last part of the survey.A. 2. were almost completely depressed. In contrast. density in diatom cultures sampled at the stationary phase ranged between 104 and 1. except on the 20th. At Dichato. Pictures were taken at the same magnification (× 200) using a digital Spot RT cooled CDD camera. Poulet et al. 1998). 4 and 5. We expected that this food-enrichment protocol could improve the reproductive responses simply by decreasing a potential food deficiency in diatom diets. 2. Calanus chilensis. 3.2 M in seawater 20% and sucrose 0.05). During the first half period of observation. Variations with time of (EPR). 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). the majority of hatched larvae were morphologically abnormal as shown by mean AL values always close or equal to 100% (Figs. HS values.45 M. EPR. EPR decreased and slightly increased during the second half. These values were relatively high in comparison with optimum rates estimated in others co-generic Calanoid species (Mauchline. 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. (HS) and (AL) reflecting the negative effects of NDA diets on the reproductive responses of females incubated 4–6 days under laboratory conditions. 2006).. The mean in situ EPR values varied between 18–28 eggs/female/day.2) until arrival to Roscoff. n = 5. In order to understand the impact of diatom-enriched diets on EPR. Poulet et al.

In Fig. At the end of the PM feeding regime. This test confirmed the negative influence of the past-feeding history (e. Doubling the diatom concentrations in diets had a significant (nonparametric Wilcoxon signed-rank test. or following a 24 h incubation period in filtered sea water (data not shown). 5 EPR values decreased significantly between days 1 and 2 (non-parametric Wilcoxon signed-rank test. which had been fed either with NDA4 diet during 4 days (Fig. EPR had partially recovered from the negative NDA4 diet effect (Fig. pale-brown blastomers. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 193 to in situ estimates. Two independent assays were conducted with two different cohorts of 20 females. High values shown in Table 2 suggested that these diets were ingested by copepods. 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. ressembling those shown in Fig. p b 0. natural diets consumed in the field before day 1) on both HS and production of morphologically abnormal larvae (AL). However. 1) . TR and SJ: Fig. respectively. 5). Those eggs were qualified as “pseudo-normal”. Deleterious effects of NDA 4 were not modified by PM diet.01) and almost completely depressed with the other NDA diets most of the time. the EPR values decreased significantly below 10 eggs/female/day.01). 2B. morphologically abnormal larvae. shape and colour of their blastomers. D. it turns out that eggs had to be classified according to the size. In contrast.g. Malformed. day 1: Fig. 5 illustrated the negative and irreversible effects of dense diatom diets on HS and AL. 1. 4. Because of the different types of eggs. egg-type 3: dark. HS was strongly affected by NDA diets. p b 0. 2 and 3 diets. homogeneous matrix and egg-type 4: enormous blastomers associated with smaller ones. egg-type 2: numerous small blastomers with irregular sizes. non-hatched eggs (Fig. 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. Photographs of the faecal pellets taken by SEM reveal that the majority of ingested diatoms belong to the bloom forming species (e. From day 1 to 6. neither HS nor AL values could return to in situ rates nor be improved with PM diet.01) higher adverse effect on EPR and HS than diets with natural diatom concentrations. 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. Poulet et al. Once again hatched larvae were scarce and morphologically abnormal.). Table 1). Spines were present on all egg membrane. except egg-type 3. Daily faecal pellet production was estimated with NDA1. Egg development and nauplius larvae (development stage N1) were monitored daily during each bioassay under a light microscope. Bioassay showing the reproductive responses of females fed successively in the field (arrow: in situ). Egg-type 1: Pseudo-normal eggs had equal size. ⁎: no values. although only differences with NDA1 diet were significant between day 1 and the following days (non-parametric Wilcoxon signed-rank test.g.S. because they did not tend to hatch. same as in Fig. p b 0. 2A: 2 3 4) were compared to unobtrusive eggs (Fig. NDA 4 diet and PM (control). or gave birth to Fig. Calanus chilensis. 5.01. C. Majority of . 2). 1). Each group was further fed the same PM diet for 10 and 6 days.A. p b 0. Results in Fig.

B: similar section in another female. F). in which egg production was arrested. observed in between cell membranes of oocytes and follicular cells. Morphology of normal nauplius larva shown in Fig. E: Abundance of small vesicles (v). 6. and 3). Fig.g. mitochondria) in normal oocyte cytoplasm. 2E. characterised anomaly no. OS1–3: oocyte development stages. Heterogeneous distribution of granules and organelles (go). characterised anomaly no. Cytological examination of gonads in females fed NDA3 and 4 diets in the laboratory (see Figs. OO: oogonia. Poulet et al. abnormal larvae shown in Fig. . Calanus chilensis. shown in the samples A and B. D: OS3 in a sterile female (EPR value was zero at time of sampling). / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 egg-type 1 could hatch. very low hatching success. 2C. 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). non-symmetrical body and appendages. high production of abnormal larvae) and B (characterised by low egg production rates and extremely low hatching success) are shown in Fig. 6. D. The same types of egg and larval morphological anomalies were observed in all field and bioassay samples (Fig. 2B were characterised by symmetrical body and appendages. are focused in the pictures C–E. Cell anomalies “go” (vitellus granules and cell organelles) and “v”(unidentified vesicles). 1 and 3). 2. C–D–E: semi-thin sections of female oviducts. characterised by deformed. Homogeneous distribution of vitellus granules and organelles (e. n: nucleus. By contrast. 1. but most of them gave birth to abnormal nauplii (Figs. A–B scale: ×200. C–D–E scale: ×800. with a tendency to aggregate around nucleus in abnormal cytoplasm. 2C and D presented several morphological symptoms.A.194 S. C: OS3 in females characterised by high EPR-low HS-high AL. Pictures of longitudinal sections in gonads and oviducts of females. belonging to egg-types A (characterised by pseudo-normal egg production rates. Proportions of egg-types were not monitored.

Dabob bay: North Pacific.S. 1996). 2007) and thus. coastal waters off Rroscoff: English Channel.and strain-dependent (Pohnert et al. were observed in many OS3.A. both field and laboratory observations are coherent among these two co-generic copepod species. chilensis females in the Dichato coastal waters (Chile) were achieved. Wichard et al. 2006. allowing safe comparison of results between the two copepod species and regions. D). Mauchline. around 9 h AM. It means that different diatoms occurring in areas located at the antipodes can exert severe impacts on the reproduction. 6A).... Alternatively. 2007. and N sp 1 did not release PUA. C sp. PUA production by isolated and unialgal cultured diatoms was around one order of magnitude lower than samples of mixed diatom assemblages.. Observations of the reproductive responses of C. submitted for publication). “pseudo-normal” OS3 had a uniform colour because “go” were homogeneously scattered in the cytoplasm (Fig. These species occupy similar ecological niches in the Southern and Northern Hemispheres. when . The cumulative effects of both diatom concentration and duration of female exposure have also been already documented for EPR and HS (Chaudron et al. Vargas et al. or areas. following the same protocols as with C. C. In contrast. Whereas PUAcomposition was similar in all samples.. Histological examination of gonads provides valuable information about the reason why reproductive factors varied between different seasons.. 4. E). Results in Fig. We already have mentioned such a phenomenon in C. Discussion Results in Figs. The two dominant. Wichard et al. At two occasions the PUA production of phytoplankton samples (NDA 3 and 4) were investigated (Table 3). as shown before with C. helgolandicus females always returned to normal EPR. as well as deficiency of specific nutrients in diets could be the causes of such irreversibility. due to the heterogeneous distribution and concentration of “go”. C. (around 33 eggs/female/day: Peterson et al. low HS values and high larval morphological anomalies (AL) were not reversible when females were fed PM diet during 6 or 10 days (Fig. 2007). 6A. Apparently. while “v” seemed to be more frequent (defining anomaly no. chilensis during upwelling driven summer dense diatom blooms in the field. 2007). chilensis to extremely abundant deleterious diatoms in nature. E). We first assumed that irreversibility was due both to highest diatom concentrations and to longest exposure of C. Poulet et al. resembling normal specific values in Calanus sp. OS2. respectively) a diatom driven reduction of reproductive success can be observed. Halsband-Lenk et al. HS and AL values when fed PM diet. helgolandicus (Poulet et al. Wichard et al. chilensis females were offered diatoms in NDA diets twice their concentrations in nature (Table 1).. Minor cell anomalies in OS3 were occurring in females fed NDA and PM diets (see “go” and “v” in Fig 6A.. 1988. 2006.. OS3 were always observed in these samples (for definition of OO and OS: see Niehoff. when C. Since polyunsaturated aldehydes are supposed to affect the copepod reproductive response. and 3 showed that EPR. 6B) colour of oocyte cytoplasm was not uniformed.. 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. Egg production rates decreased significantly in comparison to day 1. while AL was close to 100% (Fig. prior to bioassays in the laboratory. EPR values.. Koski. submitted for publication). because the majority had been spawned earlier in the morning. 2006.. 5). C). upwelling system: South Pacific. or by semi-artificial NDA diets. 2006. HS remained abnormally low in the field with mean value ≤ 50%. Norvegian fjords: Ianora et al. whereas the three investigated less abundant species. Exceptionally long periods of reproductive breakdown have been already observed with C.. scattered in the cytoplasm (“go”: vitellus granules and cell organelles) and vesicles (“v”: unidentified vesicles) sandwiched between oocyte and follicular cell membranes. 2. different detoxification mechanisms and different diatom toxicity levels. 2006. 1 indicate that mean EPR values for C. 1995a. 2005). chemical analysis of the Chilean phytoplankton samples and isolated diatom species were conducted. In nonspawning. B. D. 2007. In all investigated systems (Adriatic sea. helgolandicus females exposed to NDA diets.. 6B. 2005. 2002. blooming species SJ and TR were PUA producers. 1: Fig. oldest oocyte development OS4 stages were scarce. 1. aggregated around the nucleus (defining anomaly no. Wichard et al. Poulet et al. 6B. HS and AL were impaired in C. At time of sampling. chilensis varied between 20–30 eggs/female/day during the summer bloom. In spawning females (Fig. 2: Fig. 1998). 2003). 2006. Micro-structures. 2004. helgolandicus fed diatoms at much lower concentration (Poulet et al. helgolandicus in the Roscoff coastal waters (Laabir et al. Poulet et al.. Because PUA production is diatom species. CD. when feeding periods were ≥ 7 days (Poulet et al. 1998. 1).b. respectively.. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 195 Normal oogonies (OO) and oocyte development stages OS1. or low-spawning females (Fig. the change of PUA-proportion indicates the variable pool of those polyunsaturated fatty acids transformed into PUA (Table 3.

. Poulet et al. These results suggest that inhibitory mechanism (2) was also prevailing at Dichato at time of sampling. This third reproductive pattern was typically linked to a food shortage and nutrient deficiency. Poulet et al. 2005). when diatom diets were seasonally replaced by non-diatom preys comprising mainly nanoflagellates. Ianora et al. 2006). suggesting that they were heavily fed upon by copepods (Fig. ciliates and dinoflagellates. high and/or low HS–AL values. was due to low biomass of non-toxic preys. low HS and/or high AL. It has been recently demonstrated that diatomPUA producers do not impair EPR (Poulet et al. Since their field survey and assays lasted a complete year. 2007). 2007). Nitzschia sp. Tables 1. As such. (2007) and characterised by the presence of severe cell anomalies in oocytes matching low EPR. (Figs. rotula (b 40% HS anomalies). helgolandicus (b 40%: Pohnert et al. Fig. we assumed that TR and SJ Chilean strains were affecting only HS and AL in C. chilensis females (Fig.. 2. due to highly nutritious diatoms occurring in the field and fed upon by A. HS and AL at Roscoff (Wichard et al. First. Ianora et al. 1996. At Roscoff. defined by Poulet et al.. Skeletonema costatum and to a minor extend to T... inhibitory . This conclusion was supported by complementary results obtained by Vargas et al. this inhibitory pattern was influenced by several single diatom species in diets.. Wichard et al. Thalassiosira pseudonana. helgolandicus (corresponding to inhibitory mechanism (2): normal/high EPR.. 2007).. EPR. thus inducing a typical food limitation linked to the relative decrease of PUFA and HUFA per cell known to support high EPR (Verity and Paffenhöfer. Stephanopyxis turris. Poulet et al. Guinardia delicatula... (2007). These authors observed the same inhibitory mechanism (2). because they resemble TR and SK activities in C. 1. concentration of diatoms at Dichato was 2–5 times higher than Roscoff. tonsa. another type of inhibition was identified corresponding to inhibitory mechanism (1). These results support three conclusions. 2006). However. Paffenhöfer et al. submitted for publication). striata. As reported earlier by Ban et al. chilensis. it was related to ingestion of Navicula sp. 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. helgolandicus (T. Second. 2005. the reason why only minor oocyte anomalies and strong inhibition in HS were observed may be due to three causes: 1. Results with C. 2005). Poulet et al. several TR strains known as strong PUA producers are capable to induce either very low or medium hatching failure in C. At Roscoff. C sp.196 S. brachiatus during diatom spring–summer blooms. This pattern was already observed with C. Vargas et al. TR and SJ remains were extremely abundant in faecal pellets. it can be defined as a passive inhibitory mechanism (3). P. Third. 2007.. 2006.. other toxic oxylipins. (2006).. apparently resembling inhibitory mechanism (1). production of PUA is fuelled with PUFAs acting as precursors. TR and SJ were PUA producers and favoured high egg production. while the other species. defined by Poulet et al. and the less abundant N sp. and 3. However.. non-toxic.. parvus and C. non-diatom preys were progressively replacing high biomass of diatoms prevailing during spring–summer. 2002.. Odontella regia. 2004. even though no significant correlations could be found in the field between PUAs production. minor cell anomalies observed in gonads coincided with normal/high EPR. 2003. In C. 2007). whereas they can partially or strongly depress HS and/or increase AL (Pohnert et al.. which did not impair EPR: Wichard et al. were not (Table 3. normal high HS and low AL values). 6B and C. Ask et al. With C. 2006.. submitted for publication. 4). Wichard et al.. It was shown that the level of cell degradations in oocytes and reproductive breakdown were diatom species dependent (Poulet et al. as shown by Wichard et al... these authors could also notice that inhibitory mechanism (2) was replaced by another reproductive inhibitory pattern (lower EPR. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 females feed on diatom-rich diets. Poulet et al. (2006) showed that this pattern. chilensis. rotula and S. 2005.A. 2004. submitted for publication).. chilensis. 1. Poulet et al. low HS and high AL values (Figs.. 3 and 6. Wichard et al. 2005. Succession of reproductive inhibitory patterns (2) and (3) occurred during the summer–fall and winter–spring transitions. 2002. Poulet et al. (1997). 4). 1). because TR and SJ were the most abundant diatoms and heavily ingested by C.. submitted for publication). Therefore. 2007. 1 and 3). thus providing high food supply (Tables 1 and 3. in particular Chaetoceros calcitrans. absence of cell degradations in OS3 coincides with high and normal EPR values (Figs. the concentration of which decreases with time and thus induces indirect fatty acid deficiency in diet. G. 2007). 2. Rhizosolenia setigera. costatum strains assayed at Roscoff known as PUA producers. Niehoff. when low biomass. chilensis further showed that inhibitory mechanism (1) was not directly involved in the Chilean coastal waters at time of sampling (Fig. Wichard et al. 3). metabolised along the PUA production pathways might be involved in these inhibitory mechanisms. CD.

they can be understood by histology of female gonads. 2006). S. Thus..S.E. chlorophyll a. Wichard et al.. Recent results with Eurytemora affinis (Ask et al. Marc Blondel for sharing his Olympus microscope and to Dr. 2006. reproduction of four co-occurring Calanoid copepods was deeply impaired by diatoms. various phytoplankton blooms occur in different ecosystems with similar diatom genus composition but different species offering distinct chemical properties. Niehoff. (2005). G. It may be the reason why Irigoien et al. Ecol.. A. 2001. 2004).. Thanks are due to Dr. [SS] References Arendt. 2007. J... 1998.... Castel. 3: see incubation time ≥ 4 days). which can avoid deleterious Thalassiosira sp.. Guerrero Ruiz. (2006). Y.. J. Shi. Poulet et al.. Y. Poulet et al. Lampert.. The chemical compounds responsible for the deleterious variability have to be further investigated. Prog. 2005. 2007. R. U. The paradox of diatom–copepod interactions. A. brachiatus (Vargas et al. Kang. Uye. M. pacificus. Calanoides acutus. These mechanisms are not directly related to the concentration of phytoplankton expressed by the number of cells. Runge. helgolandicus (Ianora et al. may be because they could be post-diaposing and thus. chilensis was fed very dense NDA diets (Fig. in relation to abundance and clonal difference of diatoms. Escribano. X. Oceanog. another common small-size copepod.A. M. Therefore.. Laabir.. these inhibitory mechanisms were not correlated to PUAs production with C. Ning. Fonda Umani. W.. 2005. C. Plankton Res. 2006. 1997. 1–5) support the idea that reproductive failure in several Calanoid copepods is primarily linked to the ingestion of specific deleterious diatoms.. Figs.. Extrapolating to copepods results obtained with Daphnia sp.. C.. 2005). Mar.. 287–293... Rodriguez. Kosobokova and Hirche. 2001.. Accumulating evidences on the deleterious influence of diatom-rich diets fed upon by both C. Limnol. Effects of dietary fatty acids on the reproductive success of the calanoid copepod Temora longicornis. parvus and C.. neither by PUFA nor HUFA deficiency in diets (Laabir et al. chilensis (Tables 1 and 2. 449–454. helgolandicus and C. 2004). to Dr.. Wichard et al... (Carotenuto et al. Variation in hatching success and egg production of Eurytemora (Calanoida.. Ban. Mar. which coexisted in the Dichato coastal waters. Starr.. 28. submitted for publication)... was not affected by deleterious diatoms (unpublished data). classification of egg-inhibition and morphological aspect of larvae. (Leising et al. Biol.. calcitrans.. M. K. (2002) does not apply to every marine ecosystems.. Burns.. When different inhibitory mechanisms are involved.K. P. In conclusion. Oithona nana. Chaudron. S. chilensis. or live preys belonging to the microbial-food web (nanoflagellates b 10 μm: Vargas and González. Jónasdóttir.J. The same reasoning applies to C. might be metabolically relying on their lipid reserves for spawning at time of sampling (Hagen and Auel. 2005. 2006). J. Poulet et al. Gasparini. Moreover. E. T. 2006). Halsband-Lenk et al. P. 683–694.. J.. Lacoste et al. usually selects different food resources like detritus and faecal pellets (González and Smetacek. 1994). Lacoste.. pacificus (Halsband-Lenk et al. or Pleuromamma sp. Copepoda) from the Gulf of Bothnia. Vargas et al. Wichard et al. submitted for publication).. 157. 2005) and C. Adrianna Zingone for identification of diatoms (SJ). Carmen Morales for permission to use her chlorophyll data. Vargas et al. . helgolandicus at Roscoff (Wichard et al.. 2004. which were selected and ingested by those (Fig. Miralto. 2005). Expression of mechanism (1) was assumed to be due to the artificial increase of CD and C sp. which can express inhibitory mechanism (1): see Poulet et al. by Max Planck Institute and by the COPAS FONDAP Center. M. Gätner. Wang. Hansen. Carotenuto. Similarly. In contrast. V. Pohnert... They were sharing same inhibitory pattern as another cogeneric species C. Calanus finmarchicus and C.. POC and PON. tonsa. chilensis plead for the expression of inhibitory mechanisms (1) and (2) by diatoms in nature. conclusion raised by Irigoien et al. 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. Reinikainen.. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187–199 197 mechanism (1) was probably latent in the field. positive or negative activities on the reproductive responses can be observed following post-ingestion of diatoms by copepod females. Båmstedt. 2006. Ianora. submitted for publication). because Oithona sp. A. (2002) did not find any inhibitory patterns with Metridia sp. S. It could be expressed in the laboratory and was superimposed to inhibitory mechanism (2). Acknowledgements This work has been partly funded by a CONICYTCNRS exchange programme and by the French Biodiversity programme. the reason could be a better detoxification mechanism in these four species. helgolandicus or C. when C.. 2007. S.. C. H. 513–530. S. Ser. S. Y.. 50. marshallae could be much less influenced by diatoms than C. 2001. S. 146. Wichard. A. 4.. Rhincalanus gigas. relative to TR and SJ. Poulet. J. Christou. F. because these copepods are carnivorous. 2006. Lifehistory responses of Daphnia pulicaria to diets containing freshwater diatoms: effects of nutritional quality versus polyunsaturated aldehydes. because chemical properties and biological activities expressed by diatoms are globally variable... Hoffmeyer. Baltic Sea. Ask.H. a non-PUA producer..

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