2007, Poulet. Collapse of Calanus Chilensis Reproduction in a Marine Environment With High Diatom Concentration. (CHI)

8/13/2019 2007, Poulet. Collapse of Calanus Chilensis Reproduction in a Marine Environment With High Diatom Concentration. (CHI)

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Collapse ofCalanus chilensis reproduction in a marine environment

with high diatom concentration

S.A. Pouleta ,, R. Escribano b, P. Hidalgo b , A. Cueff a, T. Wichard c, V. Aguilera b ,C.A. Vargas b , G. Pohnertd

a Station Biologique de Roscoff, CNRS, INSU, UPMC Paris VI, UMR 7150-Unit Mer et Sant, Roscoff 29682, Franceb Center of Oceanography for the Eastern South Pacific (COPAS), Universidad de Concepcion, P.O. Box 160 C, Concepcion, Chile

cMax Planck Institute for Chemical Ecology, Hans-Knll-Str. 8; D-07745 Jena, Germany

d 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-

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 byIanora et al.,

2003; Paffenhfer et al., 2005) found, with combinations

of different copepods fed high diatom concentrations

Journal of Experimental Marine Biology and Ecology 352 (2007) 187199

www.elsevier.com/locate/jembe

Corresponding author.

E-mail address:[email protected](S.A. Poulet).

0022-0981/$ - see front matter 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2007.07.019
mailto:[email protected]://dx.doi.org/10.1016/j.jembe.2007.07.019http://dx.doi.org/10.1016/j.jembe.2007.07.019mailto:[email protected]


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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 breedingseason 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 recentlyestablished 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 highvariance 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 environ-

ments, such as those surveyed byIrigoien et al. (2002)and temporarily occurring in upwelling environments.

To do so, the reproductive response ofCalanus 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

parvusand Centropages brachiatus: seeVargas et al.,

2006).

C. chilensisis 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 ofC. 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 continuouslyyear 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 phyto-

plankton 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 25g/l (Vargas et al., 2006), which

are in the same range as the diatom richest regions

monitored byIrigoien et al. (2002)and about 2

5 timeshigher 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 copepodmaternal diets (supposedly responsible for the repro-

ductive 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 b2

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 experi-

ment were sorted and incubated individually in dishes

188 S.A. Poulet et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187199


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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 121 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 (seeFig. 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 testedwith 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 achievedaccording 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 year-

to-year spring blooms observed in the Dichato coastal

waters.

2.3. Experiments with diatom-enriched diets

Samples of mixed species in natural phytoplankton

assemblages (N11 m) (NDA1, 2, 3, 4) were collected atfour 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 (seeFig. 2). Error bars are standard deviations. Sample size

wasN=20 females maximum at each sampling date. : no values in

relation to zero hatching rate. Same symbol as inFigs. 3and 5.

189S.A. Poulet et al. / Journal of Experimental Marine Biology and Ecology 352 (2007) 187199


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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 withLugol'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 re-suspended 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). Micro-

scopic observations of NDA diets indicated that they

were dominated by chain-forming diatoms mixed with

other microorganisms, belonging to unidentified auto-

trophic 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 inPoulet et al., 2006;Wichard et al., submitted for publication) was tested as a

favourable diet at concentrations corresponding to 104

cells ml1 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 dateof bioassays with NDA1-4 diets are given inTables 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 chilensisfaecal pellets production

Date 02/12/2004 06/12/2004 16/12/2004 20/12/2004

Diet NDA1 NDA2 NDA3 NDA4-PM

Mean S.D. Mean S.D. Mean S.D. Mean S.D.

Day

1 93 46 61 20 87 35

2 106 39 82 37 121 23

3 58 31 68 24 170 35

4 68 26 42 21 167 22

5 211 31 48 18

6 37 26

7 44 26

8 61 32

9 54 22

10 68 32

Values are means of faecal pellet production standard deviation

measured daily during assays with NDA1, 2 and 3 diets, and with PMdiet following pre-incubation with NDA4 diet (: not measured).

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

Total diatoms

(cells/ml)

Total non-diatoms

(cells/ml)

Chlorophyll a

Inshore (g/l)

09/11/2004 ? ? 5.8301/12/2004 7.5 103 ? 23.19

02/12/2004 15103 ? ?06/12/2004 0.32103 6.2 9.9

09/12/2004 0.93103 34.8 14.211/12/2004 ? ? 10.78

15/12/2004 ? ? ?16/12/2004 0.22103 10.62 ?20/12/2004 8.11 6.93 ?

28/12/2004 0.74103 8.3 ?

Diatom species TR SJ C sp. 1 CD N sp. 1

Proportion (%) 40 35 21 12 2

: Date of sampling at sea and start of assays with NDA diets are same

as inFigs. 1 and 3.



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incubator (Table 2). The methods used for faecal pellet

examination have been described earlier byLaabir 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 theproportions 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 awas calculated throughLorenzen's (1966)

equation. Complementary information on phytoplank-

ton biomass for the area during this period was available

from the time series study off Concepcin carried out bythe 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 diatom-

derived polyunsaturated aldehydes (PUA) in phyto-

plankton. 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 byWichard 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 PUA-

producers. 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, 5

6 h old. C

D: photos of abnormal larva, same age as B. Scale:100 m.

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. 1and Table 2)

Strain Total PUA

fmol/cell

S.D.

Category of PUA

Heptadienal % Octadienal % Octatrienal % Decadienal % Decatrienal %

SJ 0.10 0.02 56 30 14 0 0

TR 1.41 0.22 23 77 0 0 0

CD 0

C sp. 0

N sp. 0

Mixed diatom diet

NDA3 3.12 0.48 67 15 18 0 0

NDA4 11.12 2.52 45 19 35 0 0

Five category of toxic PUA were analysed.

http://www.copas.cl/http://www.copas.cl/


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density in diatom cultures sampled at the stationary

phase ranged between 10

4

and 1.210

6

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 inseawater 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 concen-

tration, except on the 20th. The mean in situEPR 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, exceptthe 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 (Ttest, 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 inFig. 3. Mean values at day 1 corresponded

Fig. 4. SEM photos of faecal pellets produced by copepods during

bioassays with NDA1

4 diets. A: frustule remains of TR (Thalassiosirarotula). B: frustule remains of SJ (Skeletonema japonicus).

Fig. 3. Calanus chilensis. Variationswith time of (EPR),(HS) and(AL) reflecting thenegativeeffects of NDA diets on thereproductiveresponses of females

incubated 46 days under laboratory conditions. Sampling dates for NDA14 are given inFig. 1. Arrows give estimated field values at day 1. : no value.



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to in situ estimates, same as in Fig. 1. Doubling the

diatom concentrations in diets had a significant (non-

parametric Wilcoxon signed-rank test, pb0.01) 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. HSwas 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, pb0.01) and almost completely

depressed with the other NDA diets most of the time.

Once again hatched larvae were scarce and morpholog-

ically abnormal, ressembling those shown inFig. 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 Calanusspp.).

Daily faecal pellet production was estimated withNDA1, 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) , o r

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, pb0.01). Each group wasfurther 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, pb0.01; day 1: Fig. 1). In

contrast, neither HS nor AL values could return toin situ

rates nor be improved with PM diet. Results inFig. 5

illustrated the negative and irreversible effects of dense

diatom diets on HS and AL. Same result was obtained indifferent 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

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 blas-

tomers associated with smaller ones. Spines were present

on all egg membrane, except egg-type 3. Majority of

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.



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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 inFig. 2B were characterised by

symmetrical body and appendages. By contrast, abnor-

mal larvae shown in Fig. 2C and D presented severalmorphological 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 (char-

acterised by pseudo-normal egg production rates, very

low hatching success, high production of abnormallarvae) 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. A

Bscale: 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: seeNiehoff, 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 non-

spawning, or low-spawning females (Fig. 6B) colour

of oocyte cytoplasm was not uniformed, due to theheterogeneous 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 singlediatom 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 PUA-

composition was similar in all samples, the change of

PUA-proportion indicates the variable pool of thosepolyunsaturated fatty acids transformed into PUA

(Table 3,Wichard et al., 2007).

4. Discussion

Results inFigs. 1, 2, and 3showed 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. helgolandicusfed diatoms at much lower concentra-

tion (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 reproduc-

tion. In all investigated systems (Adriatic sea, Dabob bay:

North Pacific, coastal waters off Rroscoff: EnglishChannel, 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 ofC. chilensis females in the Dichato coastal

waters (Chile) were achieved, following the same

protocols as with C. helgolandicusin 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. 1indicate 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. helgo-

landicus (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 concentrationsin 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. chilensisto extremely abundant deleterious diatoms in

nature, prior to bioassays in the laboratory. We already

have mentioned such a phenomenon inC. helgolandicus

females exposed to NDA diets, when feeding periodswere 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 valu-

able information about the reason why reproductive

factors varied between different seasons, or areas, when



10/13

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, definedby 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(b40% HS

anomalies).

As reported earlier byBan 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 influ-

enced by several single diatom species in diets, inparticularChaetoceros calcitrans,Guinardia delicatula,

G. striata, Rhizosolenia setigera, Thalassiosira pseu-

donana, 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 diatom-PUA 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 (b40%:

Pohnert et al., 2002, Wichard et al., 2005, submitted for

publication, Poulet et al., 2007). These results support

three conclusions. First, other toxic oxylipins, metabo-

lised 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 (b2) below values requested to sustainnormal 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. rotulaand S. costatumstrains 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 complemen-

tary results obtained byVargas et al. (2006).

These authors observed the same inhibitory mecha-

nism (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 completeyear, 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



11/13

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., relativeto TR and SJ. These twoChaetocerosspecies 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 co-

generic species C. calcitrans, a non-PUA producer,

which can express inhibitory mechanism (1): seePoulet

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 foodresources like detritus and faecal pellets (Gonzlez and

Smetacek, 1994), or live preys belonging to the

microbial-food web (nanoflagellates b10 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 whyIrigoien et al. (2002)did not

find any inhibitory patterns with Metridia sp. orPleur-

omamma sp., because these copepods are carnivorous.

The same reasoning applies to C. pacificus, which can

avoid deleteriousThalassiosirasp. (Leising et al., 2005;Halsband-Lenk et al., 2005). Similarly, Calanoides

acutus, Rhincalanus gigas, Calanus finmarchicus and

C. marshallaecould be much less influenced by diatoms

than C. helgolandicus orC. chilensis, may be because

they could be post-diaposing and thus, might be

metabolically relying on their lipid reserves for spawn-

ing at time of sampling (Hagen and Auel, 2001;

Kosobokova and Hirche, 2001; Niehoff, 2004). Extrap-

olating to copepods results obtained with Daphnia sp.

(Carotenuto et al., 2005), the reason could be a better

detoxification mechanism in these four species. Accu-mulating evidences on the deleterious influence of

diatom-rich diets fed upon by bothC. helgolandicusand

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 cor-

related 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)andC. 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 respon-

sible 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 copepodfemales. When different inhibitory mechanisms are

involved, they can be understood by histology of female

gonads, classification of egg-inhibition and morpholog-

ical 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 CONICYT-

CNRS exchange programme and by the FrenchBiodiversity 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 identifica-

tion of diatoms (SJ). [SS]

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2007, Poulet. Collapse of Calanus Chilensis Reproduction in a Marine Environment With High Diatom Concentration. (CHI)