measuring production and viability of eggs in calanus helgolandicus

Journal of Plankton Research Vol.17 no.5 pp.1125-1142, 1995

Measuring production and viability of eggs in Calanushelgolandicus

Mohamed Laabir, Serge A.Poulet and Adrianna Ianora1

Station Biologique, CNRS et Universiti P. & M. Curie, Roscoff 29680, France and1Stazione Zoologica, Anton Dohm, Villa Comunale, 80121 Naples, Italy

Abstract The effect of several biotic and abiotic factors on the fecundity and hatching success ofCalanus helgolandicus was tested during short- and long-term incubations. The results show that thevariations of the reproductive responses of C.helgolandicus are time dependent and rely on the typeof factor tested. When standardized over a 24 h incubation period, estimates of in situ production andegg viability can be obtained with good accuracy.

Introduction

The production and viability of eggs are now considered as practical biologicalcriteria to study the functional reproductive responses of copepods toenvironmental hydrographic and phytoplankton food conditions (Ki0rboe etal., 1988; ICES, 1992; Ianora et al., 1994). These two variables give accuratemeasures of a fraction of secondary production and recruitment in the field(Poulet et al., 1994a), since they both correspond to the gonadic growth of adultfemales and reflect two basic responses (feeding and reproduction) of the adultsto their changing environment. Also, the time lag between causes (physical andchemical conditions, onset of phytoplankton biomass and shift in speciescharacteristics) and effect (incorporation of algal organic matter by adultsproducing eggs) is minimal (hours to days; Krause and Thrams, 1983; Tester andTurner, 1990; Hirche et al., 1991). In this sense, these variables are site specificand time dependent.

Several approaches to calculate secondary production have been suggested inthe past, but none has been routinely adopted by marine planktologists: cohort(e.g. Boysen-Jensen, 1949), growth rate (e.g. Ricker, 1946; Allen, 1951),physiological (e.g. Winberg, 1971) and mathematical model (e.g. Zurlini et al.,1978; Wroblewski, 1980) methods. An alternative approach, known as the 'eggproduction method', is based on the measurement of in situ fecundity. Itsprinciples and practical aspects have been discussed by Berggreen et al. (1988),Peterson et al. (1991) and Poulet et al. (1994a).

To calculate in situ fecundity, Edmondson et al. (1962), Checkley (1980) andBeckman and Peterson (1986) proposed the 'egg-ratio method', given by theequation F = El AD, where F is fecundity (eggs female"1 h"1), E is egg density infield samples (eggs m"3), A is adult female density in seawater (females m~3) andD is egg development time (h). This approach underestimates fecundity sinceeggs in the water column are exposed to dispersion, cannibalism and sinking.Consequently, planktologists have utilized instead the 'egg incubation method'to evaluate in situ copepod fecundity through incubation procedures (e.g. Dagg,

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1978; Ambler, 1985; onward). Although very promising, some confusion stillremains in the interpretation of this method regarding the effect of biotic andabiotic factors controlling, or influencing, the reproductive responses ofcopepods over short (hour-day) and long (day-month) periods of time. Thismatter must be clarified in order to define a reliable method. The aim of thiswork was to test some basic parameters against the reproductive responses, inorder to standardize an egg production method in Calanus helgolandicus.

Method

Zooplankton were collected in 1993 at a fixed station in coastal waters nearRoscoff (Western English Channel) by gently towing a 500 n,m mesh netobliquely from 10 m to the surface. Samples were kept in an insulated box untilthey reached the laboratory, usually within 1-2 h after collection. Calanushelgolandicus adult females and males were identified, sorted and pipetted intoincubators. Tests were conducted under specific physical and biologicalconditions. In each experiment, the daily egg production rate (number female"1

day"1), including crumpled membranes, and hatching success (per cent of totaleggs) were monitored, using a Wild M5 stereomicroscope, as indices of thecopepod's reproductive responses. Incubation time to hatching varied between24 and 72 h, depending on the temperature.

Physical conditions

Except for the partition experiment, which was conducted in June at 13 ± 0.5°C,all the physical factors were tested during August when in situ temperature was16 ± 0.5°C.

Temperature. Batches of five females per litre each were incubated in triplicates,at three different temperatures (5,15.7 and 22.3 ± 0.5°C). After a 24 h incubationperiod, eggs were counted. Freshly spawned eggs were incubated in crystallizingdishes filled with natural seawater at various temperatures ranging from 5 to23°C. Development time, corresponding to the time interval necessary to reach50% hatching, was recorded for each temperature. The percentage of viableeggs, corresponding to those that hatched ^72 h, was also determined for eachtemperature.

Light The effect of light on egg production was tested. Three replicate samplesof five females each, kept in 1 1 of seawater, were incubated in parallel underthree different light conditions [total darkness, continuous artificial light: 200M-E m"2, and 12 h light (L):12 h dark (D) cycle].

Stirring. In order to test the effect of turbulence on copepod spawning, fourreplicate samples of five females each were kept for 24 h in stirred water,whereas an equal number of samples was incubated in still water for comparison.Stirring was performed by gently bubbling air into the incubators or manually5-9 times a day.

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Volume of incubator. In order to evaluate the effect of incubator volume on thereproductive responses of females, three replicate samples of one female each,kept in 50, 200, 500, 1000 and 2000 ml of natural seawater, were incubated for24 h.

Partition. Comparisons between four samples of 5, 10, 20 and 40 females each,kept for 24 h in two different types of incubators, were made. The first type had apartition (a 300 p,m mesh sieve fixed at the bottom) through which eggs couldsediment before being eaten, whereas the second type had no partition.

Duration of incubation. The spawning pattern of C.helgolandicus was monitored,at three different times, during a complete diel cycle. Five batches of 10 femaleseach were incubated in unfiltered seawater, kept at ambient temperature andunder a natural light cycle (12 h L:12 h D). Eggs were removed and countedevery hour for 24 h.

Biological conditions

The experiments were conducted at temperatures corresponding to thoseprevailing in situ at the time of capture of the copepods, with a natural light cycle.The diatoms Thalassiosira rotula (157.6 pg carbon cell"1) and Nitzschiaclosterium (118.8 pg carbon cell"1) were cultured in f/2 medium (Guillard andRyther, 1962). The dinoflagellate Gyrodinium aureolum (1435.7 pg carboncell"1) was cultured in K medium (Keller et ai, 1987). These phytoplanktonspecies were grown at 17°C and on a 12 L (200 JJLE m"2):12 D cycle andprovided to copepods in the exponential phase of growth.

Experiments related to number of females were carried out in March andApril when in situ temperature was 10.5 ± 0.5°C, whereas the past feeding historyexperiment was conducted on three different dates, March, September andNovember, when ambient temperatures were 10.5 ± 0.5, 15.5 ± 0.5 and 12.5 ±0.5°C, respectively. All the others factors were tested in August and Septemberat 15.5 ± 0.5°C.

Presence of males. Reproductive responses were compared between fourreplicate samples of five females alone and four replicates of four females inthe presence of one male, incubated in natural seawater. After 24 h incubation,eggs were counted and incubated in crystallizing dishes with filtered seawater todetermine hatching success.

Food quantity. The effect of food concentration on fecundity was tested over aconstant incubation period (24 h). Batches of five females per litre each wereincubated in four replicates, under three different food conditions: 1, naturalseawater, 2, filtered seawater (0.22 jim); 3, filtered seawater enriched with theculture of T.rotula (5 x 103 cells ml"1).

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In a second series of experiments, three replicate batches of four females eachwere fed different concentrations of T.rotula ranging from 0 to 8 x 103 cells ml"1,during a longer incubation period (4-9 days). Every day females weretransferred to new incubators, with fresh filtered seawater (0.22 u,m) enrichedwith algal medium. Eggs were counted daily.

Food type. Ten replicate samples of four females each were incubated in filteredseawater enriched with variable concentrations of either N.closterium (<105 cellsml"1), T.rotula (lOMO5 cells ml"1) or G.aureolum (2-4 x 104 cells ml"1). Everyday copepods were removed and placed in new incubators with fresh media.Eggs were counted daily and then incubated in natural seawater to determineand compare fecundity and hatching success with different types of food.

Number of females. Six experiments were conducted to examine the effect offemale density on spawning. In each experiment, batches of 2,5,10, 20,40 and 60females each were incubated in parallel, triplicate samples in 1 1 containers withpartitions. After 24 h, eggs were counted in each incubator.

Cannibalism. Copepods are known to eat their eggs. This aspect was examinedunder specific conditions. Three replicates of several females and males (n = 12individuals per incubator), initially starved for 48 h in filtered seawater (0.22(xm), were incubated for 24 h with a known number of eggs, artificially colouredwith neutral red, added at the start of the experiments in incubators withoutpartitions. Females or males that had eaten coloured eggs were identified bytheir red bodies.

Variability between replicate samples. The variability in egg-laying and hatchingrate was tested by 14-15 replicate incubations, with five females each for 24 h, fordetermining its consequence on the level of error of the estimated reproductiveresponses.

Past feeding history of females. Triplicate batches of four females each were keptin incubators containing filtered seawater (0.22 ftm) enriched with T.rotula (5 x103 cells ml"1). Females were removed daily and incubated in fresh algal medium.Observations were conducted for 7 days on three occasions in March, Septemberand November. At the time, both in situ chlorophyll (Chi) a (^0.5 p,g I"1; anindex of food) and mean fecundity (^6 eggs female" day"1) were low. Thisallowed for the comparison of field and laboratory reproductive responses,mediated by the incorporation of food and duration of feeding.

Results were tested either by the Student's f-test or by ANOVA.

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Fig. L Effect of different physical experimental conditions on egg production rates of C.helgolandicusover a 24 h incubation period. (A) Temperature; (B) light regime; (C) turbulence; (D) volume ofincubator. Results correspond to means and SDs (bars) of 3-4 replicate samples.

Results

Response to physical conditions

Spawning. At the end of the 24 h incubation period, no significant effects oftemperature (one-way ANOVA, F = 0.39, P = 0.69, Figure 1A), light (one-wayANOVA, F = 0.26, P = 0.78, Figure IB), turbulence (T-test, t = 0.77, P = 0.49,Figure 1C) and volume of incubators (one-way ANOVA, F = 0.67, P - 0.63,Figure ID) could be detected on the fecundity of C.helgolandicus females.

Only two of the six physical variables tested during this short incubationperiod (24 h) had a significant effect on fecundity. The numbers of eggsproduced were significantly different (P < 0.05) between incubators equippedwith and without partitions (Figure 2A), which contained an increasing densityof females, ranging from 5 to 401"1. Also, the fecundity of females was not stableover a diel light cycle (Figure 2B). Spawning activity in summer showed twomajor peaks, occurring around midnight and midday.

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Fig. 2. Relationship between egg production rates in C.helgolandicus and the type of incubator (A).Diel spawning cycle of C.helgolandicus females (B).

Hatching. When eggs were incubated for 24-72 h under the same conditionsof temperature and light as those set up for females, no significant (n = 22,r = 0.22, P < 0.05) effects were detected on hatching success (Figure 3A).Temperature only influenced development time, as shown by the equationY = 82.89 x lO^312 + 2X) (n = 22, r = 0.94, P < 0.001), which fits aclassical Belehradek function, where Y is development time (h) and X istemperature (°C) (Figure 3B).

Response to biological conditions

The reproductive responses of C.helgolandicus to the biological variables set upin our experiments were not as direct as those obtained under the physical

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conditions. Clear distinctions were made between significant and non-significantreproductive responses for the short-term incubation period and those whichbecame significant beyond 24 h.

Spawning

Presence of males and feeding. The results in Figure 4 A show that during a 24 hincubation period the presence of males had no significant effects (T-test, t -1.65, P = 0.19) on fecundity. Similarly, the quantity of food, characterized in ourexperiments by the presence of algae (T.rotula at 32 u.g Chi a T1) in theincubator, the absence of food (filtered seawater, 0.22 jim) or natural food in

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Fig. 4. Egg production rate of C.helgolandicus females in the presence or absence of males (A) andwith different food regimes (B), during a short incubation (24 h). Mean egg production for femalesincubated with N.dosterium (6-9 x 10* cells ml"1) and T.rotula (6-8 x 103 cells ml"1) for 4 days (C).Results correspond to means and SDs (bars) of 3-4 replicate samples.

seawater (0.14 jig Chi a I"1) had no significant effect (one way ANOVA, F =3.01, P - 0.1) on spawning (Figure 4B).

The effect of food quaKty, characterized by two different types of diatoms, wastested on C.helgolandicus fecundity. Although the concentration of N.dosteriumwas 10 times higher than that of T.rotula, there was no significant differencebetween these two diets (T-test, t = -0.86, P = 0.44; Figure AC). However, asshown in Figure 5, when copepods were fed the diatom T.rotula at

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Fig. 5. Effect of the algal concentration on the egg production rate of C.helgolxmdicus. Females werefed the diatom T.rotida during 9 days.

concentrations varying between 0 and 8 x 103 cells ml"1 for 9 days, a significantand positive correlation was found between food concentration (X) and eggproduction (Y), given by the relationship Y - 2.52^ - 0.9 (n = 23, r- 0.84).

Density of females and cannibalism. During a short incubation period (24 h),major decreases were observed in egg production due to copepod density. Thenumber of females significantly (one-way ANOVA, F = 2.32, P < 0.01)controlled egg production rates in incubators (Figure 6A). The regression foundbetween the density of females {X) and the mean production rate (Y) was Y -6.67 - 1.04A" (n - 6, r = 0.94). Nevertheless, when this relationship was examinedwith a lower density of females (=sl0 per litre), the mean fecundity was notsignificantly different, as indicated by the regression equation Y = 8.29 - 0.44^(n = 9, r = 0.42).

Cannibalism of male and female C.helgolandicus was observed directly. Allpre-coloured eggs were ingested and copepods exhibited a red body at the end ofthe incubation. This experiment was conducted only once.

Variability between replicate samples. What we define under this general termincorporates the unknown age of females at the time of capture, individualspecific fecundity, potential laboratory stress, past feeding history and behaviour.The influence of these individual female characteristics on the mean fecundityand hatching success of each sample is shown in Figure 6B and C. For a givennumber of samples incubated at the same time, the variations in the replicatevalues corresponded to ± 20-50% of the mean (Figure 6B and C).

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Fig. 6. Variations of the estimate of the mean egg production rate of C.helgolandicus in relation to thenumber of females in the incubators (A) and to replicate samples for egg production (B) and hatcing(C) rates. Results in (A) correspond to the means of three replicate samples and SDs (bars), in (B)they correspond to the mean of five females and in (C) they correspond to the mean of 30-110incubated eggs.

Past feeding history of females. Females collected in winter, early spring and lateautumn often did not spawn under laboratory conditions. These results werepuzzling since it was thought that immature, unmated, diapausing or starvingfemales had been selected and incubated, instead of fertile well-fed females; thevisual distinction being difficult between these categories of females. The resultsin Figure 7 show that these low spawning rates were not related to any of thesefemale characteristics, but rather to low food concentrations prevailing in thefield. The fecundity of females, corresponding to low or zero egg productionobserved after 24 h incubation under normal conditions, was modified, in theabsence of males, simply by adding food to the incubators (Figure 7). Egg

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Rg. 7. Influence of the past feeding history of C.helgolandiats females on egg production ratesmeasured before (0 = in situ estimate) and after addition of algal food (T.rotula: 5 x 103 cells ml"1).Each dot corresponds to the mean of triplicate samples of four females each.

production increased significantly 2 days after feeding on T.rotula at constantconcentrations (5 x 10̂ cells ml"1), as shown by the regression equation Y -3.WX + 2.083 (n = 8, r - 0.93), where Y is egg production rate (number female"1

day"1) and X is duration of incubation (days).The same experiment was repeated three times, during the low spawning

seasons. In each case, the production of eggs was always triggered by addition offood. Egg production rates at the end of these incubations (^7 days) reachedhigh values, similar to in situ fecundity observed in June and July 1993,corresponding to periods of maximum annual egg production observed in thecoastal waters off Roscoff (Laabir et al., 1995).

Hatching

Among the biological factors tested, none had a detectable effect on the hatchingsuccess of eggs during a short incubation period (24 h). Beyond this time, foodtype had a significant effect on the hatching rate. As shown in Figure 8A,hatching was normal and stable with a G.aureolum (2-4 x 104 cells mT1) diet,whereas it was significantly lower with a T.rotula (3-7 x 103 cells ml"1) diet (two-way ANOVA, F = 7.03, P < 0.05). With T.rotula, hatching success not only variedsignificantly with time (two-way ANOVA, F = 7.74, P < 0.01), but also with algalconcentration (two-way ANOVA, F - 11.05, P < 0.01), as shown in Figure 8B.

The other biological factors were not tested for incubation periods exceeding24 h.

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Fig. 8. Consequence of the type of food on the hatching success of eggs spawned by C.helgolandicusfemale! fed a diatom (T.rotula: 3-7 x 103 cells mT1: o) and a dinoflagellate diet (G.aurcolum, 2-4 x104 cells mT1: •) during 4 days of feeding (A). Consequence of the increased concentration ofT.rotula (3-4 xlO3 cells ml"': •; and 1.1-1.6 x 104 cells mT1: o) on the hatching success ofC.helgolandicus eggs over 3 days (B). Results are means of 2-3 replicate samples ± SDs (bars). 0 =control, start of experiment

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Discussion

Copepodologists have often wondered if the functional responses observedduring the incubation of copepods were reflecting natural conditions or ratherlaboratory artefacts. Intensive work achieved during the past decades on thereproduction of copepods has not really addressed the problem of standardizingthe so-called 'egg method' (i.e. ICES, 1992). Results in Figures 1 and 4 show thatduring 24 h incubation, temperature, light regime, stirring, volume of incubator,quantity and type of food, and presence or absence of males had no significanteffects on the egg production rate of C.helgolandicus. Similarly, hatching successwas not affected by either temperature or food conditions prevailing during theincubations (Figures 3A and 8A). Also, the presence or absence of males andlight regime had no effect on the hatching success of eggs. Only the number offemales (Figure 6A) and the presence of a partition in the incubator (Figure 2Aand 6A) efficiently affected the values obtained for fecundity. Although therewas no significant effect of the incubator volume on fecundity within the rangetested (Figure ID), it appears that it is necessary to have a sufficient watervolume for each female in the incubator (Figure 6A).

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The reason for selecting a uniform incubation time (24 h) was dictated by thebimodal response of C.helgolandicus during a diel cycle (Figure 2B). The dailyspawning cycle of Calanus finmarchicus (Marshall and Orr, 1952) was alsocharacterized by unimodal or bimodal responses in relation to its latitudinaldistribution. Thus, it is obvious that for shorter incubation periods fecunditywould be underestimated in Calanus, as well as in any other species havingsimilar diel periodicity. The other important reason for selecting a 24 hincubation is related to the time lag required by copepods to produce an egg(Tester and Turner, 1990). In other words how long does it take to incorporatenew food into eggs during vitelogenesis. According to Tester and Turner (1990),this lag is -10-90 h to reach maximum incorporation, depending on copepod sizeand species. Before this phase, new food uptake is not fully incorporated intoeggs, indicating that fecundity is still influenced by the past feeding history of thefemales. Beyond this time threshold, it is the reverse (Figures 7 and 8B; Hassett,1993; Bautista et al., 1994). Thus, the incubation time selected in the eggproduction method must be less than the time lag necessary to convert food intooocytes and sufficient to take into account the diel periodicity of spawning.

The problem of obtaining accurate and comparable estimates of in situ eggproduction and hatching rates of pelagic copepods through incubation methodsrelies on the variability of female samples, as shown in Figure 6B and C. At leastn > 30 females incubated individually or in batches of five females each arenecessary to estimate in situ fecundity. Monitoring the in situ seasonal variationsof the fecundity of C.helgolandicus collected off Roscoff (Poulet et al., 1994a;Laabir et al., 1995) revealed that the highest variability calculated for themonthly mean values coincided with periods of minimum egg production,meaning that during those periods the number of replicate samples had to beincreased accordingly, so that the precision of the measurements remainedsatisfactory (e.g. Morin et al., 1987). Similar considerations apply to themeasurement of in situ hatching success, which should be based on theobservation of at least n a: 30 eggs (Figure 6C).

Cannibalism in C.helgolandicus was observed through two different protocols(Figures 2A and 6A; and by neutral red experiments). It has been also reportedfor other species of copepods (e.g. Ki0rboe et al., 1988). Results in Figures 2Aand 6A further show that fecundity decreased when females were too crowded inincubators even equipped with a partition, suggesting that the egg productionrate could be substantially underestimated due to this 'crowding effect', and thatthe number of females per incubator must be kept at <10 per litre. Cannibalismalso increased with the level of starvation of the copepods (i.e. neutral redexperiment), corresponding in the field to females collected during periods oflow food concentration (^0.5 u.g Chi a P1). Cannibalism was reduced when foodwas added to the incubators, or during periods of phytoplankton blooms. In anycase, more accurate measurements of fecundity can be made simply by adding apartition to the incubator and by counting crumpled membranes (Ianora et al.,1994).

The past feeding history of females is a key factor for the interpretation andmeasurement of both the fecundity and viability of eggs. In 24 h incubation,

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fecundity and hatching were not affected by any food characteristics (Figures 4Band C, and 8A and B). Results obtained through this short incubation methodthus reflected the past feeding history of females in the field rather than theeffects induced by the laboratory conditions, which must be kept low and non-significant. This is exactly the objective of the egg production method, whose aimis to estimate instantaneous responses and thus must be site specific. Beyond thistime, reproductive responses were not further influenced by the past feedinghistory of females, as shown by the results in Figures 5, 7, and 8A and B. Theywere indeed significantly modified by the new laboratory conditions. Then,because copepod reproductive responses are time dependent, estimates of fieldfecundity and egg viability can be accurately obtained only through a shortincubation period not exceeding 24 h. A subsidiary effect of the past feedinghistory was observed with females collected during periods of low foodconcentration. It was thought that they were either sterile or resting. In fact,lack of food in the field was blocking or reducing the egg production rate, whichcould be triggered in the laboratory shortly after addition of food (Figure 7),suggesting that C.helgolandicus adult females were neither resting nor unmated,but rather waiting for favourable food conditions allowing induction of eggproduction. Similar observations were reported for C.finmarchicus by Plourdeand Runge (1993) and for Calanus glacialis by Hirche (1989). Rapid switchingfrom starvation to normal fecundity is considered as a prerequisite for a quickresponse (i.e. days to weeks) to pulsed food in the field (Hirche, 1989; Hassett,1993). Past feeding history of females also significantly affected hatching success(Figure 8A and B). Initial high hatching success was significantly reduced by thetype and concentration of algae ingested by the females within a long-termfeeding experiment. Seasonal observations of both C.helgolandicus (Laabir et al.,1995) and Centropages typicus (Ianora et al., 1992) indicated that successions intime (e.g. week, month intervals) of low and high hatching rates, or vice versa,naturally occurred, depending on the occurrence, type of phytoplankton andfood selectivity of females.

Beyond 24 h, most of the physical and biological factors obviously have aneffect on the reproductive performances, growth and survivorship of copepods(e.g. Gaudy, 1971; Elmore, 1982; Hirche, 1989; Uye and Shibuno, 1992). Ourresults show that temperature and the development time of embryos are relatedthrough a Belehradeck function (Figure 3B), whereas viability (i.e. hatchingsuccess) of eggs is not temperature dependent (Figure 3A). Fecundity, althoughan expression of adult female growth (Ki0rboe and Johansen, 1986; Berggreen etal., 1988), is not directly temperature dependent during 24 h or beyond, withinthe range of temperatures to which copepods are acclimated (Figure 1A; e.g.Corkett and Zillioux, 1975; Uye and Shibuno, 1992), even though this point isstill debatable (e.g. Bautista et al., 1994).

Light is a more controversial and a less studied factor, although its importancefor the spawning periodicity and strategy of females was reported by a fewauthors (i.e. light of the rising sun induced spawning in C.helgolandicus: Mullin,1968), others did not (i.e. C.typicus: Valentin, 1972). These contradicting resultsrather suggest that some copepod species are probably 'discrete clutch' spawners

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(i.e. C.typicus: Valentin, 1972; Paracalanus: Uye and Shibuno, 1992; Temorastylifera: Ianora and Poulet, 1993; Acartia tumida: Hassett, 1993). However, inthese small copepods diel periodicity of egg production is unimodal. In contrast,large size copepods are 'intermittent' spawners (i.e. C.helgolandicus: Figure 2B;Mullin, 1968; C.finmarchicus: Marshall and Orr, 1952), characterized by bimodalegg production, strongly influenced by diel light cycle, or by a latitudinal lightregime.

Uye and Fleminger (1976) reported that Acartia tonsa eggs hatched normallyin complete darkness, as in C.helgolandicus (Figure IB). Photoperiod andtemperature act together to induce transformation of subitaneous to diapauseeggs in Labidocera aestiva (Marcus, 1980, 1982). The formation of diapause,resting and quiescent eggs in several copepod species belonging to theTemoridae, Centropagidae, Pontellidae, Acartidae and Tortanidae (e.g. Sazhina,1968; Grice and Gibson, 1975; Uye, 1985) remains a problem for estimating eggviability through the egg production method, since such eggs will not hatch in 24-72 h. In that case, alternative methods must be found to measure their viabilityrate. Dead eggs are easily differentiated from normal subitaneous or diapauseeggs, since they are generally disintegrating in a few hours (^72 h) or can beidentified using trypan blue, which specifically colours DNA of dead cells (Pouletet al., 1994b). For distinguishing diapausing eggs, other methods exist or must bedesigned (e.g. Lohner et al., 1990).

Hatching success is directly affected by male fecondation. Two cases areknown in copepods. Some species request only one fecondation (e.g.C.helgolandicus; Figure 4A), whereas in some others continuous remating isnecessary to maintain fertility (e.g. A.tonsa; Wilson and Parrish, 1971). In theabsence of males, females can lay oocytes (e.g. Watras, 1983; Williamson andButler, 1987; Ianora et al., 1994), which can be potentially confused with deadeggs. In that case, underestimation of hatching success is possible because thehatching rate cannot be monitored per se. However, specific colouration of maleand female pronuclei by Hoechst 33342 may confirm if eggs have been fertilized(Ianora et al., 1989).

Food level is generally considered as a limiting factor for egg production innature (i.e. Checkley, 1980; Hirche, 1989). Such an interaction applies in the longrun (e.g. day to week intervals; e.g. Bautista et al., 1994), but is not relevant tomeasurements of the daily instantaneous fecundity at a given site, obtained bythe egg production method.

Food quality, defined by the type of algae and their chemical nature, is alsoconsidered as a key factor determining the production and viability of eggs(Ianora and Poulet, 1993; Ianora et al., 1994; Kleppel, 1994; Poulet et al., 1994b;Laabir et al., 1995; Figure 8B). However, our results show that this parameter isnot important in short (24 h) term incubation (Figure 8A and B).

Oxygen concentration and salinity may interfere with the fecundity andhatching success of copepods. Extreme levels of both have been reported asbeing detrimental to their reproductive responses in relation to time or artificialconditions simulated under laboratory conditions (Uye and Fleminger, 1976;Lutz et al., 1992; Miliou, 1993). However, these parameters are not strictly

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relevant to the egg production method, since incubations of both females andeggs are necessarily conducted in seawater collected at the same time ascopepods, having salinity and oxygen levels to which they are acclimated and,thus, which are presumably compatible with normal functional responses.

Acknowledgement

This work was partially supported by PNOC, and by Roscoff and Naples MarineStations.

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Received on July 14, 1994; accepted on January 19, 1995

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