Growth and development of Calanus chilensis nauplii reared under laboratory conditions: testing the effects of temperature and food resources

Journal of Experimental Marine Biology and Ecology 294 (2003) 81 – 99 www.elsevier.com/locate/jembe

Growth and development of Calanus chilensis nauplii reared under laboratory conditions: testing the effects of temperature and food resources Cecilia G. Torres a, Ruben Escribano b,* a

Departamento de Oceanografı´a, Universidad de Concepcio´n, P.O. Box 160 C, Concepcio´n, Chile Oceanographic Center for the Eastern South Pacific (COPAS), Departamento de Oceanografı´a, Universidad de Concepcio´n, P.O. Box 42, Dichato, Concepcio´n, Chile

b

Received 22 March 2002; received in revised form 28 April 2003; accepted 23 May 2003

Abstract We assessed growth and development of naupliar stages of Calanus chilensis Brodsky 1959, under a combination of three temperatures and two food levels in laboratory conditions. Both food supply and temperature significantly affected naupliar growth and development. High food, measured as chlorophyll a (Chl a) concentration, was 40 Ag l
1, on average, and yielded temperature-dependent growth rates in the range of 0.13 – 0.17 day
1. Low food was about 1.2 Ag chlorophyll a l
1 and retarded or arrested development and drastically reduced the growth rate to the range of 0.05 – 0.09 day
1. To test whether these experimental results were consistent with field data, we used published information on temperature and chlorophyll a variability in northern Chile and developed a combined temperature/food-dependent model to diagnose naupliar growth in the field through a 2-year seasonal cycle including the 1997 – 1998 El Nin˜o conditions. We concluded that in the upwelling region off northern Chile, C. chilensis might seldom encounter conditions of food shortage, as those applied in the laboratory. Thus, naupliar growth of this species may be primarily controlled by environmental temperature and

* Corresponding author. Tel.: +56-41-683247; fax: +56-41-683902. E-mail addresses: [email protected] (C.G. Torres), [email protected] (R. Escribano). 0022-0981/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-0981(03)00261-2

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this might also be the case for the dynamics of the entire population inhabiting the coastal upwelling zone. D 2003 Elsevier B.V. All rights reserved. Keywords: Copepods; Food supply; Nauplii; Northern Chile; Temperature; Upwelling

1. Introduction Growth and development of marine copepods have been extensively studied in the field and laboratory. Studies have concluded that both physiological rates are strongly dependent on temperature (Huntley and Lopez, 1992) and food supply (Hirst and Lampitt, 1998). Several works have described how copepods grow and develop under the effects of varying temperature and food quantity and quality. However, the research has been mostly focused on copepodite stages, with very few parallel studies for the naupliar phase (Mullin, 1988; Hygum et al., 2000), despite the fact that nauplii are usually more abundant than copepodites in the field and that their success in the plankton will ultimately determine recruitment into the copepodite phase and, consequently, the population dynamics. In the coastal upwelling region off northern Chile, the endemic and very abundant copepod Calanus chilensis (Heinrich, 1973; Boyd et al., 1980) has received increasing attention lately. Studies on its population dynamics, however, have only dealt with copepodites and eggs and nauplii have seldom been considered (Escribano, 1998; Giraldo et al., 2002). This has limited reliable estimates of population growth and a clear understanding of the species life cycle (Escribano and McLaren, 1999). C. chilensis reproduce and grow year round at northern Chile, under apparently lack of food limitation (Escribano, 1998; Ulloa et al., 2001), such that copepodite growth might be primarily determined by temperature (Escribano and McLaren, 1999). In the laboratory, however, development and growth rates of copepodites C. chilensis become greatly reduced under low food (Escribano et al., 1997), suggesting that food supply in the field must remain sufficiently high to maintain maximal growth rates. This possibility has not yet been assessed for nauplii in this species. In other Calanus species, naupliar growth has been found to be less dependent on food supply compared to copepodite growth (Vidal, 1980; Hart, 1990; Hirst and Lampitt, 1998; Hygum et al., 2000). This could also be true for nauplii of C. chilensis, whose development may depend on temperature as well. Such possibility still needs to be examined. Nevertheless, because of their very small size ( < 0.5 mm) and usually fast moulting rates, in situ growth rates of nauplii are difficult to estimate. To solve this, laboratory experiments can be considered as a useful tool because they may provide insights on how individual growth responds to varying temperature/food regimes; this can help understanding naupliar growth in nature. Calbet et al. (2000) provided a method to study naupliar growth by estimating changes in body volume with a digital image analyser system. Using this method, we studied the growth and development of nauplii reared in the laboratory under controlled conditions of temperature and food. We aimed at determining the relative importance of temperature and food supply on regulating naupliar growth and development and further assess the

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hypothesis that in the rich upwelling region off northern Chile, nauplii may indeed develop into copepodites at temperature-dependent rates.

2. Methods 2.1. Experiments Three experiments were performed during August – September 2001, corresponding to the austral winter – spring conditions at the upwelling region off northern Chile. Zooplankton were captured in the Bay of Mejillones (23jS), which is a highly productive coastal embayment, under the influence of wind-driven coastal upwelling most of the year (Marı´n et al., 2001; Escribano, 1998). Zooplankton were captured near the center of the bay with a 0.5 opening diameter, 200-Am-mesh-size plankton net, which was vertically towed in the upper 60 m on several occasions. Zooplankton samples were immediately diluted with surface seawater, placed in coolers and transported to the laboratory within 2 h. Females C. chilensis were sorted under the microscope and maintained in filtered seawater at about 15 jC, until the experiments. We used a combination of three nominal temperatures, 10, 15 and 18 jC, and two levels of food quantity, measured as concentration of chlorophyll a (Chl a). The temperature range was that usually observed in the upper 50 m of Bay of Mejillones through seasons (Escribano and McLaren, 1999). As food, a mixture (in variable proportion) of microalgal cultures of Chaetoceros calcitrans (10 –20 Am) and Isochrysis galbana (3.3 –5.8 Am) was added until a greenwish color was attained. This high Chl a was assumed as excess of food. Low food level was obtained after 1/16 dilution of the high level. Algal cultures were used during their exponential phase of growth for all the experiments. The microalgae I. galbana have proven to be an adequate food for C. chilensis copepodites. This microalgae has been suscessfully used to grow nauplii and copepodites of this species in the laboratory (Escribano et al., 1998) and to induce egg production (Escribano et al., 1996). Chaetoceros spp. are common and abundant diatoms in the upwelling zone off northern Chile (Rodriguez et al., 1996). Chl a was measured by the spectrophotometric method (Parsons et al., 1992). For this, aliquots of the food media (100 –300 ml) were obtained, filtered in GF/C Whatman filter and pigments extracted in 90% acetone for 24 h. Pigment absorbance was measured at 665 and 750 nm and Chl a concentration calculated according to Greenberg et al. (1992). Table 1 summarises the experiments and associated conditions of food and temperature. The first experiment (Exp. 1) was carried out at a constant temperature of 10 jC, under excess of food. For this, 10 females were placed in each of four 1-l glass jars, which contained the food media at high level and a 450-Am sieve near the bottom to prevent eventual cannibalism. Females were then left undisturbed and after 24 h of incubation at 10 jC in a semidark cold room, they were removed and the jars were gently poured onto 37Am sieves and the spawned eggs collected in petri dishes. Using pasteur pippetes, the eggs were counted and transferred to 150 ml BOD glass-stopper bottles, which had previously been filled with 1 Am filtered seawater, aerated overnight and kept at the same temperature. BOD bottles were used because they allowed a rapid inspection of live nauplii, which could

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Table 1 Summary of experiments to assess growth and development rates of C. chilensis nauplii reared in the laboratory at three temperatures and under high and low food concentration Food level

Temperature

High

10 15 – 18 15 – 18 15 – 18

Low

Chl a(i)

Chl a(f)

Experiment

155.7 F 88.08 19.8 F 5.19 1.2 F 0.32

10.27 6.0 F 3.47 7.0 F 1.60 1.2 F 0.20

1 2 3 3

Food level was measured as chlorophyll a (Ag l
1). Chl a(i) is the mean (x F S.E.) of food concentration at every replacement of the media, and Chl a(f) is the mean concentration of food at the end of each experiment.

be seen through the glass under the microscope without opening the bottles; food media could be easily resuspended by turning the bottles; and also because of their small volume, desired temperature could be rapidly achieved. Caution was taken to avoid presence of bubbles, in which small nauplii can get caught. The initial number of eggs per BOD bottle was about 100, depending on their availability; however, their concentration never exceeded 900 egg l
1. A total number of five BOD bottles were obtained in these experiments. After 2 – 3 days, hatched nauplii could be observed through the glass. Thereafter, subsamples (10 –25 ml) were daily taken from the BOD bottles. The extracted volume from the BOD bottles was replaced with fresh filtered seawater. These subsamples were fixed with 5% formalin for later analysis. This experiment lasted about 2 weeks and spawned eggs came from females captured in two opportunities in Bay of Mejillones. Hatched nauplii were fed with 100% of I. galbana at the third day. The microalgae culture was added into the BOD bottles with small pipettes. Nauplii were thereafter fed at each daily observation. When larger nauplii (N4 – N6) could be observed, small amounts (f 25%) of the microalgae C. calcitrans were added along with I. galbana. Temperature was recorded every 12 h, at the time when the BOD bottles were also carefully rotated to allow algal become well mixed. A single measurement of Chl a was made during the experiment using the remaining food media after nauplii had been fed. A second experiment (Exp. 2) was performed 1 week later. In this case, two temperatures (15 and 18 jC) were used, under excess of food. Chl a was measured every 2 days, when fresh food media was prepared. Additional measurements of Chl a were made at the end of the experiment in the remaining media after all nauplii had been removed (Table 1). In this experiment, the eggs were initially incubated in the BOD bottles, which had already been filled with food media, containing only I. galbana. However, after 2 days, when nauplii stage N3 were observed in the subsamples, the food media was replaced by a mixture of 30% C. calcitrans and 70% I. galbana. For this experiment, a total number of four BOD bottles were obtained for each temperature containing about 100 eggs per bottle. Subsamples for nauplii were obtained every 12 h, removing 25 ml from one BOD bottle per treatment. The volume of the subsample was increased up to 100 ml in some cases, when too few nauplii were observed. At every removal of subsamples, the bottles were refilled with fresh food media. After 10 days, all remaining nauplii had been removed and the experiment was terminated. A third experiment (Exp. 3) used the same procedure as Exp. 2; however, in this case, the two food levels, high and low, were used. As in Exp. 2, Chl a was measured every 2

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days (both high and low food), in the freshly prepared media, as well as in the remaining media, at the end of each treatment. In this experiment, three BOD bottles, containing between 50 and 100 eggs bottle, were obtained for each combination of temperature and food. Subsamples for nauplii in this case were obtained every 24 h, removing 50 ml from one BOD bottle per each treatment. This volume was increased (up to100 ml) in some cases to obtain at least five nauplii per subsample. This experiment was finished when no nauplii were obtained in the subsamples (after 10 days). Preserved samples from the three experiments were analysed 1 month later. The naupliar stages were identified and counted under the microscope to examine the stage frequency through time, allowing estimations of naupliar development times. Naupliar growth was studied by analysing daily changes in naupliar volume, regardless of developmental stage. The naupliar volume was estimated using a microscope (40  magnification) equipped with a digital camera and connected to an image analyser system. Digital images of nauplii were used to calculate naupliar volume, according to Calbet et al. (2000), such that, 4 w V ¼ A ð1Þ 3 2 where V = volume (Am3), A = naupliar area (Am2) in a dorsal view, w = naupliar width (Am). 2.2. Data analysis Naupliar growth was described by an exponential model, such that, V ¼ Vi egt

ð2Þ

where Vi = initial volume (Am3), g = volume-specific instantaneous growth rate (day
1), and t = time (day). From this Eq. (2), g between observations was estimated as, g ¼ ln ðViþ1 =Vi Þ=t

ð3Þ

where Vi + 1 and Vi are naupliar volumes (Am3) between two subsequent observations and t the time interval (day) between observations. Development rates were evaluated for two time intervals during the naupliar phase: (1) the duration between egg laying and first appearance of nauplii stage N3 (tegg – tN3), and (2) the time at initial stage N3 and the time of first appearance of stage N6. The former was assumed as purely temperature-dependent because nauplii are just starting to feed at this stage N3, and the latter assumed as depending on both temperature and food conditions. The time at egg laying (tegg) was assumed as the median time between two subsequent observations during the incubation period of females (f 24 h) when they had laid eggs. Time of initial stages N3 and N6 was assumed as the time when they first appeared in the daily, or twice a day samples. Temperature-dependent development times were described using the Beˇlehra´dek equation (McLaren, 1995) as, D ¼ aðT þ aÞ
2:05

ð3Þ

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where D = development time (day), T = temperature (jC), a and a are parameters that define the size dependence and the temperature adaptation of development, respectively. We used a fixed value of 2.05 as the exponent, which can be assumed as constant among copepod species (McLaren, 1995). Beˇlehra´dek equation for embryonic time of C. chilensis was determined by Escribano et al. (1998) as, D ¼ 947:7 ðT þ 11:0Þ
2:05

ð4Þ

Eq. (4) may be used to estimate temperature-dependent time of later stages assuming the equiproportional rule of development (Corkett et al., 1986), which states that each stage occupies a fixed proportion of the total development time. Estimated proportions at a given temperature can thus be used to calculate stage durations at any other temperature. We used this method to calculate the parameter a of Eq. (4) and hence obtain temperaturedependent equations for durations of the phases egg– N3 and egg– N6. Temperature –food effects on naupliar size were tested by ANOVA, whenever homogeneous variance among treatments could be demonstrated by Barlett’s test, or when this became homogeneous after log transformation. Curve fittings were performed by nonlinear estimates of parameters using the quasi-Newton algorithm (Wilkinson, 1990). Parameters comparisons were made using the Student’s t-test.

3. Results 3.1. Temperature and food conditions Controlled temperatures varied in narrow ranges in the cold room (mean F S.D.): 10.3 jC F 0.65, 14.95 jC F 0.30 and 17.95 jC F 0.10. Nauplii were fed with considerably high amounts of food in terms of Chl a (Table 1). Concentration of Chl a also showed sharp decreases after 2 days in treatments of high food levels, suggesting that nauplii were actively feeding (Table 1). For all treatments under high food levels, the Chl a concentration was, on average, more than 6 Ag l
1 whereas low food level never exceeded 1.8 Ag l
1. The low food level may represent an extreme condition of low phytoplankton Table 2 Proportion of total C. chilensis nauplii obtained after incubation of eggs under five different combinations of temperature  food treatments Treatment

Proportion (%)

Temperature (jC)

Food level

10 15

High High Low High Low

18

15.5 21.7 91.0 47.3 76.0

This proportion represents the eggs that successfully hatched and survived until the end of each experiment from the total of eggs initially incubated.

Food resource

Volume (  105 mm3) Egg

N1

N2

N3

N4

10

High

15

High Low

18

High

1.73 F 0.507 (24) 1.66 F 0.313 (22) 1.67 F 0.376 (5) 2.24 F 0.572 (8)

18

Low

1.18 F 0.103 (12) 1.22 F 0.234 (13) 1.11 F 0.073 (15) 1.31 F 0.225 (9) 1.19 F 0.098 (11)

3.15 F 0.555 (19) 2.84 F 0.648 (55) 2.80 F 0.406 (87) 2.89 F 0.637 (81) 2.94 F 0.578 (51)

6.44 F 1.466 (5) 6.90 F 1.441 (59)

15

1.85 F 0.276 (20) 2.05 F 0.220 (59) 1.91 F 0.207 (3) 2.09 F 0.313 (6) 1.85 (1)

Temperature

N5

N6

n 80

9.05 F 0.801 (12)

11.34 F 0.597 (2)

222 110

6.70 F 1.667 (66) 4.68 F 0.730 (14)

9.88 F 2.062 (38)

Nauplii stages are N1 through N6. Numbers in parentheses are sample sizes, and n is total number of eggs and nauplii per treatment.

12.63 F 2.582 (12)

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Table 3 Estimates of egg and naupliar volumes (mean F S.D.) of C. chilensis reared in laboratory conditions under two food levels (high and low) and at three temperatures

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biomass in Bay of Mejillones in terms of Chl a (Escribano, 1998). The total number of nauplii analysed for each experimental treatment depended on the initial number of incubated eggs, on the proportion of egg that hatched and on naupliar mortality. From the initial number of incubated eggs per treatment and the total number of sampled nauplii thereafter, an estimate of the proportion of eggs that hatched, turned into nauplii and

Fig. 1. Growth in body volume of C. chilensis nauplii reared in the laboratory under three temperatures and two food levels. The curves are nonlinear fittings of exponential model of growth. High food was assumed as excess of food, measured as concentration of chlorophyll a, and low food was 1/16 of the high one.

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Table 4 Parameter estimates after nonlinear regression of growth in body volume of C. chilensis nauplii reared in the laboratory at three temperatures and two levels of food T (jC)

10 15 18

Food level

Vi (Am3  105)

SE

High High Low High Low

1.2 1.5 1.9 2.8 1.8

0.11 0.13 0.10 0.24 0.15

g(d
1)

0.13 0.17 0.05 0.14 0.09

SE

95% Confidence limits

0.011 0.011 0.007 0.012 0.012

Lower

Upper

0.103 0.150 0.039 0.111 0.070

0.147 0.193 0.068 0.160 0.118

Vi = estimate of initial volume (Am3  105), g = volume-specific growth rate (d
1) and SE = asyntotic standard errors.

survived until sampling is shown in Table 2. These proportions are significantly different among treatments ( G test, v2 = 144.1, p < 0.001), suggesting treatment-dependent mortalities. It should be noted that lower survival was obtained at high food, and it is difficult to establish which factor (temperature or food) caused more mortality. 3.2. Naupliar growth Although naupliar volume was estimated independently from stages, they were actually identified for subsequent analysis of temperature/food effects on development. However, not all the treatments allowed complete development of naupliar stages. In fact, the final nauplii stage N6 was only reached at 15 and 18 jC under high food. The low-temperature (10 jC)/high food condition allowed development only up to stage N4 despite high food. The number of eggs and nauplii available to obtain volume measurements was also variable, depending on treatment. Table 3 summarises sample sizes and mean estimates of naupliar volume per treatment. Temperature effects on naupliar volume can be examined by comparing the treatments under which nauplii developed up to stage N3, assuming that potential food effects are not yet noticeable, as they are just starting to feed (Marshall and Orr, 1955). One-way analysis of variance (ANOVA) showed that naupliar volume of stages N2 and N3 are significantly affected by temperature ( F2,56 = 4.88, p < 0.05 and F2,29 = 3.32, p < 0.05, respectively). Table 3 shows that mean body volume of N3 cultured under low temperature (10 jC) is greater than that obtained for N3 reared under 15 and 18 jC. Food effects on naupliar Table 5 Values of Student’s t-test to compare growth rates ( g) of nauplii C. chilensis reared in the laboratory under three temperatures and two food levels (high food = HF and low food = LF)

10j 15j 15j 18j

HF HF LF HF

15j HF

15j LF

18j HF

18j LF

2.09**

4.00** 6.56**

0.48 1.52 4.37**

1.35 3.35** 2.16** 1.75

Values of Student’s t-test are at 5% level of significance (**) and infinite degrees of freedom.

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volume could only be tested by comparing stages N4 reared under high and low food at 18 jC. In this case, body volume of N4 was significantly reduced (Table 3) under the low food condition ( F1,140 = 26.10, p < 0.001).

Fig. 2. Daily changes in the volume-specific growth rate ( g) of C. chilensis nauplii reared in the laboratory under three temperatures and two food levels. g was estimated from the exponential model of growth between subsequent daily observations. High food was assumed as excess of food, measured as concentration of chlorophyll a, and low food was 1/16 of the high one.

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Fig. 3. Daily changes in relative abundance of naupliar stages of C. chilensis reared in the laboratory under three temperatures and two food levels. High food was assumed as excess of food, measured as concentration of chlorophyll a, and low food was 1/16 of the high one.

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When no nauplii could be observed in the samples, the experiments were terminated. The experiments lasted between 10 and 11 days and this time was similar for all treatments. Growth patterns, as shown by changes in body volume and fitted curves, are illustrated in Fig. 1. Food effects on naupliar growth seem clear when comparing the high and low food at 15 and 18 jC. The fitted exponential models, judging by the determination coefficients (r2), are all significant ( p < 0.05). Estimated rates of growth ( g) and their associated statistics are shown in Table 4. Confidence limits (95%) of g indicate not only potential effects of food but also a temperature influence. There are significant differences in g between temperatures and between food treatments although the most remarkable difference is between high and low food at 15 jC (Table 5), suggesting that at least at this temperature low food substantially reduced g. Daily changes in g between consecutive observations for each treatment are shown in Fig. 2. The growth rate, g, was more variable under high food condition, except for the low temperature of 10 jC. Fig. 2 suggests a tendency of g to decrease during the first 3– 4 days, becoming negative in some cases, but overall means of g were all positive and greater than 0.08 day
1 at all treatments. 3.3. Naupliar development Attempts to estimate duration of single stages were made from changes in relative abundance of stages through time. However, the lack of synchrony in moulting times, as shown by Fig. 3, precluded the possibility of obtaining reliable estimates. In C. chilensis under laboratory conditions, the lack of synchrony of development is even present in the hatching time of eggs spawned in a single clutch within females (Escribano et al., 1998). We thought that the partition of total naupliar time into two phases, egg –N3 and N3 –N6, may help reduce individual variation. The proportion of time occupied by each phase at a given treatment was also estimated as well as the parameter a of the Beˇlehra´dek equation (Table 6). These estimates should be considered as temperature-dependent because they were all obtained under conditions of high food. Using the mean value of the parameter a

Table 6 Duration (day) of the development phases egg to N3 (tegg – tN3), N3 to N6 (tN3 – tN6) and egg to N6 (tegg – tN6) of naupliar stages of C. chilensis, reared in the laboratory under three temperatures and excess of food Developmental phase tegg – tN3 tN3 – tN6 tegg – tN6

a (egg – N6) a (egg – N3)

Temperature (jC) 10

15 4.24 6.91 11.15

5728 2179

Mean time proportion

18 2.58 6.00 8.58

6826 2053

2.56 3.00 5.56

5533 2548

0.38 0.62 1.00 Mean a 6029 2660

Durations are estimates of stage initial times. The value tN3 – tN6 at 10 jC was not available from the experiments, but was estimated using the mean proportion of time from the other temperatures, according to the equiproportional rule (Corkett et al., 1986). The parameter a is that derived for the Beˇlehra´dek equation.

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from Table 6 and Eq. (4), development time (D) between egg and N6, as a function of temperature (T), can be described as, D ¼ 6029 ðT þ 11:0Þ
2:05 ;

ð5Þ

Eq. (5) may thus be used to examine temperature-dependent development of nauplii and combined with Eq. (2), the growth rate ( g) could be estimated for field conditions provided some measurements of egg and N6 weights, carbon content or body volume as in this study.

4. Discussion Changes in body volume of copepod nauplii primarily occur at each moulting so that growth should be considered as discrete. However, the incorporation of new somatic tissue takes place continuously through development, giving rise to increases in size (biovolume), even within stages, as reflected in changes in naupliar volume from initial stage to end stage (Calbet et al., 2000). Therefore, although body volume may not necessarily represent body weight, its relative changes, both within and between stages, allowed us to examine the growth pattern and estimate a volume-specific growth rate, which can be considered as an index of the mass-specific growth rate. Indeed, our estimates of naupliar growth ( g), in the range of 0.05 – 0.17 day
1, do not considerably differ from g estimated for copepodids as 0.114 day
1 at 15 jC, under excess of food (Escribano et al., 1998). Furthermore, g did not show abrupt changes during the experiments (Fig. 2). These results suggested that growth was rather uniform throughout naupliar development. Stability of g, however, was accompanied by a large variation in moulting times, possibly originated from between and within treatment variation in egg development. Females were incubated for 24 h, but egg spawning did not occur before 12 h of incubation, possibly after acclimation to conditions and feeding have taken place. Females in C. chilensis also produce eggs in small groups, at variable times within a few hours (Escribano et al., 1996, 1998). Thus, high variability of egg development may even occur within clutches (Escribano et al., 1996, 1998). This might give rise to lack of synchrony in naupliar development, precluding the possibility of obtaining reliable estimates of mid-stage moulting times as in other studies with different Calanus species (e.g. Peterson, 1986). The estimate of average moulting times may not represent this individual variation either. Thus, in order to make estimates for temperature-dependent development rates, we suggest that initial stage times, assumed as the time for stage first appearance, may be a recommended approach when dealing with continuously reproducing species. As shown in a previous study (Escribano et al., 1997), both growth and development rates of copepodids C. chilensis are highly sensitive to low food supply when reared in the laboratory. Naupliar stages seem to respond similarly. Low food not only reduced the growth rate but also retarded or even arrested naupliar development at stage N4; this was independent of temperature. As at 10 jC, development stopped at N4 despite high food, we think this temperature might be too low for C. chilensis development because in the field, individuals tend to aggregate in the upper 20-m layer (Escribano et al., 2001), in

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which they may not experience temperatures below 12 jC (Escribano and Hidalgo, 2000). Furthermore, the parameter a of the Bı`lehra´dek equation has been estimated as 11.0 (Eq. (3)), suggesting that the population at northern Chile is mostly adapted for developing at temperatures higher than 11 jC. This might not be the case for populations of the species distributed at coastal areas of Central/South of Chile (Marı´n et al., 1994). In this region, temperatures are colder and it is likely that the temperature adaptation parameter may be lower than 11 jC. This possibility, however, has not yet been assessed. Phytoplankton seems to be the main food item for C. chilensis in northern Chile (Boyd et al., 1980). The species was also successfully reared from egg to adult using mixtures of phytoplankton in the laboratory (Escribano et al., 1997). The microalgae used in these experiments, however, may not fully represent food resources available in the field, which may comprise a rich variety of diatoms, flagellates and microheterotrophs in this area (Gonza´lez et al., 2000). Also, pigment concentration (measured as Chl a) should only be considered as an index of food quantity. The actual organic carbon available as food may greatly vary with Chl a. In fact, the carbon/Chl a ratio of diatoms is known to range widely from 30 to 150, depending on culturing conditions (Omory and Ikeda, 1984). This means that our low food condition could have been in the range of 0.03 –0.18 mg C l
1, whereas the high food (about 40 Ag Chl a l
1 on average) exceeded 1.2 mg C l
1; that is, high food was at least one order of magnitude higher than low food. We do not know if this carbon content is sufficient (high food level) to allow maximal development rates of C. chilensis nauplii; however, using Eq. (4), the estimated development time between egg and N6 is about 7.58 days at 15 jC. This value does not differ from 7.4 days estimated by Escribano et al. (1998), who used a mixture of natural phytoplankton as food. This suggests that the applied food media, at least under high food, could have been adequate to allow growth at temperature-dependent rates. The drastic effects of low food on development and growth of C. chilensis nauplii contrast with experimental results in nauplii of Calanus finmarchicus (Hygum et al., 2000), in which nauplii were shown to obtain nearly maximal development rates at low food (f 1 Ag Chl a l
1, or 0.2 mg C l
1). However, C. finmarchicus may not necessarily respond like C. chilensis to low food. C. finmarchicus is a high-latitude species, subjected to strong seasonaliy in food supply, such that at times, individuals must cope with extremely low food, being forced to use alternate food resources such as microzooplankton (Ohman and Runge, 1994). On the other hand, C. chilensis, exhibiting continuous reproduction throughout the year, may not be adapted to withstand low food because such condition is seldom found, at least in coastal waters off northern Chile (Escribano, 1998; Ulloa et al., 2001), with very high year-round primary production rates (Daneri et al., 2000). Nevertheless, food resources, in terms of phytoplankton biomass, and also water temperature may show much spatial and temporal variability in the upwelling zone off northern Chile (Thomas et al., 2001) in association with the spatial structure of cold upwelled waters (Giraldo et al., 2002). In order to assess how this variability in food resources and temperature may influence growth of C. chilensis nauplii in the field, we made use of a time series of temperature and Chl a concentration obtained between June 1996 and January 1998 off Mejillones (23jS) at a fixed station (Hidalgo and Escribano, 2001). We chose data from 10 m because this depth seems to represent well the actual

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habitat of this species (Escribano and McLaren, 1999). We assumed that under nonlimiting conditions of food and combining Eqs. (2) and (4), maximal temperature-dependent growth rate of naupliar stages may be expressed as, gmax ¼ ½ln ðSN6 =Segg Þ=½6029ð11:0 þ T Þ
2:05 

ð6Þ

where gmax = temperature-dependent growth rate (day
1); SN6 and Segg are sizes (weight, carbon, or volume) of naupliar stage N6 and egg, respectively; and T is temperature (jC). An equivalent equation to describe g as a function of food concentration has not been yet developed. However, we may approximate one using the half-saturation model, as the one used by Mullin et al. (1975) to describe the ingestion rate as a function of food concentration as follows, g ¼ gmax ðC
C0 Þ=½k þ ðC
C0 Þ

ð7Þ

where g = size-specific growth rate (day
1), C = food resource concentration, C0 = parameter to define a threshold of food concentration to initiate feeding (Parsons et al., 1967), and k = the half-saturation food concentration, equivalent to a half of the concentration to attain gmax. If we substitute gmax of Eq. (6) into Eq. (7), the combined dependence of g on temperature and food can be expressed as, g ¼ ½ln ðSN6 =Segg Þ=ð½6029ð11:0 þ T Þ
2:05 ½ðC
C0 Þ=ðk þ ðC
C0 ÞÞÞ

ð8Þ

Using Eq. (8), we can now examine how g responds to Chl a concentration and temperature in the field. To do that, we first need to make estimates of k and C0. Escribano et al. (1997) found that copepodids C. chilensis retarded their development at Chl a concentration lower than 2.5 Ag l
1. In our experiments with nauplii, the low food never exceeded 1.8 Ag Chl a l
1 although the mean value was about 1.2 Ag l
1. It seems that below 2.5 Ag Chl a l
1, development is retarded so that we used this value as a critical concentration needed to achieve gmax. The threshold parameter (C0) indicates the minimal concentration required for initiating feeding and might not considerably affect g so that we arbitrarily assume a value of 0.5 Ag Chl a l
1. As size measurements of eggs and N6, we used the overall means of volumes from the experiments, estimated as 2.0  105 and 12.0  105 Am3 for egg and N6, respectively. Potential body size variations of N6 and perhaps of eggs in the field is certainly a shortcoming to fully account for temporal changes in g and this may need further exploration. The time series of temperature and Chl a obtained at time intervals of about 15 days (Hidalgo and Escribano, 2001) included conditions before and during the 1997 –1998 El Nin˜o, which caused abnormally warm waters in the coastal area (Ulloa et al., 2001). This can be seen in the plot of temperature at 10-m depth (Fig. 4a). During the entire period, Chl a exhibited much variability in the range of 1.0 Ag Chl a l
1 in June 1996 to about 16 Ag l
1 in May 1997 (Fig. 4b). The combined temperature/food-dependent growth rate ( g) estimated with Eq. (8) and maximal temperature-dependent gmax with Eq. (6) are shown in Fig. 4c. Although gmax always appears higher than g due to the dependence upon Chl a concentration, it seems that both rates ( gmax and g) follow the pattern of temperature and

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Fig. 4. Expected seasonal variation in the temperature-dependent ( gmax) and temperature/food-dependent ( g) volume-specific growth rate of C. chilensis nauplii in Bay of Mejillones, northern Chile (upper panel); seasonal changes in temperature at 10-m depth (mid panel); and concentration of chlorophyll a at 10-m depth (lower panel). The shaded area represents the 1997 – 1998 El Nin˜o conditions at northern Chile.

they even show a positive trend with increasing warming during El Nin˜o period (Fig. 4c). In fact, Ulloa et al. (2001) showed that C. chilensis increased in numbers during El Nin˜o period, suggesting an acceleration of development and growth rates upon elevated temperatures, in the absence of food shortage. In any case, it became clear that temperature explained much variability of g, accounting for a 44% of total variance, as compared to Chl a concentration, which accounted for 26% (Fig. 5). Thus, growth of nauplii seems primarily determined by temperature. In coastal upwelling systems, copepods are multivoltine species, with 10 or more generations a year, which can be sustained mostly because food may be high-throughout seasons (Peterson, 1998). Upwelling zones are also advective habitats and circulation may

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Fig. 5. The relationship between the expected volume-specific growth rate of C. chilensis nauplii in Bay of Mejillones, northern Chile, and the temperature and chlorophyll a concentration at 10-m depth. The growth rate was estimated as a function of temperature and chlorophyll a. Linear regressions and r2 illustrate the relative importance of temperature and chlorophyll a quantity on the growth rate. Dotted lines are 95% confidence limits of the regression lines.

impose physical forces with which copepods must deal to maintain their populations nearshore. Peterson (1998) has suggested that such forces may be more important than food supply and temperature for population dynamics of copepods in upwelling systems. Off northern Chile, upwelling circulation may indeed affect abundance, distribution and population growth of C. chilensis (Escribano et al., 2001; Giraldo et al., 2002). However, our findings suggest that naupliar growth, which directly determine copepodid recruitment, is mostly governed by temperature variability. Furthermore, the direct effect of temperature on seasonal abundance (Escribano and Hidalgo, 2000), secondary production (Escribano and McLaren, 1999), population size and body size (Ulloa et al., 2001) as well

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as on individual traits, such as reproductive state, lipid content, body weight and length (Giraldo et al., 2002), altogether may be considered as sufficient evidence to support the view that temperature is the driving force of population dynamics of C. chilensis at the upwelling region off north Chile.

Acknowledgements This work has been funded by FONDECYT-Chile, grant 199-0470. Partial support has also been provided by the Oceanographic Center for the Eastern South Pacific (COPAS), funded by FONDAP-CONICYT of Chile. We thank P. Hidalgo, J. Rodriguez and D. Ferna´ndez for helping during sampling and with the experiments. Valuable comments from two anonymous reviewers have greatly helped clarify ideas and improve the work. [RW]

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