Development, growth, and egg production of Neocalanus flemingeri in the eastern subaractic pacific: a synthesis of laboratory and field approcaches Hui. Liu, Laura M. Slater, Cheryl Clarke, and Russell R. Hopcroft Institute of Marine Science, University of Alaska Fairbanks Fairbanks, AK USA Abstract: Neocalanus flemingeri is one of the dominant copepods in the subarctic Pacific, yet there are few estimates of the rates at which many processes occur during its life-cycle. Growth and development of N. flemingeri CI to CIV copepodites were determined in the field by both the artificial cohort method, and the incubation of single stages, each March, April and May over 3 years at ~5°C. Development, growth, and egg production were also determined at 5°C in the laboratory at saturating food concentrations. In the field, CI to CIV stage durations ranged from 7 to more than 100 days, dependent on chlorophyll concentration, with the duration of each stage ~10 days under optimal conditions. Stage durations varied from 5-24 days for NI to NVI, and 6-15 days for CI to CIV in the laboratory, with development time from egg to CV of 117 days. Weight-specific growth rates ranged from 0.23 d -1 to close to zero in the field, but was typically between 0.20 and 0.08 d -1 under more optimal conditions. This compares favorably to 0.07 to 0.27 d -1 obtained in the laboratory. In both cases, growth rate typically decreased with increasing stage. Fecundity of N. flemingeri was 534 ± 43 (mean ± S.E.) eggs female -1, representing ~70% of the female’s initial weight. Results are compared to rates of other copepods determined concurrently in this ecosystem. Introduction: Of the ~15 common species of copepods in the Gulf of Alaska (GoA), the three Neocalanus species (N. plumchrus, N. flemingeri, N. cristatus) frequently dominate the zooplankton community biomass over the entire spring. Their abundance and large size make them important prey species for higher trophic levels. Although we have an overall picture of the life cycles of the large-bodied copepods in the Northern Pacific (Mackas & Tsuda, 1999), the details are largely inferred. Despite the presumed importance of Neocalanus, there are only three estimates of development rate and one for growth rate in copepodites, and only two studies of egg production or naupliar development. Here we present results to address this deficiency for the copepodites of N. flemingeri with field experimental results from the 2001,2002 and 2003 spring seasons and that from the laboratory. Discussion: Field estimated stage duration for the earlier copepodites appear surprisingly consistent. Our lab-determined values of days (Table 1) were comparable to durations of days estimated by Miller (1993) from examination of natural field cohorts. The only directly determined stage durations of days for CIII & CIV copepodites (Miller and Nielsen, 1988) is also within the range observed here, although even longer stage duration appears common for CIV during lipid accumulation. The more rapid development of CIV in May 2002 corresponds to highest chlorophyll concentrations in the larger phytoplankton (Fig.3). Based on the field results presented here, the first 4 copepodite stages would be completed in days assuming conditions remained comparable to those experienced during these incubations, consistent with the lab estimate of 44 days (Table 1). Saito & Tsuda (2000) estimated the duration of naupliar stages for N. cristatus to be days at 4°C. Our lab values for N. femingeri was 56 days, close to the 55 days for N. plumchrus (Mackas and Tsuda, 1999). Our lab estimated development time from eggs lain to CV was 117 days. For growth rates at earlier stages, our field and lab results matched very well with a similar pattern of decreased growth with increased stage. Vidal & Smith (1986) estimated the growth rate of a mixture of N. plumchrus and N. femingeri at CI to CIV in the Bering Sea ranging between d -1, similar to our result for CII and CIII, but below our estimates for CI. For CIV the lab determined estimate was more similar to 0.05 d -1 estimated by Miller and Nielsen (1988). Field results by both of picked method and artificial cohort method were consistent and comparable, validating the application of the artificial cohort technique for the study of larger copepods in subarctic Pacific. The estimates for N. femingeri were consistent with that of others species in this area; average growth rate of 0.17 d -1 for Centropages abdominalis (Slater and Hopcroft, in review), in situ specific egg production rate of 0.12 and 0.09 d -1 for Pseudocalanus newmani and P. mimus respectively (Hopcroft, unpublished data) and in situ specific egg production rate of 0.10 and 0.11 d -1 for Metridia pacifica and M. okhotensis respectively (Hopcroft et al, in press). Further analysis to couple field and lab estimates for growth with food resource and in situ temperature is underway. Fig.4,5.6 (above, top-down) results of field experiments in 2001, 2002 and 2003, stage duration(upper panels) and growth rates (lower panels) of N. flemingeri in the northern Gulf of Alaska verses initial developmental stage. Results from single stage populations (bars) and artificial cohorts (circles) referenced to the ‘average’ copepodite stage present at the start of the experiment. Fig.3. Distribution of size-fractionated chlorophyll for the northern Gulf of Alaska, References: Hopcroft, R.R., Pinchuk, A.I., Byrd, A. & Clarke, C. (2004) The paradox of Metridia spp. egg production rates: A new technique and measurements from the coastal Gulf of Alaska. Mar. Ecol. Prog. Ser., in press. Kiorboe, T. & Sabatini, M. (1994) Reproductive and life cycle strategies in egg-carrying cyclopoid and free-spawning calanoid copepods. J. Plankton Res., 16, Liang, D., Uye, S. and Onbe, T. (1994) Production and loss of eggs in the calanoid copepod Centropages abdominalis Sato in Fukuyama Harbor, the Inland Sea of Japan. Bull. Plankton Soc. Jap., 41, Mackas, D.L. & A. Tsuda Mesozooplankton in the eastern and western subarctic Pacific: community structure, seasonal life histories, and interannual variability. Prog. Oceanogr. 43: Miller, C.B Development of large copepods during spring in the Gulf of Alaska. Prog. Oceanogr. 32: Miller, C.B. & R.D. Nielsen Development and growth of large, calanid copepods in the ocean subarctic Pacific, May Prog. Oceanogr. 20: Runge, J.A. & Roff, J.C. (2000) The measurement of growth and reproductive rates. pp In: Harris, R.P., Weibe, P.H., Lenz, J., Skjoldal, H.R. and Huntley, M. (ed.) ICES Zooplankton Methodology Manual, Academic Press, London. Saito, H. & A. Tsuda Egg production and early development of the subarctic copepods Neocalanus cristatus, N. plumchrus and N. flemingeri. Deep-Sea Res. I. 47: Slater, L.M. & R.R. Hopcroft (in press) Development, growth, and egg production of Centropages abdominalis in the subarctic Pacific. J. Plankton Res. Gomez-Gutierrez, J. and Peterson, W. T. (1999) Egg production rates of eight calanoid copepod species during summer 1997 off Newport, Oregon, USA. J. Plankton Res., 21, Vidal, J. and Smith, S. L. (1986) Biomass, growth and development of populations of herbivorous zooplankton in the southeastern Bering Sea during spring. Deep-Sea Res., 33, Results: Three years field experiments showed that both the artificial cohorts method and picked-stage method have similar pattern (Fig.4,5,6), although the artificial cohort method appears to produce more variable results. In March, only the first two copepodite stages are present, but stage duration is long and growth rate slow ( d -1 ). In April, the first 4 copepodite stages are common, with the first 3 stages being of similar duration (~10 days), but CIV generally develops more slowly while accumulating lipid stores. During April, growth rate appears to decline with increasing stage. In May, only CIV and CV are common, but CIV growth is generally more rapid than experienced during the previous month. Again, we were unable to observe molting or growth in CV copepodites, due to that stage’s long duration of lipid accumulation prior to diapause. Overall, in situ estimated growth rate fall between ~ d -1 during spring season in this area. Fig.8. Daily cumulative percent at stage of N. flemingeri nauplii and early copepodite stages reared in the laboratory at 5°C. Solid lines show statistically significant linear regressions (P<0.01). Dashed line indicates a change in methodology from population sub sample to non-destructive population census. Fig.1.Sampling area. Typical experimental sites indicated in large red dots. Fig.7 Fecundity of N. flemingeri females reared in the laboratory at 5°C. Some first clutches may contain more than one clutch. Fig.2 Relationship between prosome length (PL, mm) and dry weight (DW, mg) for N. flemingeri CI to CV (depicted by different symbols). Table 1 Median stage durations and instantaneous growth rates (g) of N. flemingeri reared in the laboratory at 5°C Method: Field experiments were executed at stations GAK1, 4, 9, 13 and Prince William Sound (PWS) ( Fig.1). Copepods were collected from the upper 50 m with a 64 µm net and sorted into size classes of “artificial cohorts” by serial passage through mesh sizes from 1300 µm down to 400 µm. Half of each fraction was preserved immediately as the time zero, and the remainder equally divided among several 20L carboys filled with µm prescreened seawater. Carboys were incubated on-deck in large tubs at sea surface temperatures with ship movement providing constant ‘mixing’ of the carboys. After 5 days, carboys were screened onto a 45 µm mesh, pooled by size fraction, and preserved. Parallel experiments were executed simultaneous for N. flemingeri by picking single stages of copepodites from an additional net and incubating under the same conditions. Later, in the lab, copepods were identified to species, staged and the prosome lengths (PL-mm) were measured. The progression of the cohort was determined by changes in the mean size. The dry weights (DW-mg) were predicted from the relationship: Log(DW)=3.56*Log(PL)-2.32 (Fig.2). The calculation of in situ weight–specific growth rate was based on the g =(InDW t - InDW 0 ) t -1. Copepods for lab experiment were collected from m deep in PWS December, 2002 using a 100 µm MOCNESS net. For individual fecundity estimates, 35 females were placed individually into 1 L PETE bottles, checked every 3-4 days for eggs and preserved once spent. Females that failed to produce eggs or died prior to “spawning-out” were removed from analysis. Egg weight was calculated as 0.69 µg DW based on diameter (Kiørboe and Sabatini, 1994). An additional 10,000 eggs were harvested from ~100 females, and incubated at 5°C in multiple 9L flasks at ~70 eggs L -1. Containers were subsampled daily to monitor stage progression. Once hatched they were maintained on cultures of Isochrysis sp., Pavlova lutheri, and Chromonas sp. Naupliar mortality was high, so by CI, experiments required restarting from ~350 field-collected CIs incubated individually in 70 ml flasks checked daily for stage. After molting to CIII, they were transferred to 270 ml flasks. Observations stopped after molt to CV, because the stage is long-lived and development to adulthood occurs during N. flemingeri’s descent from the surface layer (Miller and Clemons, 1988) and we could not mimic such conditions in the laboratory. Stage duration was calculated as the median number of days between stages. Instantaneous growth rates (g) were calculated from the predicted dry weight (DW) of the mean length for each copepodite stage (i) and that stage’s duration (D i ): g i = (lnDW i+1 - lnDW i ) D i -1 (Runge and Roff, 2000). Fecundity of N. flemingeri was high with 535 ± 258 female -1 (mean ± S.D.) and a maximum of 1036 female -1. There were up to 4 clutches of eggs could be identified with an average clutch size of 225 ± 154 eggs (mean ± S.D.), and a trend of decreasing clutch size with successive clutches (Fig.7). Egg production appears to represent 70% of the females initial body weight. Egg hatching was nearly synchronous with over 90% of the eggs hatching between Day 2 and 3. Development of stages was also relatively synchronous and least-squares regressions of CPS were significant (P<0.005) (Fig.8). The estimated median development time from eggs lain to copepodite CV was about 117 days. Lab estimated weight-specific growth rates of CI to CIV averaged 0.15 d -1, and decreased with stage from 0.28 to 0.07 d -1 (Table 1).