Kam W.Tang, Hans G.Dam and Leah R.Feinberg Department of Marine Sciences, University of Connecticut, Groton, CT06340, USA

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1 Journal of Plankton Research Vol.20 no.0 pp , 998 The relative importance of egg production rate, hatching success, hatching duration and egg sinking in population recruitment of two species of marine copepods Kam W.Tang, Hans G.Dam and Leah R.Feinberg Department of Marine Sciences, University of Connecticut, Groton, CT06340, USA Abstract. The disappearance of spawned copepod eggs can, at times, approach 00% day-' and may be a bottleneck to population recruitment of marine copepods. We examined the egg production rate and egg hatching success of Centropages hamatus and Temora longicomis (Copepoda: Calanoida) on natural diets, and the role of delayed hatching combined with high sinking rates in removing their eggs from the water column. Cumulative hatching success within IS days was consistently high from March to June 996: -80% for Chamatus and 70% for T.longicornis. Minimum hatching time for C.hamatus was S days in winter and -4 days in spring. Minimum hatching time for T.longicornis was days in winter and -2.S days in spring. We measured the diameter and density of eggs of both species to estimate sinking rates from Stokes' law. Sinking rates ranged from 4.6 to 22.4 m day for Chamatus and from 3.4 to 2S.4 m day~' for T.longicornis. In shallow waters like Long Island Sound (S20 m), in most cases virtually all the eggs may sink out of the water column before hatching starts. Therefore, we suggest that delayed hatching combined with a high sinking rate can be a significant cause for the high apparent egg mortality in copepods. We also discuss the consequences of egg sinking and asynchronous hatching in the estimation of in situ egg production. Introduction Recruitment is determined by the difference between the birth rate and the agespecific mortality rate. For copepods, naupliar recruitment is a function of egg production rate, egg hatching success and egg mortality rate. It has been shown that copepod eggs can suffer an apparent mortality rate as high as almost 00% (Table I). The apparent egg mortality rate in marine copepods is usually calculated as the difference between the egg production rate measured by bottle incubations and the egg production rate estimated in situ (references in Table I). Thus, the apparent egg mortality rate is indeed how fast the eggs are removed from the water column. Although several mechanisms to remove eggs from the water column have been proposed, such as predation, cannibalism and horizontal advective loss (Beckman and Peterson, 986; Liang et al, 994; Peterson and Kimmerer, 994), these hypotheses have rarely been tested empirically. Since copepod eggs usually have higher density than sea water, egg sinking has also been suggested as a major mechanism to remove eggs from the water column (e.g. Marcus and Fuller, 986; Peterson and Kimmerer, 994). Before we can assess the importance of any egg removal mechanisms, several precautions have to be taken, (i) The importance of any egg removal mechanism must be assessed against egg hatching success. Only the disappearance of viable eggs will have an impact on population recruitment. If the egg hatching success is low (e.g. Ianora et al, 992; Ianora and Poulet, 993; Laabir et al, 995), removal of the eggs will have little impact on the immediate naupliar recruitment of copepods. (ii) The importance of egg sinking rate has to be interpreted in conjunction with egg Oxford University Press 97

2 K-W.Tang, H.G.Dam and L-R.Feinberg hatching duration and depth of the water column. Egg hatching duration is the time required for an egg to h^tch into a nauplius and is known to be affected by temperature (McLaren, 966; McLaren et al., 969) and salinity (Uye and Fleminger, 976). If eggs can hatch before they reach the sea floor (the egg deposition time), sinking will not be a limiting factor for recruitment. On the other hand, if eggs reach the sea floor before they hatch, they will disappear from the water column, contributing to the apparent egg mortality. Although copepod egg densities and sinking rates have been studied for years (Salzen, 956; Uye, 980; Marcus and Fuller, 986; Miller and Marcus, 994), little has been done to compare egg hatching and egg deposition times directly. We conducted experiments to measure the seasonal natural egg production rates, hatching success, hatching duration, egg size and egg densities of two coastal calanoid copepod species: Centropages hamatus and Temora longicornis. Our goal was to assess the relative importance of egg production rate, hatching success, hatching duration and sinking in limiting the seasonal naupliar recruitment in these two species of marine copepods. Method Sample collection Samples for egg production and hatching experiments were collected from a station (depth <20 m) in eastern Long Island Sound, USA (72 03'27"E; 4 8'26"N) on 2 cruises between 9 March and 26 June 996. Eggs for density measurements were collected in spring of 996 and winter of 997. Water samples were collected from the surface with a bucket and from the middle and bottom of the water column with a Niskin bottle. Water temperatures were measured with a thermometer and salinities with a hand refractometer. Animals were collected.by drifting near the surface a 0.5-m-diameter plankton net fitted with a 200 um mesh and a solid cod end. The contents of the tow were diluted into a 5 thermo-container filled with surface sea water. Water analysis Water samples from different layers were mixed in equal proportions and the water mixture was used for all subsequent work. Two replicates (50-00 ml) of Table I. Apparent in situ egg mortality for marine copepods. Egg mortality is expressed as a percentage of the expected (= bottle incubation method) egg production per day. For details, see Introduction Reference Beckman and Peterson, 986 Ki0rboe etal, 988 KiOrboe and Nielsen, 994 Liang etal, 994 Peterson and Kimmerer, 994 Species Acartia tonsa Acartia tonsa Paracalanus parvus Small calanoid copepods Centropages abdominalis Temora longicornis Egg mortality (% day-') 4-88 >

3 Population recruitment of marine copepods size-fractionated (total, <20 um and <0 urn) chlorophyll a concentrations were measured with the fluorometric method (Parsons et al., 984). Two replicates ( ml) of the size fraction <63 um were collected on combusted (500 C, 24 h) GF/F filters for particulate organic carbon (POC) and nitrogen (PON) analyses (Carlo-Erba EA08 elemental analyzer). The remaining sea water was filtered through either a 63 um sieve for animal incubation or GF/F filters for egg incubations (see below). Animal incubation Incubations and hatching experiments were conducted, within an hour of collection of animals, in a walk-in environmental chamber with temperature control (±0.5 c C variation from a set temperature) and a light-dark cycle of 2 h L:2 h D. The temperature of the chamber was set at the average water column temperature on the day of sample collection (lowest attainable temperature = 6 C). Animals were sorted by species using a dissecting microscope. Only mature and actively swimming adult copepods with intact appendages were used for incubation. Five females (with spermatophores in C.hamatus and dark ovary strips in T.longicornis) and one male (with fully developed fifth legs) were incubated (2-4 replicates) in polycarbonate bottles filled with sea water filtered through a 63 um sieve. This filtering procedure removed any copepod eggs in the sea water while retaining most of the food particles. Occasionally, when the daily egg production rate was low, up to 30 females were incubated in 2 bottles in order to obtain enough eggs for experiments. All bottles were mounted on a plankton wheel (2 r.p.m.) to maintain particles in suspension. After 24 h, animals and eggs were collected gently on a 200 and a 63 um sieve, respectively. Eggs were then rinsed into Petri dishes, pipetted out and briefly rinsed in GF/Ffiltered sea water. All eggs and nauplii were counted. When available, the diameter of at least 20 eggs was measured with a stereomicroscope equipped with an ocular micrometer. Cumulative hatching success and hatching duration When the egg production rate was high, at least 30 eggs from each bottle were incubated for monitoring hatching success. When the egg production rate was low, eggs from all bottles were pooled and used for monitoring hatching success. Occasionally, eggs from the 2 bottles were used. Eggs were incubated individually in Corning 96-well cell plates. Each well (6.4 mm diameter X mm depth) was filled to -80% of its volume with GF/F-filtered sea water. A bottle of GF/F-filtered sea water was gently bubbled with air and kept in the environmental chamber for subsequent use. Eggs were examined every 24 h and our observations were classified into five categories: (i) unhatched eggs; (ii) normal nauplii: defined by normal morphology and motility; (iii) abnormal embryos and nauplii: defined as partially hatched eggs (Poulet et al., 995), nauplii with deformed appendages, or dead nauplii; (iv) empty egg shells: defined as transparent egg shells without nauplii; (v) missing eggs: defined as the disappearance 973

4 K-W.Tang, H.G.Dam and LJLFeinberg of eggs; probably a result of disintegration of unhatched (and possibly unfertilized) eggs (Laabir et al, 995; W.T.Peterson, personal communication). Eighty percent of the incubation sea water for the unhatched eggs was renewed every day to minimize oxygen depletion (Uye and Fleminger, 976). Egg examination continued up to 5 days or until no new hatching occurred for 2-3 days. Cumulative hatching success is defined as the percentage of eggs hatched to normal nauplii within the observed period; it thus indicates the portion of eggs which could potentially contribute to the copepod population within that period of time. The first hatching event marks the minimum time required before any of the eggs will hatch. Hatching duration is defined as the time required to attain the cumulative hatching success, which indicates how long it takes to recruit nauplii from the viable portion of the eggs. The time gap between the first and the last hatching events reflects synchronicity of the hatching process among viable eggs: the larger the time gap, the less synchronous hatching among eggs. Note that our definition of minimum hatching time is different from that of McLaren (966) and McLaren et al. (969). McLaren (966) reported the time to hatch 50% of the eggs. In our study, we were more interested in the minimum hatching time, which allows us to compare the egg deposition time to the egg hatching time. Egg density and sinking rate Egg density was determined with a density-gradient centrifugation method with a mixture of NALCO060 colloidal silica and sucrose solution (Butler and Dam, 994). The density-gradient centrifugation method has been commonly used to measure the density of copepod eggs (Marcus and Fuller, 986; Miller and Marcus, 994). Densities of the gradient were calculated as mass/volume; mass was determined with a Mettler PM 2000 electronic scale (accuracy 0.0 g) and a standard volume of 000 ul (Eppendorf pipette) was used. We did not buffer the ph of the gradient (Schwinghamer et al., 99; Butler and Dam, 994) since variation in gradient ph did not affect the density measurements or egg recovery (personal observations). Five different densities (at an increment of g ml", or 3-9%) were used. Solutions of the gradient were carefully pipetted into 5 ml centrifuge tubes (2-6 replicates) to make up five layers of increasing density, from top to bottom. At least 20 eggs were concentrated in a small drop of sea water and then transferred onto the surface of the density gradient with a mouth pipette. A small amount of sea water was inevitably added to the gradient, but it never exceeded 0.5 ml. The tubes were centrifuged at r.p.m. for min. After centrifugation, each layer was pipetted out, from top to bottom, with a peristaltic pump at a rate of.8 ml min". Eggs retained in each density layer were counted. The density of each layer was re-confirmed as mass/volume. Only the final densities were used for calculations, although there was little difference between the initial andfinal densities of each layer. Eggs settled in a particular layer were assumed to have equal density as that layer. Egg sinking rates were calculated with Stokes' law. Seawater density and kinematic viscosity for Stokes' law were estimated from water temperatures and salinities (Sverdrup et al., 942) of the corresponding dates. 974

5 Population recruitment of marine copepods All density measurements were made at room temperature (~20 C) and we did not control the osmotic potential of the density gradient. Miller and Marcus (994) demonstrated a significant change in egg density when the eggs were exposed to media of different osmotic potential, although the change was very small (a <% change in egg density for a 00% change in ambient osmotic potential; Table 3 in Miller and Marcus, 994). Our eggs might thus have changed their density when they were transferred from the incubation bottles (filteredsea water of in situ osmotic potential) to the density gradient (experimental osmotic potential). Based on Miller and Marcus (994), we estimated the error in density measurements due to this osmotic potential difference and how it might affect our calculations of egg sinking rate (see Results). Results Environmental conditions The temperature of the entire water column increased and water salinity decreased throughout the study period (Figure A and B). Since the temperature of the water column increased sharply and thermal stratification started to occur after 9 May, for convenience we designated the periods before May 9 as 'winter' and after 9 May as 'spring'. Two periods of relatively high chlorophyll concentration were observed during the study period: the first one from late March to early April and the second one in June (Figure C). The size-fractionated chlorophyll concentration data indicate that the first period was dominated by large phytoplankton (>20 um), whereas the second period was mainly composed of smaller (<20 um) phytoplankton. POC and PON concentrations were also highest during these two periods (Figure ID). Egg production rate A high egg production rate of Chamatus was observed during the first chlorophyll peak (late March to early April; Figure 2A). After thefirstchlorophyll peak, the egg production rate decreased throughout the study period. An increase in chlorophyll, POC and PON in June was not matched by an increase in egg production rate. Temora longicornis was scarce until early April. The egg production rate of T.longicornis was lower than that of Chamatus in the present study (Figure 3A). Peaks of egg production rates coincided with the peaks of ambient chlorophyll concentrations. Egg morphology and size The eggs of Chamatus appeared pale with a spiny surface under a stereomicroscope (X28). Egg diameter without spines for Chamatus was consistently in the range of um with small variation (SD < 2 um), except for 5 June when the mean egg size was 82.9 um and the SD was 8.3 um (Figure 2B). The eggs of T.longicornis appeared as a sphere of dark embryonic biomass 975

6 ICW.Tang, H.G.Dam and L.R.Feinberg Fig.. Environmental variables during the study period (9 March-26 June 996). (A) Water temperature; (B) water salinity; (C) size-fractionated chlorophyll concentrations for the water column; each point represents the mean concentration from two replicated fluorometric measurements (see the text for details); (D) POC and PON concentrations and C:N ratio of particles <63 um. Each point represents the mean of two replicates. enveloped by a thick, clear outer layer (stereomicroscope at X28). The outer layer of the eggs appeared red under strong light; the outer membrane was smooth. The egg size of T.longicornis was more variable (SD < 5 um) and generally larger than that of C.hamatus. Mean egg diameter ranged from 79 to 90 um without a notable seasonal trend (Figure 3B). Cumulative hatching success and hatching duration The cumulative hatching success of eggs of Chamatus ranged from 65 to 95% without notable seasonaiity (Figure 2C). In other words, given enough time, eggs 976

7 Population recruitment of marine copepods 88- S S 72 - m 68 - I I I.. r. M ',,, i i, i i ),, i i i, I, O fi« Q - - \ A C -A-Jli' 'I ' ' ' ' A Days to first hatching event -V- Days to cumulative HS i V [ * B D i ' ' Rg. 2. Reproductive variables of Chamatus during the study period (9 March-26 June 996). (A) Egg production rate (EPR; mean ± SD); (B) egg diameter (mean ± SD); (C) cumulative hatching success (HS) within IS days (mean ± SD); (D) duration of hatching events (points are mean values). produced in winter had similar chance to hatch as eggs in spring. Cumulative hatching success was not correlated with chlorophyll concentrations, POC, PON or particulate carbon:nitrogen (C:N) ratio. Minimum hatching time and hatching duration, on the other hand, changed sharply throughout the study period (Figure 2D). At the beginning of this study, no hatching was observed until the fifth day (minimum hatching time = 5 days) and hatching duration was >0 days. Both minimum hatching time and hatching duration decreased towards June when hatching was completed within 3 days. The time gap between the first and the last hatching events was -6 days in winter and -2 days in spring. Cumulative hatching success of eggs of T.longicomis ranged from 70 to 90% throughout the study without a distinctive seasonal pattern (Figure 3C). No hatching experiments were carried out between 9 April and June, when we did not find enough adults or eggs for incubation. The hatching experiments showed that 977

8 K-W.Tang, H.G.Dam and L.R.Feinberg Days to first hatching event V Days to cumulative HS Fig. 3. Reproductive variables of T.longicomis during the study period (9 March-26 June 996). (A) Egg production rate (EPR; mean ± SD); (B) egg diameter (mean ± SD); (C) cumulative hatching success (HS) within 5 days (mean ± SD); (D) duration of hatching events (points are mean values). Between 9 April and June, there were not enough animals or eggs for experiments. eggs produced in different months had similar potential to hatch to normal nauplii. Cumulative hatching success was not correlated with chlorophyll concentrations, POC, PON or particulate C:N ratio. Hatching did not start until day 3 in April (minimum hatching time = 3 days), compared with day in June (minimum hatching time = day; Figure 3D). However, in both April and June, the first and the last hatching events were separated by only <.5 days. Egg size and egg hatching success were not correlated in either of the species, which suggests that the hatching process was not regulated by food reserve in the eggs (cf. Guisande and Harris, 995). Abnormal nauplii or missing eggs (Laabir et al., 995; Poulet et al., 995) accounted for <5% for Chamatus and <0% for T.longicomis over the entire study period. 978

9 Population recruitment of marine copepods Osmotic potential and egg density measurements We estimated the in situ osmotic potential from in situ salinities and the experimental osmotic potential based on the composition of the density gradient. Assuming that the change in egg density is linearly correlated with the change in ambient osmotic potential (Miller and Marcus, 994), we estimated that the error in density measurements due to this osmotic potential difference was <% for C.hamatus in both spring and winter, and for T.longicomis in winter. For T.longicomis in spring, the egg density could have been overestimated by at most 2%. Since our analytical error in mass measurements was %, the error associated with osmotic potential would only be a concern for T.longicomis in winter. Also, our estimates of the error were likely at the higher end since our eggs were exposed to the density gradient for only min, compared with the 3 h exposure time in Miller and Marcus (994). Nevertheless, we corrected the mean egg density for T.longicomis in winter accordingly and used it to calculate the egg sinking rates. Egg density and sinking rate Egg density measurements for C.hamatus were made in spring of 996 ( June, and 2 and 4 August) and winter of 997 (3,9 and 25 March). Ninety-six out of 0 eggs were recovered (87.3% recovery) in spring and 339 out of 42 eggs were recovered (80.5% recovery) in winter. In spring, egg density ranged from.07 to.9 g cm" 3, with most of the eggs found in the range g cm" 3 (Figure 4A). Mean density for spring was.0 g cnr 3 with a SD of 0.03 g cm~ 3. In winter, egg density was generally lower, ranging from 0.99 to. g cm" 3 (Figure 4B). Mean density for winter was.06 g cm" 3 (SD = 0.03 g cm" 3 ). The mean densities for the two periods were significantly different (Wilcoxon two-sample test, P < 0.00). We applied the mean winter egg density to calculate egg sinking rates for dates before 9 May and the mean spring egg density for dates after 9 May. Egg sinking rate for C.hamatus ranged from m day in winter to >22 m day in spring (Table II). Variation in sinking rates within each period was driven by variation in egg size, seawater density and viscosity. Egg density measurements on T.longicomis were also made in spring of 996 (,2, 20 and 26 June) and winter of 997 (9 March, and 3 and 7 April). Two to six replicates were run on each date. Egg recovery was 64% (503 out of 789) in spring and 80% (262 out of 327) in winter. In spring, egg density ranged from.04 to.45 g cm" 3, but the distribution was skewed towards the lower end: g cm" 3 (Figure 4C). Mean density for spring was.3 g cm" 3 (SD = 0.06 g cm" 3 ). Egg density for winter was less variable (mean =.0 g cm" 3, SD = 0.02 g cm" 3 ; Figure 4D) and the mean density for this period was significantly different from that for spring (Wilcoxon two-sample test, P < 0.00). Mean density for spring corrected for osmotic effect was. g cm" 3. Using the mean winter density and the corrected mean spring density, the egg sinking rate for T.longicomis was estimated to be m day before 9 May and m day after 9 May (Table II). 979

10 ICW.Tang, H.G.Dam and L.R-Feinberg o c (D 3 CT (D ou ou Centropages hamatt JS June-August 996 A C, II. hi III Temora longicomis June 996 n= Centropages hamatus B Temora longicomis March March 997 n=339 n=26 fill I, T i ' i Density (g cm ) Fig. 4. Frequency distribution of egg densities. (A) and (B) Chamatus; (C) and (D) T.longicomis. Data are not corrected for osmotic effect (see the text for an explanation). Table D. Egg sinking rates of C.hamatus and T.longicomis in winter (before 9 May) and spring (after 9 May). Egg sinking rates (ESR) were estimated from Stokes' law. Egg deposition time is the time to sink 20 m; minimum hatching time is the time to the first hatching event Date March 9 March 27 April 8 April 9 April 25 May 7 May 6 June S June June 2 June 9 June 20 June 26 Centropages hamatus ESR (m day ) i Egg deposition time (days) n/d, no data. Values corrected for osmotic effect. Minimum hatching time (days) Temora longicomis ESR (m day ) * 25.43* 24.44* 22.32* 24.35* 23.6* Egg deposition time (days) Minimum hatching time (days) n/d n/d 2 980

11 Population recruitment of marine copepods Source of missing eggs in density measurements In order to identify the cause for missing eggs in the density measurements, we carried out the following experiments: we placed a sample of 35 eggs (C.hamatus, duplicates) in the lowest density layer used (.02 g ml" sucrose solution), which presumably had the highest osmotic potential. After 30 min (duration of centrifugation in the density measurements), 97-00% of the eggs were recovered. We then transferred the eggs to centrifuge tubes and spun them at 3000 r.p.m. for 30 min. Eighty to 85% of the eggs were recovered and some debris was found in the solution. We repeated the experiment with T.longicomis (triplicates of 5-20 eggs in.07 g ml" sucrose solution) and observed higher loss of eggs after centrifugation (90-95% recovery before and 6-86% recovery after centrifugation). These observations led us to conclude that missing eggs were mostly the results of mechanical damage during centrifugation. We also compared two centrifugation schemes (3000 r.p.m. for 30 min and 500 r.p.m. for 60 min) with eggs (T.longicomis, duplicates). We obtained a slightly higher recovery (80% versus 75%) with the lower spinning speed, whereas the patterns of egg segregation were similar. Therefore, we recommend a low spinning speed and long spinning time in future studies to minimize mechanical damage to the eggs. Discussion Egg production rate and environmental conditions The maximum egg production rates observed in this study were 5 eggs copepod" day for C.hamatus and 7 eggs copepod" day" for T.longicomis. The maximum rate for Chamatus was similar to that observed by Ki0rboe and Nielsen (994) in their seasonal study in the Kattegat, Denmark (mean chlorophyll concentration at the time of maximum egg production rate in the Kattegat ~5 mg m~ 3, with -80% of the chlorophyll > um). However, the maximum egg production rate for T.longicomis in this study was much lower than the typical maximum rates (40-50 eggs copepod" day ) reported for T.longicomis during spring blooms (Peterson and Bellantoni, 987; Ki0rboe and Nielsen, 994; Peterson and Dam, 996). Since this study started in mid-march, we missed the peak of the typical spring bloom in early March in Long Island Sound. The total (~4 mg nr 3 ) and >20 um (-2 mg nr 3 ) chlorophyll concentrations measured in this study at the time of maximum egg production by T.longicomis were 4-5 times lower than those previously reported for early March in Long Island Sound (Dam and Peterson, 99), when egg production of T.longicomis is typically highest (Peterson and Kimmerer, 994). Peterson (985) showed that cohorts of T.longicomis in Long Island Sound were initiated by bursts of egg production during phytoplankton blooms in late February to early March and the fecundity of T.longicomis became limited by phytoplankton abundance after the blooms. Thus, relatively low concentrations of large-sized phytoplankton may have accounted for the relatively low egg production rates observed in T.longicomis during our study period. 98

12 K.W.Tang, H.G.Dam and L.R.Feinberg Egg size and hatching success Guisande and Harris (995) found that egg hatching success increased with egg size in Calanus helgolandicus since larger eggs may contain more food reserves. Cumulative hatching success for Chamatus and T.longicomis was independent of egg size in the present study. However, the egg size of Chelgolandicus varied by as much as 20% (Guisande and Harris, 995), compared with <2% in the present study. The narrow size range of the eggs of Chamatus and T.longicomis indicates that food reserves in the eggs of these two species are almost invariant. Egg hatching duration, egg density and sinking We observed that the egg hatching potential for Chamatus and T.longicomis, in terms of cumulative hatching success within 5 days, remained consistently high throughout the length of the study. However, the minimum hatching time and hatching duration were longer in winter. This can be explained by the fact that the hatching process of copepod eggs is greatly delayed at low temperature (Cooley and Minns, 978). Also, since the lowest temperature of our environmental chamber was 6 C, which was still higher than the average ambient water temperature in March (<4.3 C), the hatching process might have been artificially accelerated in our laboratory conditions. Given the low ambient water temperature in winter and early spring, the minimum hatching time and hatching duration may be even longer in the field. Delayed hatching in winter will increase the probability of egg loss by predation, advection and sinking. In shallow regions such as at our station in Long Island Sound, sinking to bottom sediments can be a major loss of the egg population in the water column. Assuming an average depth of Long Island Sound to be 20 m (Kim and Bokuniewicz, 99), we estimated the time required to remove an egg from the water column by sinking, and how it compares with the minimum time required for hatching (Table II). For Chamatus, eggs would reach the bottom just before hatching began in winter (except 7 and 6 May). Since the hatching process in Chamatus was more asynchronous in winter (Figure 2D), many of the eggs would, in fact, take >5 days to hatch and could sink to the bottom before hatching began. Egg sinking rates for Chamatus were much higher in spring due to higher egg density and lower water viscosity. As a result, it would take less than a day to remove an egg from the water column by sinking, whereas the minimum hatching time was -4 days. The eggs of T.longicomis were generally larger and denser, and egg sinking rates were accordingly higher. In all cases, the eggs of T.longicomis would sink to the bottom before hatching began in the water column (Table II). Our comparison of the minimum hatching time and the time required to deposit the eggs implies that even though the eggs had a high potential to hatch, in most cases virtually all the eggs would sink out of the water column before hatching occurred, contributing to the apparent egg mortality in the water column (Table I) even in the absence of such other factors as predation, cannibalism and horizontal advection. High egg sinking rates have also been reported for other copepod species (Marcus and Fuller, 986; and references therein) and may 982

13 Population recruitment of marine copepods explain the common occurrence of copepod eggs in coastal sediments (Marcus, 990,995; Marcus et al., 994; and references therein). The significance of fast settlement of copepod eggs is that the post-settlement fate of the eggs instead of water column processes will determine the true egg mortality. Depending on the sea floor environment, if settled eggs continue to hatch at the sea floor and return to the water column as nauplii, the apparent egg mortality will overestimate the true egg mortality. On the other hand, if eggs are buried and survive in bottom sediments, they will contribute to future recruitment (De Stasio, 989; Marcus et al., 994; Hairston, 996). Marcus and Fuller (986), who also used a similar density gradient method to determine copepod egg densities and sinking rates, observed a significant difference in the density between subitaneous and diapause eggs (.29% difference between the modal densities). We observed a more pronounced difference in the mean egg density between winter and spring (4.9% for C.hamatus, 2.6% before correction for osmotic potential and 0.9% after correction for T.longicomis). Although the higher egg densities in spring may suggest that most of the eggs were diapause, this hypothesis was not supported by the high hatching success observed in spring. Therefore, our density data may reflect a true seasonal variation in the density of subitaneous eggs. It has not escaped our attention that the egg sinking rates presented here are the theoretical rates for a laminar fluid environment. The actual sinking rate of eggs in the water column can be expressed as W = W + W, where W is the mean sinking rate and W is the sinking rate associated with vertical turbulent motion in the fluid. We assume that the mean sinking rate is given by our estimates derived from Stokes' law. The magnitude of W may be estimated, from dimensional considerations, as K z IZ', where K z represents the vertical eddy diffusion coefficient {L 2 /T) and Z' represents a characteristic vertical length scale for the turbulent eddies (L). In shallow systems, Z' may be approximated as 0.4Z (Fisher et al., 979), where Z is the depth of the water column. In estuaries, the magnitude of K z depends on whether the water is mixed or stratified. In the former case, K z = 2.5 X 0-3 ZV and in the latter, K z = (2.5 X 0" 3 ZV Ri)~ m, where V is the mean flow velocity in the water column and Ri is the Richardson number (Dyer, 973; Fisher et al., 979). For a tidally driven estuary like Long Island Sound, V = 0.5 m s", and assuming Z = 20 m, yields W ~ 300 m day- for a mixed water column and values /0-/00 smaller during stratified conditions. With such values of turbulent vertical velocities, if eggs were to stick to the sediments once they reached the bottom, the net effect of vertical turbulent motion would be to shorten the egg deposition time, sometimes quite considerably. Whether eggs are resuspended from the bottom depends on whether W, as defined above, is greater than /*, the bottom shear velocity. U* = (KV 2 ) m, where K = 2X 0" 3 (Dyer, 973). Again, assuming V = 0.5 m s" yields U* = 900 m day. Thus, eggs are likely to be resuspended from the bottom in most tidally driven estuaries. In the simplest terms, the flux of eggs, F, from the sediments to the water column will depend on the vertical gradient of eggs near the bottom and can be approximated by F= K z dcldz. This assumes, of course, that eggs are more abundant in the sediments than in the overlying water. If the opposite were the 983

14 K-W.Tang, H.G.Dam and L.R.Feinberg case, then the net flux of eggs would be to the sediments, regardless of the value of U*. This analysis suggests that the fate of the eggs is determined by conditions at the sediment-water interface. Future studies of copepod egg settlement should consider the vertical distribution of eggs at this interface and the physical conditions over the tidal cycle. Implications for in situ estimation of egg production rates The egg ratio method has been commonly used to estimate the in situ egg production rate of copepods (Checkley, 980; Omori and Ikeda, 984; references in Table I). To do so, one has to assume a specific egg development time for a particular water temperature (Edmondson et al., 962; McLaren, 966). However, the method is accurate only if egg development is highly synchronous. In the present study, we found that egg development in C.hamatus is highly asynchronous at low temperature. For example, from March to April, when the hatching experiments were run at 6 C, the first and the last hatching events were separated by as many as 6 days. This indicates that the development time among the eggs of C.hamatus was highly variable. As a result, employing one specific egg development time in the egg ratio method will inevitably lead to considerable over- or underestimation. On the other hand, egg development in T.longicornis appeared to be more synchronous since the gap between the first and the last hatching events was consistently <.5 days. Thus, the bias in the egg ratio method due to asynchronous egg hatching should be less severe for this species. The potential bias in using the egg ratio method due to asynchronous hatching should be explored further, but is beyond the scope of the present paper. High egg sinking rates, as estimated in the present study, may present another problem for using the egg ratio method: many calanoid copepods are known to spawn their eggs at night (Tiselius et al., 987; Uye and Shibuno, 992; White and Roman, 992; Cervetto et al., 993; Kosobokova, 994). If field samples are collected during daytime, there will be a time lag between egg spawning and egg sampling from the water column. Assuming a time lag of 2 h, for example, eggs of T.longicornis may have sunk >0 m. In shallow waters, like Long Island Sound, a good portion of the eggs may have been lost to bottom sediments before sampling. This may partly explain why the egg production rate calculated from the egg ratio method is consistently lower than that from bottle incubations (Peterson and Kimmerer, 994). Nocturnal spawning and a high egg sinking rate will require night sampling if the egg ratio method is to be used for estimating the in situ egg production rate. Conclusion In the present study, we showed the following, (i) The egg production rate of C.hamatus gradually decreased from March towards June. Therefore, a low egg production rate is one of the reasons why Chamatus usually disappears from the Sound in spring and summer. In comparison, naupliar recruitment in T.longicornis was limited by a low egg production rate throughout our study period, (ii) Egg 984

15 Population recruitment of marine copepods hatching potential, as indicated by the cumulative hatching success, remained consistently high for both species. These observations suggest that seasonal naupliar recruitment of these two species is not limited by the ability of the eggs to hatch, (iii) The whole hatching process was delayed in winter. This is indicated by the longer minimum hatching time in winter than in spring. Also, in the case of Chamatus, hatching was more asynchronous in the winter, as indicated by the larger time gap between the first and the last hatching events, (iv) Egg sinking rates for Chamatus and T.longicornis, estimated from egg size and egg density, indicate that eggs may be lost to the bottom sediments before hatching can begin in the water column. Our study suggests that a high sinking rate combined with delayed hatching can be an effective removal mechanism of copepod eggs. This mechanism counteracts a high egg production rate (e.g. Chamatus) and high hatching success (e.g. both Chamatus and T.longicornis) in naupliar recruitment. Egg sinking is perhaps as important a source of apparent mortality as predation, cannibalism and horizontal advection. Acknowledgements Research supported by grant NSF OCE awarded to H.G.D. We thank R.Banker, L.Burch, P.Cancellieri and T.Fenn for technical assistance, W.T.Peterson and T.Ki0rboe and an anonymous reviewer for comments on earlier versions of the manuscript. We thank P.Bogden and J.O'Donnell for discussions regarding the sinking rate of eggs and their resuspension. The Marine Science and Technology Center of the University of Connecticut (MSTC, UCONN) provided the research vessels. This is contribution no. 30 of the MSTC, UCONN. References Beckman,BR- and Peterson.W.T. (986) Egg production by Acartia tonsa in Long Island Sound. / Plankton Res., 8, Butler.M. and Dam,H.G. (994) Production rates and characteristics of fecal pellets of the copepod Acartia tonsa under simulated phytoplankton bloom conditions: implications for vertical fluxes. Mar. EcoL Prog. Ser, 4, 8-9. Cervetto.G., Gaudy.R., Pagano,M., Saint-Jean.L., Verriopoulous.G., Arfi,R. and Leveau,M- (993) Diel variations in Acartia tonsa feeding, respiration and egg production in a Mediterranean coastal lagoon. J. Plankton Res., 5, Checkley.D.M. (980) Food limitation of egg production by a marine, planktonic copepod in the sea off southern California. LimnoL Oceanogr., 25, CooleyJ.M. and Minns.C.K. (978) Prediction of egg development times of freshwater copepods. / Fish. Res. Board Can., 35, Dam,H.G. and Peterson,W.T. (99) In situ feeding behavior of the copepod Temora longicornir. effects of seasonal changes in chlorophyll size fractions and female size. Mar. EcoL Prog. Ser., 7, De Stasio3T. r Jr (989) The seed bank of a freshwater crustacean: Copepodology for the plant ecologist Ecology, 70, Dyer.K.R. (973) Estuaries: A Physical Introduction. Wiley, London, 40 pp. Edmondson.W.T., Comita.G.W. and Anderson.G.C. (962) Reproductive rate of copepods in nature and its relation to phytoplankton population. Ecology, 43, Fisher,H.B., List^J., Koh,R.C., ImbergerJ. and BrooksJJ.H. (979) Mixing in Inland and Coastal Waters. Academic Press, New York, 483 pp. Guisande.C. and Harris^ (995) Effect of total organic content of eggs on hatching success and naupliar survival in the copepod Calanus helgolandicus. LimnoL Oceanogr., 40,

16 ICW.Tang, H.G.Dam and L.R.Feinberg Hairston,N.G.,Jr (996) Zooplankton egg banks as biotic reservoirs in changing environments. LimnoL Oceanogr., 4, Ianora.A- and Poulet,S.A. (993) Egg viability in the copepod Temora stylifera. LimnoL Oceanogr, 38, Ianora^A., Mazzocchi.M.G. and Grottoli.R. (992) Seasonal fluctuations in fecundity and hatching success in the planktonic copepod Centropages typicus. J. Plankton Res., 4, Kim3-H. and Bokuniewicz r H.J- (99) Estimates of sedimentfluxesin Long Island Sound. Estuaries, 4, Ki0rboe,T. and Nielsen.T.G. (994) Regulation of zooplankton biomass and production in a temperate, coastal ecosystem. I. Copepods. LimnoL Oceanogr., 39, Ki0rboe,T, M0hlenberg,F. and Tiselius,P. (988) Propagation of planktonic copepods: production and mortality of eggs. Hydrobiologia, 67/68, Kosobokova.K.N. (994) Reproduction of the calanoid copepod Calanus propinquus in the southern Weddell Sea, Antarctica: observations in laboratory. Hydrobiologia, 292/293, Laabir,M., Poulet.S.A. and IanoraA- (995) Measuring production and viability of eggs in Calanus helgolandicus.j. Plankton Res., 7, LiangX>., Uye.S.I. and Onbe.T. (994) Production and loss of eggs in the calanoid copepod Centropages abdominalis Sato in Fukuyama Harbor, the Inland sea of Japan. Bull. Plankton Soc. Jpn, 4,3-42. Marcus.N.H. (990) Calanoid copepod, cladoceran and rotifer eggs in sea-bottom sediments of northern California coastal waters: identification, occurrence and hatching. Mar. Biol., 05, Marcus J4.H. (995) Seasonal study of planktonic copepods and their benthic resting eggs in northern California coastal waters. Mar. Biol., 23, Marcus^N.H. and Fuller.C.M. (986) Subitaneous and diapause eggs of Labidocera aestiva Wheeler (Copepoda: Calanoida): Differences in fall velocity and density. /. Exp. Mar. BioL EcoL, 99, Marcus,N.H., Lutz,R., Bumett.W. and CableJ". (994) Age, viability and vertical distribution of zooplankton resting eggs from an anoxic basin: Evidence of an egg bank. LimnoL Oceanogr., 39, McLaren.I.A. (966) Predicting development rate of copepod eggs. BioL Bull., 3, McLaren.I.A., Corkett.C.J. and Zillioux.EJ. (969) Temperature adaptations of copepod eggs from the Arctic to the tropics. BioL BulL, 37, Miller JD.D. and Marcus,N.H. (994) The effects of salinity and temperature on the density and sinking velocity of eggs of the calanoid copepod Acartia tonsa Dana. /. Exp. Mar. Biol. EcoL, 79, Omori,M. and Ikeda.T. (984) Methods in Marine Zooplankton Ecology. Wiley, New York. Parsons.T.R., Maita.Y. and Lalli.C.M. (984) A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, New York. Peterson.W.T. (985) Abundance, age structure and in situ egg production rates of the copepod Temora longicomis in Long Island Sound, New York. BulL Mar. Sci., 37, Peterson.W.T. and Bellantoni.D.C. (987) Relationships between water column stratification, phytoplankton cell size and copepod fecundity in Long Island Sound and off central Chile. 5. Afr. J. Mar. ScL, 5,4-42. Peterson.W.T. and Dam.H.G. (996) Pigment ingestion and egg production rates of the calanoid copepod Temora longicomis: implications for gut pigment loss and omnivorous feeding. / Plankton Res., 8, Peterson.W.T. and Kimmerer.WJ. (994) Processes controlling recruitment of the marine calanoid copepod Temora longicomis in Long Island Sound: Egg production, egg mortality and cohort survival rates. LimnoL Oceanogr., 39, Poulet.S.A., Laabir,M., Ianora.A. and Miralto.A- (995) Reproductive response of Calanus helgolandicus. I. Abnormal embryonic and naupliar development. Mar. EcoL Prog. Sen, 29, Salzen,E. A. (956) The density of eggs of Calanusfinmarchicus.J. Mar. BioL Assoc UK, 35, Schwinghamer,P., Anderson J).M. and KulisJJ.M. (99) Separation and concentration of living dinoflagellate resting cysts from marine sediments via density-gradient centrifugatioa LimnoL Oceanogr., 36, SverdrupJi.U., Johnson,M.W. and Fleming,R.H. (942) The Oceans: Their Physics, Chemistry and General Biology. Prentice Hall, New Jersey. TiseliusJP., Berggren.U, Bamstedt.U., Hansen3., Ki0rboe,T. and M0ehlenbergJF. (987) Ecological and physiological aspects of propagation in marine herbivorous copepods (a preliminary report). Rep. Mar. Pollut Lab. Charlottenlund,,

17 Population recruitment of marine copepods Uye.S. (980) Development of neritic copepods Acartia clausi and A. steuri. I. Some environmental factors affecting egg development and the nature of resting eggs. Bull Plankton Soc. Jpn, 27,-9. Uye.S. and Fleminger,A. (976) Effects of various environmental factors on egg development of several species of Acartia in southern California. Mar. BioL, 38, Uye.S. and Shibuno,N. (992) Reproductive biology of the planktonic copepod Paracalanus sp. in the Inland Sea of Japan. J. Plankton Res., 4, WhiteJ.R. and Roman,M.R. (992) Egg production by the calanoid copepod Acartia tonsa in the mesohaline Chesapeake Bay: the importance of food resources and temperature. Mar. EcoL Prog. Ser., 86, Received on January 2,998; accepted on June 8,