Marja Koski

Sigrun Huld Jonasdottir

Espen Bagøien

Sigrun H Jónasdóttir

Kristine Arendt

Frank Hansen

Louisa Poulsen

Sanna Gärtner

Biological processes in the North Sea: Vertical distribution and reproduction of neritic copepods in relation to environmental factors 1)

Keywords: copepod, Dogger Bank, reproduction, food quantity, food quality, vertical distribution, secondary production Copepod, Dogger Bank, reproduction, secondary production, food quantity and quality, vertical distribution

come

Biological processes in the North Sea II. Vertical distribution and reproduction of neritic copepods in relation to environmental factors

Introduction

Coastal areas and many productive frontal areas in the North Sea are dominated by small neritic copepod species, such as Temora longicornis, Pseudocalanus spp., Paracalanus parvus, Oithona spp., Acartia spp. and Centropages spp. [START_REF] Fransz | Metazoan plankton and the structure of the plankton community in the stratified North Sea[END_REF][START_REF] Nielsen | Zooplankton diversity and the predatory impact by larval and small juvenile fish at the Fisher Banks in the North Sea[END_REF]. At the Dogger Bank in the central North Sea, these species can form the majority of zooplankton biomass especially in summer [START_REF] Fransz | The zooplankton of the North Sea[END_REF][START_REF] Nielsen | Structure of plankton communities in the Dogger Bank area (North Sea) during a stratified situation[END_REF], contributing substantially to the diet of larval fish [START_REF] Nielsen | Zooplankton diversity and the predatory impact by larval and small juvenile fish at the Fisher Banks in the North Sea[END_REF]. Small copepods are also major grazers of phytoplankton and microzooplankton in the area, with the estimated grazing rates of 15-30% of the daily primary production and a potential to control the biomass of microzooplankton [START_REF] Nielsen | Structure of plankton communities in the Dogger Bank area (North Sea) during a stratified situation[END_REF].

The frontal areas of the North Sea, including the Dogger Bank, are characterised by strong subsurface phytoplankton maxima at depths between 15 and 30 m during the stratified season [START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF][START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF]. These subsurface peaks are suggested to be centres for high zooplankton production, where new production of large phytoplankton (mainly dinoflagellates) is efficiently transferred to copepod production [START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF]. The primary production of subsurface peaks over the year is estimated to exceed that of the spring bloom [START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF], emphasising the potential importance for small neritic copepod species, with their annual biomass peak in late summer [START_REF] Nielsen | Zooplankton diversity and the predatory impact by larval and small juvenile fish at the Fisher Banks in the North Sea[END_REF]. The perceived importance of subsurface chlorophyll maximum for copepod secondary production is based on observations of peak biomass of Oithona spp., Temora longicornis and Pseudocalanus spp. at the pycnocline [START_REF] Nielsen | Structure of plankton communities in the Dogger Bank area (North Sea) during a stratified situation[END_REF], as well as a typically higher secondary production at the depth close to the subsurface chlorophyll maxima [START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF].

Secondary production of copepods is formed by biomass and growth, and mostly measured in terms of egg production. Egg production is influenced by factors such as temperature [START_REF] Halsband | Reproductive cycles of dominant calanoid copepods in the North Sea[END_REF], food concentration [START_REF] Bunker | Fecundity of marine planktonic copepods: global rates and patterns in relation to chlorophyll a, temperature and body weight[END_REF], composition, mineral quality [START_REF] Anderson | Stoichiometric theory extended to micronutrients: Comparison of the roles of essential fatty acids, carbon, and nitrogen in the nutrition of marine copepods[END_REF] or biochemical quality [START_REF] Jónasdóttir | Copepod egg production in Long Island Sound, USA, as a function of the chemical composition of seston[END_REF]. Copepod biomass is also influenced by mortality, mainly assumed to be due to predation [START_REF] Ohman | Mortality estimation for planktonic copepods: Pseudocalanus newmani in a temperate fjord[END_REF]. Normal diel vertical migration behaviour of copepods, with an ascent to surface waters for feeding during night time and descent during day time, has been linked to predation risk from visual predators, such as planktivorous fish [START_REF] Hays | Interspecific differences in the diel vertical migration of marine copepods: The implications of size, color, and morphology[END_REF]1996). As the subsurface maximum layer occurs in the deepest part of the photic zone where light levels are low (Richardson et al., Formatted: Font color: Auto 4 1998), one may assume that vertical migration to greater depths during daytime to avoid visual predators should not be very rewarding. A high copepod biomass in the subsurface chlorophyll maximum layer could thus be due to low predation pressure, while the high production rate could be related to increased food concentration, favourable food composition, superior food quality or low energetic costs of (no) vertical migration.

Our study concentrated on the stratified summer season in the area of the Dogger Bank, although to broaden the range of environmental conditions, we also included transects at other frontal areas in the coastal and central North Sea during different seasons. Dogger Bank is an important feeding ground for planktivorous fish [START_REF] Daan | Ecology of North Sea fish[END_REF], and has been studied intensively during the last few decades [START_REF] Nielsen | Structure of plankton communities in the Dogger Bank area (North Sea) during a stratified situation[END_REF][START_REF] Fransz | Metazoan plankton and the structure of the plankton community in the stratified North Sea[END_REF][START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF], including studies focusing on subsurface phytoplankton blooms [START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF].

We studied the vertical distribution and reproduction of dominant neritic copepod species in this area with 3 questions in mind. First, we wanted to know if these species are concentrated in the chlorophyll maximum layer, and if they perform diel vertical migration. Second, we wanted to know if egg production rates and hatching success are significantly higher in the chlorophyll maximum layer than in the surface. Third, we wanted to identify the most important environmental factors determining the vertical distribution, egg production and hatching success of neritic copepods in the North Sea. To be able to separate effects of temperature, body size and previous feeding history from the effects of food quantity and quality in the subsurface chlorophyll maximum, we included a bio-assay approach (see [START_REF] Müller-Navarra | Seasonal patterns of food limitation in Daphnia galeata: Separating food quantity and food quality effects[END_REF], where cultured copepods were fed with in situ water to estimate their reproduction.

Method

Sampling stations

The study took place over four years : 2001, 2002, 2003 and 2005. The experiments in 2001 were designed to study the seasonal succession of in situ egg production and hatching of small copepods (represented by Acartia clausi and Centropages typicus) and their dependence on environmental variables (hereafter referred to as 'in situ' experiments), while the sampling in 2002, 2003 and 2005 focused on the late summer vertical distribution and biomass of all dominant neritic copepods. The bio-assay study in 2003 was conducted to evaluate the value of subsurface chlorophyll maximum vs. surface water for copepod (represented by Acartia tonsa) reproduction (hereafter referred to as the 'bioassay' experiment). The sampling stations in 2001 were situated 1). In all years, the water column was sampled for temperature, salinity and fluorescence profiles (CTD) and microplankton species composition and biomass. In addition, in 2001 and 2003 fatty acid composition and concentration was measured. An overview of experiments, measurements and stations is presented in Table I, while the details of the different methods are described below.

Hydrography, microplankton and fatty acid analysis

Vertical distributions of temperature, salinity and fluorescence were measured using a SeaBird SBE11 CTD equipped with an in situ fluorometer. Fluorescence profiles were not taken on the Alkor cruise in September 2001 but standard chlorophyll measurements were made at the surface and within the well mixed water column. Fluorescence was calibrated against chl-a, measured in water samples from selected depths and stations during each cruise. The linear regression between fluorescence and chl-a typically had an R 2 value > 0.7 (data not shown).

In 2001, microplankton concentration and composition were measured from the water samples collected at the surface (ca 4 m), in the chlorophyll maximum layer, and close to the bottom, and integrated over the whole water column. In 2003, phytoplankton was sampled from the surface and from the chlorophyll maximum layer. The water was sampled using Niskin bottles on the CTD rosette. Microplankton samples were preserved in 4% acidic Lugol's solution for phytoplankton and ciliates, and gluteraldehyde for separation of autotrophic versus heterotrophic organisms. Lugol's samples were allowed to settle in a 50-ml settling chamber (Uttermöhl, 1958;[START_REF] Hasle | The inverted-microscope method[END_REF] and gluteraldehyde samples handled according to Haas [START_REF] Haas | Improved epifluorescence microscopy for observing planktonic microorganisms[END_REF]. Phytoplankton and ciliates were enumerated and geometrical axes of 10 cells per taxon were measured under inverted microscope (Lugol's samples) or epifluorescence microscope (gluteraldehyde samples).

Volumes were calculated using the software program Planktonsys 3.11 from BioConsult A/S, and carbon content calculated according to Edler [START_REF] Edler | Recommendations on methods for marine biological studies in the Baltic Sea -Phytoplankton and chlorophyll[END_REF] and [START_REF] Mullin | Relationship between carbon content, cell volume and area in phytoplankton[END_REF]. A minimum of 400 cells (in total) were counted from each sample: a minimum of 50 cells sample -1 were counted for the most common taxa, while all cells in the 50-ml sample were counted for the less abundant taxa. were filtered under a low pressure on precombusted GF/C (Whatmann) filters, folded into cryovials and immediately frozen at -80 °C in nitrogen atmosphere. In 2001, known water volumes (0.5-60L) were first size fractionated by successive filtration through 80, 40 and 20 µm mesh nylon gauze filters to obtain fatty acid composition in different size fractions. Fatty acids were extracted from filters containing the field collected seston using chloroform:methanol solution (2:1 by volume). A known amount of the fatty acid C23:0 was added to the sample and used as an internal standard for quantification of specific fatty acids. After at least 24 hours extraction at -20°C, the chloroform: methanol phase was collected and the filters were washed three times with chloroform: methanol solution. The extracts were washed according to the modified Folch method [START_REF] Hamilton | Extraction of lipids and derivative formation[END_REF].

After saponification, the samples were transmethylated to fatty acid methyl esters (FAME) using boron trifluoride (BF3) and then stored in air-tight vials in an argon atmosphere at -80°C until GC analysis. The FAME sample was injected into a gas chromatograph (Hewlett Packard 5809A, with a 30 m omegawax 320 µm column, and equipped with a split/splitless injection system) using helium as a carrier gas at 1.8 mL min -1

. Fatty acid methyl esters were identified based on comparison with retention times of several standards; Larodan PUFA standard, fatty acid from the dinoflagellate Prorocentrum minimum to locate 18:5(n-3), Matreya PUFA-3 and Supelco 18919, resulting in identification of 42 fatty acids.

Copepod biomass and vertical distribution

The sampling in 2002, 2003 and 2005 was estimated from samples collected using a zooplankton pump (ca 1200 L min -1 ), equipped with a 30 µm net, and preserved with Borax buffered formalin (ca 4% final concentration).

The zooplankton samples were counted from 1/ 4 -1/64 fraction of the sample, so that the counts exceeded 200 individuals for all dominant copepod species (see Results). Copepods were identified to the genus or species level, and divided into 7 developmental stages (nauplii + copepodite I-VI). Loose copepod eggs (not in 2005) and egg-sacs were included in the counts. [START_REF] Klein Breteler | Influence of cultivation and food concentration on body length of calanoid copepods[END_REF], Sabatini and Kiørboe (Sabatini and Kiørboe, 1994) and [START_REF] Cohen | Length-weight relationships for several copepods dominant in the Georges Bank-Gulf of Maine area[END_REF] for respectively Temora longicornis, Centropages spp. and Pseudocalanus sp., Oithona spp. and Paracalanus parvus. For Microcalanus pusillus, the length-weight regression of T. longicornis was used. Dry weight was converted to carbon assuming 40% carbon content of the dry weight [START_REF] Mullin | Production of zooplankton in the ocean: the present status and problems[END_REF].

To avoid bias due to low numbers of individuals, the vertical distribution of different life-stages was examined in detail only for those species which contributed ≥ 10 % to the total biomass for the given year. To ensure sufficient numbers of individuals all copepodite stages (I-V) were grouped together. Vertical distribution was expressed both in absolute numbers (ind. m -3 ) and as a proportion of the population at a given depth (% of total biomass).

Reproduction

In situ egg production and hatching success of Acartia clausi and Centropages typicus were measured along 4 transects during spring (March-April), summer (May/June, July/August) and autumn (September) 2001. Egg production and hatching experiments were carried out wherever A. clausi and / or C. typicus were present, which resulted in a total of 22 and 10 in situ egg production stations for A. clausi and C. typicus, respectively (Fig. 1, Table I). In addition, in situ egg production of C. typicus was measured in one occasion in 2003. The bio-assay in 2003 was conducted at 4 stations along one transect, which was repeated 6 times, so that each station was sampled during 6 subsequent days at approximately same time of the day.

In situ reproduction: Individuals for the in situ experiments were collected by vertical hauls from below the thermocline, with a 180 µm plankton net (WP2) equipped with a 5 L nonfiltering cod end. Healthy active females were placed individually into 250 ml bottles containing 64 µm sieved ambient seawater from 4 m depth (15-30 replicates) and the bottles were placed in a dark temperature-controlled room (6-12°C). The sieving removed any ambient copepod eggs, while parts of the small size plankton community passed through. After 24 hours, eggs were collected by filtering the samples gently onto a 20 µm mesh and immediately counted using a stereo microscope.

The prosome lengths of the females were measured. Eggs from all replicates from a specific station were pooled and incubated in 650 mL bottles containing 20 µm sieved ambient water, at in situ 8 temperature (6-12°C) appropriate for the station where they were collected. After an additional 72 hours, the bottle contents were filtered onto a 20 µm mesh, and fixed in 5% Lugol's solution. Eggs, nauplii and empty egg shells were later counted under a binocular microscope. As the incubation time in April-May was too short to obtain complete hatching at the low temperature (6-7 °C), hatching success during these months could not be estimated. Weight-specific egg production was calculated based on the length-carbon regression of [START_REF] Berggreen | Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for determination of copepod production[END_REF], assuming the carbon content of eggs to be ca 0.04 µg C egg -1 [START_REF] Kiørboe | Scaling of fecundity, growth and development in marine planktonic copepods[END_REF].

Bioassay: The bioassay was conducted using Acartia tonsa, which have been cultured in the National Institute for Aquatic Resources (DTU Aqua) for over 100 generations. Typically, copepods are reared at 18 °C and 33 ‰, and fed Rhodomonas sp. in excess. In the experiments, recently (≤ 3 days before) matured A. tonsa were fed with in situ water from the chlorophyll maximum layer and surface, as well as with a saturating concentration (> 400 µg C L -1 ) of Rhodomonas sp. (control for standard egg production), at a constant temperature of ca 15 °C. This bioassay approach excluded the effects of body size, maternal nutrition and temperature, so that all the changes in egg production and hatching success were supposedly due to changes in food conditions, either quantity or quality [START_REF] Müller-Navarra | Seasonal patterns of food limitation in Daphnia galeata: Separating food quantity and food quality effects[END_REF]Koski et al., 2010).

However, the use of cultured copepods introduces other problems; such as the typically low egg production, as well as the question of how well do the cultured individuals represent in situ animals (Koski et al. 2010). To reduce these problems, we included the standard food source Rhodomonas sp., to give an estimate of the close-to-maximum egg production of the culture.

The bioassay was run for a total of 6 days, of which the first 24-h period served as an adaptation time. In the beginning of the experiment ca 15 adult females and 3 adult males were sorted out from the culture and placed together into each of 5 replicate bottles of ca 0.6 L. After each 24-h period, the eggs were counted, the copepod condition checked (dead/alive), and the copepods were thereafter transferred to a new food suspension, which was collected daily. The mortality during the incubations was relatively low, typically < 10 % d -1 in both surface and chlorophyll maximum water. For estimation of hatching success, the eggs from the 5 replicate bottles were pooled into one 0.3 L hatching bottle, which was left to incubate for ca 3 days. All bottles were placed on a plankton wheel (ca 1 RPM), in the dark and at ca 16 °C. This was approximately similar to the surface temperature at the study site. At the termination of the hatching experiment, the bottle-contents were carefully filtered onto a 50 µm sieve and preserved in 4% acid Lugol's solution, for later counts of eggs and nauplii. Copepod secondary production was calculated from the depth-integrated carbon biomass and weight-specific egg production, assuming that all stages have equal weight-specific growth rates [START_REF] Berggreen | Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for determination of copepod production[END_REF]. This method has many limitations (see e.g., [START_REF] Hirst | Are in situ weight-specific growth rates body-size independent in marine planktonic copepods? A re-analysis of the global syntheses and a new empirical model[END_REF], and the secondary production rates presented here should thus be taken as rough estimates. To estimate the egg production we used the egg sacs collected with the zooplankton pump hauls for sac-spawning species, and both eggs collected with the pump and egg production measured in incubations for broadcast spawning species. The egg production of the sac-spawners was calculated by dividing the depth integrated egg sac biomass by the biomass of sac-spawning females (egg-ratio method; e.g., Sabatini and Kiørboe, 1994) and temperature-dependent development time of eggs [START_REF] Andersen | Hatching rate of the egg-carrying estuarine copepod Eurytemora affinis[END_REF]. The egg production of broadcast spawners was first estimated similarly, but since the rates obtained were very low, it seemed likely that the number of eggs in the water column did not accurately represent the egg production (due to e.g., sinking of eggs). We therefore decided to use the regression between egg production and body size (prosome length) obtained from all in situ incubations pooled together to estimate the egg production of broadcast spawning copepods. However, as the egg production of copepods < 1 mm in prosome length was not related to body size (Fig. 2), we decided to use the average egg production of all individuals < 1 mm for this size class, and only use the significant regression between prosome length and egg production for the individuals > 1 mm (R 2 = 0.84; p < 0.0001; Fig.

2).

Statistics

Egg production, spawning percentage, brood size, hatching and prosome length were tested for differences between stations or water layers (only bioassay) using 1-or 2-way analyses of variance (ANOVA); and, after pooling the stations, for differences between months (only in situ reproduction). The Tukey HSD post-hoc test was used for pairwise comparisons. If the conditions for normality and equal variance were not met, the non-parametric Kruskal-Wallis test and Dunn's pairwise comparison were used. Pearson correlation analysis was used to check for the correlations between egg production / hatching, body size and temperature, as well as between biomass / secondary production and temperature, chl-a and microplankton concentration. To investigate the dependence of egg production and hatching success on environmental variables, we ran a series of multiple regression analyses. To account for the effects of body size and temperature on the in situ reproduction, a multiple regression analysis was first run for egg production, body size and temperature. As the model showed a contribution of body size rather than temperature (see Results), we then used the weight-specific egg production in forward stepwise multiple regression analysis with the environmental variables, without correcting the egg production for temperature. Finally, we combined the significant factors from both models for a final model. For in situ reproduction, temperature and salinity were included as averaged values, while chl-a and phytoplankton concentration / composition were included as integrated values for the 0-50 m water column. For fatty acids only values from the chlorophyll maximum layer were available. As Acartia spp. in the North Sea appears to be relatively evenly distributed in the upper 40 m of the water column (P. Munk, DTU Aqua; pers. com) we consider this to be a reasonable assumption.

The environmental variables used in the multiple regression model, were chosen after data reduction by principal component analysis (PCA). The PCA was run on a correlation matrix with Varimax rotation, first separately for all the fatty acids, and then after combining the fatty acids which had the highest loading on different PCs with the other environmental variables. The PCA for fatty acids included 22 stations. In short, the PCA grouped the fatty acids according to the size fractions rather than type, so that most fatty acids in the size fraction 0-20 µm had a moderate or significant loading on PC3, most fatty acids in 20-40 µm had a significant loading on PC2, and most fatty acids in 40-80 µm had a significant loading on PC1 (data not shown). However, the be used together with the egg production and hatching in the multiple regression model. Chl-a was used as a representative of PC3 rather than dinoflagellate biomass, as this allowed more stations ( 22) to be included in the model.

As standard copepods from a culture were used in the bioassay experiment, we did not need to account for the effect of body size or temperature, and only a single multiple regression was run. For all analysis we used responses from day 4 only, as this allowed a long adaptation time for the animals combined with simultaneous measurements of all environmental variables; the total number of observations was thus 8 (4 stations with 2 water layers). Similar to the in situ reproduction, PCA was used to select for the environmental variables to be included in the model.

All fatty acids had the highest loading in PC1, while only the ratio of EPA:DHA was separated with the highest loading on PC2 (data not shown). We thus included total fatty acids and the EPA:DHA ratio for the second PCA. The PCA combining fatty acids with other environmental variables separated flagellates and dinoflagellates together with a high concentration of total fatty acids (PC1) from diatoms and ciliates with a high EPA: DHA ratio (Table IIB). We thus chose total fatty acids as a representative of PC1 and EPA:DHA ratio as a representative of PC2. All statistics were conducted using the SigmaStat 3.1 or SPSS 11.5 statistical packages.

Results

Environment

Seasonal variation at transect stations: The water column was well-mixed in March and April, while in May and July a thermocline, accompanied with increased salinity, formed below ca 20 m. There was no strong subsurface chlorophyll maximum layer, but chl-a was either equally distributed in the upper 40 m (March and July), increasing slightly with depth (April), or clearly highest in the surface layer (May) (Fig. 3). The average water column temperature (0-50 m) over the year was rather similar at all stations, 4-6 °C during March-April, close to 10 °C in May and 11-15 °C during July-September (Table III). The highest chl-a concentrations of ca 5 µg L -1 (ca 140 mg m -2 ) were observed during spring in March at T3 and May at T5, at other times and transects the average chl-a concentrations ranged between 1.6 and 3.5 µg L -1 (38-102 mg m -2 ). Diatoms dominated the microplankton composition at all stations and times and the proportion of heterotrophic organisms was typically between 10-20% of the total microplankton carbon. Polyunsaturated fatty acids (PUFA) were abundant in the layer of maximum chl-a, with concentrations up to ca 50 µg L -1 , forming between 14 and 36% of the total fatty acids (Table III).

Year-to-year variation at the Dogger Bank: In all years surface temperatures were high (ca 18 °C) and a thermocline was situated between ~ 25 and 30 m (Fig. 4). Clear chlorophyll subsurface maxima were found just below the thermocline, with peak concentrations of 2-2.5 µg chl-a L -1 . Also the biomass of heterotrophic microplankton was higher in the chlorophyll maximum layers than at the surface, up to ca 30 µg C L -1 (Fig. 4). During the bio-assay study when microplankton composition and fatty acid concentration of seston were measured in more detail, about half of the microplankton was formed by heterotrophic dinoflagellates and ciliates (Table III), with an approximately equal contribution of autotrophic flagellates, dinoflagellates and diatoms.

Both the absolute (≤ 12 µg L -1 ) and proportional (5-12%) concentrations of PUFA were lower during the bioassay study than on any of the transects sampled for in situ reproduction (Table III).

Details of the environmental conditions in the sampling area in 2002 are presented by Poulsen and Kiørboe [START_REF] Poulsen | Vertical flux and degradation rates of copepod fecal pellets in a zooplankton community dominated by small copepods[END_REF], and in 2005 by Koski et al. [START_REF] Koski | Degradation of appendicularian houses by pelagic harpacticid Microsetella norvegica and other agents[END_REF] and Jónasdóttir and Koski (Jónasdóttir and Koski, submitted).

Vertical distribution and biomass

No diel vertical migration was observed for any of the copepod species, but the different species seemed to consistently inhabit different depth layers (Fig. 5). While Pseudocalanus sp. and Temora longicornis seemed to primarily reside below the thermocline, copepod nauplii, Paracalanus parvus, Microcalanus pusillus and Oithona spp. were mainly found above the thermocline. Centropages typicus was primarily found above the thermocline in 2002, but in 2005 this species seemed to have a bimodal daytime distribution, with about half of the individuals residing below the thermocline. If the vertical distribution was separated into life-stages it appeared that all life-stages (separated to nauplii, copepodites, males and females) of Oithona spp. and P. parvus preferred the surface layers. The distribution of other species was more spread with most of the copepodites of M. pusillus and C. typicus rather evenly distributed in the upper 40 m, and most of the copepodites of T. longicornis and Pseudocalanus sp. evenly distributed below 20 m. In contrast, adults of these species were most abundant between 30 and 40 m, thus close to the chlorophyll maximum layer, with the exception of Pseudocalanus sp. females, which were most abundant close to the bottom (data not shown). If the vertical distribution was compared with the temperature and chl-a concentration, it appeared that Oithona spp., Paracalanus parvus and Centropages typicus were most abundant in the layer where temperature was > 14 °C, while Temora longicornis and Microcalanus pusillus were spread in temperatures between 6 and 15 °C and 7 and 18 °C, respectively (Fig. 6A).

Pseudocalanus spp. had a bimodal distribution with a high proportion of individuals residing either in rather cold (6-8 °C) or rather warm (> 14 °C) water layers. With the possible exception of adult stages of T. longicornis, M. pusillus and C. typicus, none of the species appeared to be strongly associated with the chlorophyll maximum layer, as the peak distribution of most life-stages of all species typically was at ≤ 1 µg Chl-a l -1 (Fig. 6B). Similarly, no effect of salinity was observed for any of the species or life-stages (data not shown).

The depth integrated biomass of small copepods varied threefold, with the lowest IV, Fig. 5).

No significant correlation was observed between the depth-integrated copepod biomass and phytoplankton (average and max. chl-a concentration) or microzooplankton concentration, neither between the total biomass and temperature (Pearson p > 0.05; data not shown). However, the biomass of sac-spawning copepods was significantly negatively correlated with temperature (Pearson correlation coefficient -0.789; p < 0.05).

Egg production and hatching

In situ reproduction: The egg production rate of Acartia clausi was highest in April (21 ± 15 eggs f -1 d -1 ; mean ± SD), due to the significantly higher number of eggs produced per brood compared to the other months (Kruskal-Wallis; H 3 = 95; p < 0.001). However, there were also significant differences in egg production between stations (1-way ANOVA, F 6 = 6.7, p < 0.001 for April; Kruskal Wallis, H 4 = 45.5; p < 0.001 for June and Kruskal-Wallis, H 7 = 64.8, p < 0.001 for July), sometimes with > 10-fold differences in egg production even between the stations on the same transect (Fig. 7A). Hatching success over the year varied between 60 and 75%, but, due to the short incubation time, the spring hatching results had to be omitted. The between-station variation in hatching success was typically less than the variation in egg production, although a few in July, respectively. The female prosome length indicated that the spring, early summer and late summer individuals belonged to different generations (Kruskal-Wallis; H 3 = 252; p < 0.001; Table V). Due to the low female abundance in spring, the high egg production in April did not result in a high number of nauplii, but the nauplii peak was observed in late summer during the peak abundance of females (Fig. 7A).

Centropages typicus egg production was highest in late summer (65 ± 56 eggs f -1 d -1 ), intermediate in spring (34 ± 24 eggs f -1 d -1 ) and low in autumn (14 ± 17 eggs f -1 d -1 ), due to the significantly lower number of eggs per brood in September than in March or July (Kruskal-Wallis; H 2 = 70; p < 0.001). Similar to Acartia clausi, also C. typicus egg production was significantly different between stations, both in July (Kruskal-Wallis; H 4 = 17.0; p < 0.01) and in September (Kruskal-Wallis; H 3 = 29.8; p < 0.001), although with typically lower between-station variation (Fig. 7B). When measured, the hatching success was relatively low (36 ± 11%). Based on the female prosome length, it appeared that the spring individuals belonged to a different generation than summer and autumn individuals (Kruskal-Wallis; H 2 = 24; p < 0.001; Table V). Similar to Acartia clausi, the high egg production of spring and summer did not result in a nauplii peak which only occurred during the high female abundance in September (Fig. 7B).

The egg production rate of Acartia clausi was significantly positively correlated with prosome length, and significantly negatively correlated with temperature (Pearson correlation; p < 0.01; Fig. 8), while hatching did not correlate with prosome length, temperature or egg production (Pearson; p > 0.5; data not shown). Egg production of C. typicus was not significantly related to body size or temperature (Pearson; p > 0.05; Fig. 8). As a multiple correlation analysis with egg production of A. clausi against body size and temperature showed that the variation in egg production was mostly explained by body size (Table VIA), we decided to use the weight-specific egg production in the multiple regression analysis with food variables, but not to correct the egg production for temperature. Based on the PCA, we chose one environmental variable representing each PC for the multiple correlation analysis with the weight-specific egg production and hatching success. The analysis showed that PC2 and PC4 represented by EPA: DHA ratio in the size class of 40-80 µm and percentage of PUFA from the total fatty acids in the 0-40 µm size fraction had a significant effect on egg production, while only the EPA:DHA ratio in the 40-80 µm size class had a significant effect on hatching success. The model implied an increase of 0.02 µg C (µg C) -1 d -1 in weight-specific egg production and a decrease of 19 % in hatching success with a unit change in VIA). Along with the EPA: DHA ratio in 40-80 µm, also diatoms, total autotrophs and total microplankton concentration had a significant loading on PC2, while flagellates had a significant loading along with the PUFA proportion in PC4 (Table IIA), so that the effect of these factors could not be separated.

Both the model with body size and the model with food variables, however, only explained ≤ 60% of the variation in egg production. We thus decided to combine these two and ran the multiple regression analysis with the egg production, body size and EPA:DHA ratio in 40-80 µm size class. The combination of body size and EPA:DHA ratio in 40-80 µm (representing the biomass of diatoms and autotrophic microplankton in general) explained 68% of the variance in egg production, with an increase of 46 eggs f -1 d -1 with a millimetre increase in prosome length and an increase of 4.8 eggs f -1 d -1 with a unit increase in EPA:DHA ratio. Due to scarcity of data, a multiple regression analysis was not attempted for Centropages typicus egg production or hatching success.

Bio-assay: Egg production of the cultured Acartia tonsa fed with in situ water was generally low, on average ca 5 and 1.5 eggs f -1 d -1 , in the chlorophyll maximum and surface layers, respectively (Fig. 9A,Table V). The cumulative egg production (day 3) was significantly different between the stations (2-way ANOVA; F 3,33 = 9.8; p < 0.001) and the water layers (F 1,33 = 61.5; p < 0.001), with significantly highest egg production at station 8 and in the chlorophyll maximum layer (Tukey; p < 0.05). The average egg production in the chlorophyll maximum layer was similar to the egg production on Rhodomonas sp. (Tukey HSD; p > 0.05). Hatching success was high, respectively 81 and 86% in the surface and chlorophyll maximum layers, and similar to the hatching on Rhodomonas sp. (Fig. 9B, Table V). The multiple regression analysis confirmed the positive effect of increasing EPA: DHA ratio (here associated with diatom and ciliate biomasses) on egg production and its negative effect on hatching success, as observed with the in situ reproduction.

Model coefficients suggested an increase in egg production by 11.4 eggs f -1 d -1 and a decrease in hatching success by 59.7 % with a unit change in EPA: DHA ratio. However, the model explained only 45% of the egg production and 55 % of the hatching success of Acartia tonsa (Table VIB).

Secondary production

The secondary production of dominant neritic copepods in July-August was typically highest at the shallow station 8 (18-19 mg C m -2 d -1 ). The secondary production at this station was IV). Secondary production thus did not always correlate with the biomass, but a high proportion of broadcast spawning species tended to lead to a high secondary production, likely due to their higher weight-specific egg production (Table IV). No significant correlations were observed between secondary production and phytoplankton (average or maximum chl-a) or microzooplankton biomass (Pearson, p > 0.05; data not shown), but secondary production was significantly positively correlated with the average water column temperature (Fig. 10).

Discussion

In summary, our results showed that the vertical distribution of small copepods was not determined by subsurface chlorophyll maximum, but only temperature seemed to have an effect on the distribution of (some) species. Secondary production, however, was highest at the most productive station 8 on top of the Dogger Bank, and egg production seemed to benefit from the chlorophyll maximum particularly at this station. Secondary production appeared to be governed mostly by the copepod species composition and reproductive rate, as the broadcast spawning calanoid species had a substantially higher weight-specific reproduction than e.g., Oithona spp.

While reproduction seemed to be controlled by the changes in body size and qualitative aspects of food (such as the EPA: DHA ratio), temperature appeared to have an influence on species composition (broadcast vs. sac-spawning species). The seasonal differences in temperature (body size) and food quality likely overrode the potential effects of food quantity, which otherwise could have manifested in a high reproduction at productive frontal stations. These findings and their relevance are discussed in detail below.

Secondary production on Dogger Bank

It has been suggested that the frontal zones in the North Sea are hot spots of biological activity, where production, food-web structure and diversity change within short distances [START_REF] Nielsen | Zooplankton diversity and the predatory impact by larval and small juvenile fish at the Fisher Banks in the North Sea[END_REF]. In these areas the elevated chlorophyll concentration would support high zooplankton production, with an associated biomass peak of copepods in the chlorophyll maximum [START_REF] Nielsen | Structure of plankton communities in the Dogger Bank area (North Sea) during a stratified situation[END_REF][START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF]. On the Dogger Bank, stations 5 and 8 could be considered the most productive, as they were located closest to the area with the upwelling of new nutrients [START_REF] Richardson | Subsurface phytoplankton blooms fuel pelagic production in the North Sea[END_REF]. Our study confirmed the high secondary production at these stations, though at station 5 only when the small copepods were dominated by broadcast-spawning calanoids. The other potential frontal stations, 5.1, 5.2 and the stations in the transect 1 (see Fig. 1), did not show a clear subsurface chlorophyll maximum, but the chl-a values were low throughout the summer. Consequently, reproduction was not elevated at these stations (Fig. 7), and reproduction on an annual scale appeared controlled by other factors than chl-a.

The highest depth integrated biomass and secondary production at stations 5 and 8 were similar to those which have been reported previously for the small copepods in the frontal areas of the North Sea, but lower than the typical biomass in the coastal areas where the small copepod species dominate [START_REF] Nielsen | Zooplankton diversity and the predatory impact by larval and small juvenile fish at the Fisher Banks in the North Sea[END_REF]. It appeared that the high biomass did not directly lead to a high secondary production (see Table IV), but species composition (broadcast vs. sac-spawners) and weight-specific reproduction determined the secondary production. Neither biomass nor secondary production were connected to chlorophyll concentration, but temperature seemed to have an effect on dominance of specific groups. It seemed that the biomass of sacspawning copepods was negatively related to temperature, while the total secondary production increased with increasing temperature, with a highest depth-integrated production occurring at temperatures > 13 °C (Fig. 10). This was likely due to the high abundance of two out of three dominant calanoid broadcast-spawning species (Centropages typicus and Microcalanus pusillus) at the warm temperatures of ca 18 °C (Fig. 6), although temperature could also have a direct influence on the reproduction rate (e.g., Koski & Kuosa, 1999; Table VI). If the secondary production of small copepods was compared to that of Calanus spp. (Jónasdóttir and Koski, submitted) it appeared that the small copepods dominated the secondary production at station 8, while at stations 3 and 5 further away from the Dogger Bank secondary production of Calanus spp. was ca 2-6 times higher than that of small copepods. However, there was a large between-year variation in secondary production of both Calanus spp. and small copepods at these stations, with a 2-3 times higher secondary production of small copepods in 2002, but 2-6 times higher secondary production of Calanus spp. in 2005 (Jónasdóttir and Koski, submitted). Our results for the vertical distribution of dominant small copepod species did not support the observations of highest biomass in the subsurface chlorophyll maximum (Fig. 5), but were more similar to the studies showing maximum abundances below or above the pycnocline [START_REF] Harris | Interactions between diel vertical migratory behaviour of marine zooplankton and the subsurface chlorophyll maximum[END_REF][START_REF] Hays | Long-term changes in the diel vertical migration behaviour of zooplankton[END_REF]. It appeared that only adult stages of some copepod species (Temora longicornis, Centropages typicus and Microcalanus pusillus) resided in or close to the chlorophyll maximum layer, while juvenile stages and all stages of other species preferred layers above or below the chlorophyll maximum.

The effect of subsurface chlorophyll maximum on vertical distribution and reproduction

In principal the choice of the habitat layer of copepods should represent the best choice between maximum food abundance and minimum predation risk [START_REF] Fossheim | Habitat choice by marine zooplankton in a high-latitude ecosystem[END_REF]. Although visual predators (such as planktivorous fish) mainly forage in the surface layers, invertebrate predators can be abundant in the subsurface chlorophyll maximum [START_REF] Fossheim | Habitat choice by marine zooplankton in a high-latitude ecosystem[END_REF]. Some of the main invertebrate predators are chaetognaths, which have been shown to feed selectively on some small copepods, such as Pseudocalanus sp., and may control their biomass in late summer and autumn (Tönnesson and Tiselius, 2005). Although no significant connection between the abundance of different copepod species in the chlorophyll maximum and chaetognath abundance was observed in the study area (data not shown), it appeared that in 2003 the maximum abundance of chaetognaths (ca 50 ind. m -3 ) was located close to the surface, while in 2002 and 2005 when copepods were typically either above or below the chlorophyll maximum layer, chaetognath abundances were at their maximum (20-50 ind. m -3 ) in the chlorophyll maximum. It could be that the vertical distribution of invertebrate predators contributes to the vertical distribution of small copepods, along with food availability, temperature and salinity.

However, in the present study we only observed weak suggestions of the effects of temperature and invertebrate predators and no effects of salinity or subsurface chlorophyll maximum.

Harris [START_REF] Harris | Interactions between diel vertical migratory behaviour of marine zooplankton and the subsurface chlorophyll maximum[END_REF] suggested that, although copepods rarely reside in the subsurface chlorophyll maximum, they fill their guts and produce maximum amount of eggs while swimming through it during vertical migration. This behaviour was shown with Calanus helgolandicus, while in the study of Harris [START_REF] Harris | Interactions between diel vertical migratory behaviour of marine zooplankton and the subsurface chlorophyll maximum[END_REF] Acartia spp. was the only copepod species which was permanently associated with the chlorophyll maximum layer. The subsurface chlorophyll maximum layer could thus be especially profitable for these species, although other neritic copepods would neither migrate through nor reside in the chlorophyll maximum layer (see Fig. 5).

The egg production of Acartia tonsa was always higher when incubated in chlorophyll maximum water than in surface water at all stations. It has been suggested that the abundant dinoflagellates in the subsurface chlorophyll blooms would be profitable for copepod production [START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF]. However, this was not case in our study (Table VIA), although on average 55% of the phytoplankton biomass in the subsurface maximum was composed of dinoflagellates. The most abundant dinoflagellates during our study were various Ceratium species, which due to their large size and spiny morphology are typically not ingested by Acartia spp., although larger copepods such as Calanus spp. might graze on them [START_REF] Jansen | Copepod feeding behaviour and egg production during a dinoflagellate bloom in the North Sea[END_REF].

It has also been shown that phytoplankton in the subsurface chlorophyll maximum is less nutrient limited than phytoplankton in the surface layer [START_REF] Richardson | Spatial heterogeneity in the structure of the planktonic food web in the North Sea[END_REF]2000). As exponentially growing algae are generally of a better quality food than senescent ones (Jónasdóttir, 1994), a positive effect of food quality could be expected due to a higher nutrient concentrations in the chlorophyll maximum layer. If we assume that most of the phytoplankton diet of Acartia tonsa was formed by diatoms and flagellates (thus not Ceratium), and plot the egg production rate as a function of the concentration of these groups for the two layers, it indeed appears that the egg production was generally higher while feeding on chlorophyll maximum algae than on the corresponding concentration of surface algae (Fig. 11A). This apparent higher quality could have been related to the proportional PUFA content, which was higher in the chlorophyll maximum (12 ± 6%) than in the surface (5.3 ± 0.7), the high EPA: DHA ratio as suggested by the multiple regression analysis, or possibly to e.g., lower C:N ratio of the seston, which was not measured in the present study. Whatever the reason for improved quality, it did not affect the hatching, as no difference between the layers was observed for the hatching success (Fig. 11B).

Factors controlling reproduction

We observed both seasonal (between months) and spatial (between stations) variation in egg production. As observed earlier, Acartia clausi egg production was positively related to prosome length and negatively related to temperature, likely reflecting the negative effect of temperature on body size (e.g., [START_REF] Halsband | Reproductive cycles of dominant calanoid copepods in the North Sea[END_REF]. As the body size or temperature rarely differed between the sampling stations (during the same month), the effect of body size on egg production was mainly relevant at an annual scale. If the effect of body size was removed by using the weight-specific egg production, the egg production appeared to be dependent on the qualitative aspects of the food, such as proportional PUFA content and EPA: DHA ratio. This effect of food could have been due to both spatial and seasonal patchiness in the food environment and could thus EPA:DHA ratio has been observed to correlate with egg production in several studies, sometimes positively and sometimes negatively (reviewed by [START_REF] Jónasdóttir | Assessing the role of food quality in the production and hatching of Temora longicornis eggs[END_REF]. The reason for the correlation between egg production and EPA:DHA ratio is unclear, although it is assumed that it has to do with the chemical similarity of these compounds and the following competition in chemical and physiological reactions (Sargent et al., 1999). However, in our study the beneficial effect of a high EPA:DHA ratio and a high percentage of PUFA likely reflected a positive effect of autotrophic food sources in general (diatoms in particular), as a high EPA:DHA ratio also reflected high concentrations of diatoms and total autotrophic microplankton (Table II). This suggested that the body size (likely controlled by temperature) and specific fatty acids abundant in phytoplankton were the main factors controlling egg production. Our results are thus in agreement with previous field studies demonstrating a combined effect of temperature (via body size) and food (Calbet & Agusti 1999[START_REF] Bunker | Fecundity of marine planktonic copepods: global rates and patterns in relation to chlorophyll a, temperature and body weight[END_REF][START_REF] Maps | Egg production and hatching success of Temora longicornis (Copepoda, Calanoida) in the southern Gulf of St. Lawrence[END_REF], although EPA: DHA ratio (or diatom biomass) rather than bulk chl-a appeared to be a better predictor of egg production.

As observed earlier, egg production and hatching success were controlled by different factors; in fact while the high EPA: DHA ratio was beneficial for egg production, it appeared to have a negative effect for hatching success. Typically, high hatching success correlates with a high ingestion of DHA, while a negative correlation is often observed between hatching success and EPA: DHA ratio (Jόnasdόttir et al., 2009). High EPA concentration of seston is mostly associated with a high biomass of diatoms, which typically promotes high egg production but low hatching success (e.g., Peters et al., 2007). As diatoms often dominate the phytoplankton in the coastal areas of the North Sea, particularly during the spring bloom, a low hatching success, together with a low female abundance, can lead to low recruitment irrespective of the high egg production rate.

In the present study the maximum egg production of Acartia clausi occurred in April, and was similar to the rate previously recorded for this species [START_REF] Halsband | Reproductive cycles of dominant calanoid copepods in the North Sea[END_REF]Wesche et al., 2007). Due to the low number of females in the water column, the high egg production in the spring was, however, not followed by an increase in nauplii abundance, which only peaked in the summer. Similarly, the peak egg production of Centropages typicus was highest in July-August, but the peak abundance of nauplii first occurred 2 months later in September.

Although the irregular sampling and advection did not allow a detailed analysis of population dynamics; it appeared clear that the high egg production in spring (Acartia clausi) or summer (Centropages typicus) did not lead to an increase in biomass. For A. clausi it is also likely that the female peak in June did not originate from April eggs, but rather March eggs: even in optimal food conditions the generation time of A. clausi at the 6-7 °C would be > 60 days [START_REF] Huntley | Temperature dependent production of marine copepods: a global synthesis[END_REF][START_REF] Klein Breteler | Development of Acartia clausi (Copepoda, Calanoida) cultured at different conditions of temperature and food[END_REF], while the time between the sampling events was ca 40 days. The April cohort thus more or less disappeared from the water column. It seems that the success of spring generations of small copepods can be extremely variable, with high mortality rates following from for example, predation and cannibalism (Peterson and Kimmerer, 1994), food concentration (Koski et al., 2010) or temperature affecting the timing of the spring bloom [START_REF] Dutz | Life cycle and spring phenology of Temora longocornis in the Baltic Sea[END_REF]. Plankton Res., 16, 1329--1351. Sargent, J., McEvoy, L., Estevez, A., Bell, G., Bell, M., Henderson, J. and Tocher, D. (1999) Lipid nutrition of marine fish during early development: current status and future directions. , 179, 217--229. Tönnesson, K. and Tiselius, P. (2005) Feeding of the chaetognaths Sagitta setosa and S. elegans in relation to prey abundance and vertical distribution. Mar. Ecol. Prog. Ser., 289, 177--190. Uttermöhl, H. (1958) Zur vervollkommung der quantitativen phytoplankton-methodik. Temperature, (S) salinity, (Chl) chlorophyll a, (Flag) flagellates, (aut dino) autotrophic dinoflagellates, (het dino) heterotrophic dinoflagellates, (dino) total dinoflagellates, (diat) diatoms, (cil) ciliates, (aut tot) total autotrophs, (het tot) total heterotrophs, (micropl tot) total microplankton, (PUFA) polyunsaturated fatty acid concentration, (PUFA%) percentage of PUFAs from total fatty acids. All microplankton variables are biomass in carbon (µg L -1 ).

Aquaculture

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Table III. Average temperature, salinity (‰) and integrated chl-a concentration (mg m -2 ) for the 0-50 m water column (± SD), list of dominant algae groups with the number of stations in which the group dominates in parenthesis, integrated concentration of heterotrophic and total microplankton biomass (µg C L -1 ) with the percentage of heterotrophs in parenthesis, concentration of polyunsaturated fatty acids ARA (18:3n3), DHA (22:6n3), EPA (20:5n3) and total PUFA (µg L -1 ; with the percentage of PUFA from total FA in parenthesis), as well as the DHA: EPA ratio (µg: µg) in the chlorophyll maximum layer averaged over the different transects in March-September (T1-5) and August (Bio-assay study; S: surface water, M: chl-max. layer). For the location of transects and the number of stations per transect, see Fig. 1. and Table I (-) Missing data. Other abbreviations as in Table II.

Table IV. Depth-integrated biomass (Bm; mg C m -2 ) and secondary production (Sp; mg C m -2 d -1 ) of broadcast and sac-spawning copepods, as well as the average egg production (Ep; % body weight d -1 ) used to calculate the secondary production, and the species dominating the biomass of small copepods (Acartia spp., Temora longicornis, Pseudocalanus sp., Centropages typicus, Microcalanus pusillus, Paracalanus parvus and Oithona spp.) in 2002, 2003 and 2005 (see Fig. 5).

The egg production of sac-spawning copepods was calculated using the egg ratio method; the egg and egg production (Fig. 2; Methods). For station localities, see Fig. 1. Table V. Average egg production (eggs f -1 d -1 ), percentage of females producing eggs (% day -1 ) and brood size (no of eggs brood -1 ), hatching success (%) and female prosome length (mm) of Acartia A. tonsa. Weight-specific egg production is expressed as µg C (µg C) -1 d -1 , egg production in number of eggs f -1 d -1 , body size as length in mm, chl-a and total fatty acids as µg L -1 , ciliate biomass as µg C L -1 , PUFA as a percentage from total fatty acids, and EPA:DHA as a weight ration (µg : µg). The coefficients describe the expected increase / decrease in egg production or hatching per a unit change of the independent variable. (*) Denotes the significant coefficients at the p < 0.05 level, (**) significant coefficients at the p < 0.01 level. (2003) and (open circle) sampling for vertical distribution and biomass (2002, 2003 and 2005). In 2001, the first number refers to the transect, the second number to the station (e.g., 3.3 is the station 3 on the transect 3). Only species and years representing ≤ 9% of the total biomass of small copepods are included in the figure (see Fig. 5). 1.27 ± 0.07 September (4)

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  was conducted at 2-4 stations along one Dogger Bank transect, with samples taken at depth-intervals of 10 m (2002) or 5 m (2003 and 2005). The number of daily samplings was one in 2003 and 2005, and nine in 2002. Zooplankton abundance

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  percentage of PUFA from total fatty acids in 0-20 and 20-40 µm size fractions, and EPA: DHA ratio in the 40-80 µm size fraction, typically had a high loading on a different PC than the absolute concentrations. We thus chose the sum of PUFAs in different size classes (0-20, 20-40 and 40-80 µm), percentage of PUFAs in 0-20 and 20-40 µm size class and DHA: EPA ratio in 40-80 µm size class for further analysis with the other environmental factors. Due to the missing data on phytoplankton biomass, the final PCA only included 11 stations. Roughly, the PCA grouped together diatoms with the total (autotrophic) microplankton biomass and EPA:DHA in 40-80 µm size fraction (PC1), heterotrophic biomass of all kinds (PC2), dinoflagellates and chl-a (PC3), flagellates represented by a high PUFA percentage in the 0-20 µm fraction (PC4), and temperature with a high percentage of PUFA in 40-80 µm fraction (PC5; Table IIA). Based on this, we chose EPA:DHA in the 40-80 µm size fraction as a representative of PC1, Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto a representative of PC2, chl-a as a representative of PC3, percentage of PUFA in the 0-20 µm size fraction as a representative of PC4 and temperature as a representative of PC5, to

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  values observed in 2002 (90-120 mg C m -2 ), while in 2003 and 2005 the biomass ranged from 130 to 290 mg C m -2 . Typically, sac-spawners (Oithona spp.) dominated the biomass in 2002, while in 2003 all stations were dominated by calanoid broadcast spawning species (Microcalanus pusillus and Centropages typicus), and in 2005 the dominance switched between broadcast spawners (mainly Temora longicornis; station 8) and sac-spawners (Oithona spp.; station 5; Table

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  successes of 26 and 33% were observed at stations 5.7 in June and 3.4

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  . andKiørboe, T. (1994) Egg production, growth and development of the cyclopoid copepod Oithona similis. J.

  Theoretische und Angewandted Limnologie 9, 1-38. Wesche, A., Wiltshire, K.H. and Hirche, H.J. (2007) Overwintering strategies of dominant calanoid copepods in the German Bight, southern North Sea. Mar. Biol., 151, 1309--1320. Overview of the number of sampling stations, copepod species and measured variables. For the location of stations, see Fig. 1. (T) Temperature, (FA) fatty acids. Table II. Rotated component matrix (PCA with Varimax rotation) showing the loadings of different environmental variables in (A) 2001 and (B) 2003. The significant high loadings (> 0.7) for each principal component (PC) are printed in bold, the significant moderate loadings (> 0.5) are printed in italics; the variables which were chosen for the multiple regression analysis are underlined. (T)

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  clausi and Centropages typicus in March -September (in situ reproduction), and Acartia tonsa in August 2003 (bioassay reproduction; mean ± SD). The number in parenthesis after the month indicates the number of stations, the number after hatching success the number of observed eggs. The letters group the months which are not significantly different from each other (p > 0.05). (Blank) no experiment, (-) missing data. Table VI. Multiple regression coefficients and their significance for the models describing the factors influencing (A) the in situ egg production and hatching of Acartia clausi and (B) the bioassay egg production and hatching of Acartia tonsa. In (A) [Egg production = constant + (C1 body size) + (C2 temperature)], [Weight-specific egg production / hatching = constant + (C1 PUFA % in the 0-40 µm size fraction) + (C2 EPA: DHA in the 40-80 µm size fraction) + (C3 T) + (C4 Chl-a) + (C5 ciliate biomass)] and [Egg production = constant + (C1 body size) + (C2 EPA:DHA in 40-80 µm)]. In (B) [Egg production / hatching = constant + (C1 EPA: DHA) + (C2 total fatty acid concentration)]. The values represent the best fit of a multiple linear regression for egg production, body size and temperature (A. clausi) and the best fit of a forward stepwise regression for the weight-specific egg production and environmental variables (A. clausi) and for all regressions with

Fig. 1 .Formatted

 1 Fig. 1. Map of the study area showing transects and sampling stations in (A) 2001 and (B) 2002, 2003 and 2005. The stations in 2001 were situated along 4 transects in the coastal and central North Sea and sampled in March-September, while stations in 2002-2005 were all situated at the Dogger Bank area and sampled in July-August. (Cross) stations with Acartia spp. reproduction and

Fig. 2 .

 2 Fig. 2. Egg production (eggs f -1 d -1 ) as a function of prosome length (mm) in all incubations. (Solid circles) Acartia clausi, (open circles) Centropages typicus. The dashed line indicates the significant linear regression between the egg production and body size for > 1 mm copepods (see Methods). As the egg production of C. typicus in March and September was exceptionally low (five open circles in the lower right-hand corner of the graph), and clearly out of range in relation to body size, it was excluded from the regression. The regression statistics and the average egg production of < 1 mm copepods are indicated in the figure.

Fig. 3 .

 3 Fig. 3. Example of a typical vertical distribution of temperature (°C; open circles), salinity (‰; solid triangles) and chl-a (µg L -1 ; solid circles) in March (T3), April (T5), May (T5) and July (T5).For location of the transects, see Fig.1.

Fig. 4 .

 4 Fig. 4. Vertical distribution of temperature (°C; lines) and chl-a (µg L -1 ; symbols) at Dogger Bank in 2002, 2003 and 2005. (Squares and solid line) station 2, (diamonds and medium dashed line) station 4, (downward triangles and long dashed line) station 1, (circles and dotted line) station 3, (upward triangles and solid line) station 5 and (stars and dash-dot line) station 8. The numbers indicate concentration of microzooplankton and heterotrophic flagellates (µg C L -1 ± SD) in surface and in chlorophyll maximum layer. For locations of the stations, see Fig. 1.

Formatted:FormattedFig. 6 .

 6 Fig. 6. The proportional distribution of different life stages (% from total) as a function of (A) temperature, and (B) chl-a. (Open symbols with a cross) nauplii, (open symbols) copepodite I-V, (solid symbols) females, (grey symbols) males. (Triangles) 2002, (circles) 2003, (squares) 2005.

Fig. 7 .

 7 Fig. 7. Female abundance (ind. m -2 ), in situ egg production (eggs f -1 d -1 ; mean ± SE) and nauplii abundance (ind. m -2 ) of (A) Acartia clausi and (B) Centropages typicus along the North Sea transects in March-September. The transect / station numbers as in Fig. 1 and Table III. The calculated nauplii abundance refers to the amount of nauplii estimated based on female abundance, egg production and hatching success, while the observed abundance is the number of nauplii in zooplankton (pump) samples (see Methods). (MD) Missing data.

Fig. 8 .

 8 Fig. 8. Egg production (eggs f -1 d -1 ; mean ± SE) of Acartia clausi and Centropages typicus as a function of prosome length (mm; mean ± SE) and temperature (°C). The significant correlation coefficients from the Pearson correlation analysis are shown in the figure.

Fig. 9 .

 9 Fig. 9. (A) Cumulative egg production during the 5 subsequent days of incubation (eggs f -1 ) and (B) average hatching success (%) of cultured Acartia tonsa incubated in water collected from chlorophyll maximum (solid symbols, black columns) and surface (open symbols, white columns) layers at the four Dogger Bank stations, as well as in Rhodomonas sp. (grey symbols and columns; mean ± SE). In (B), 1, 3, 5 and 8 refer to the stations numbers, M and S to chlorophyll maximum and surface layers, respectively.

Fig. 10 .

 10 Fig. 10. Secondary production (mg C m -2 d -1 ) of small copepods as a function of average water column temperature. The Pearson correlation statistics are included in the figure.

Fig. 11

 11 Fig. 11. (A) Egg production (eggs f -1 d -1 ) and (B) hatching success (%) of Acartia tonsa as a function of concentration of diatoms and flagellates (µg C L -1 ) in the bio-assay experiment (mean ± SE). Only the range of concentrations occurring both in the surface and chlorophyll maximum layer are included. (Filled circles) Surface layer, (open circles) chlorophyll maximum layer.

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  Figure 1 Koski et al.

Table I .

 I Overview of the number of sampling stations, copepod species and measured variables.

	Table II. Rotated component matrix (PCA) showing the loadings of different environmental variables
	in (A) 2001 and (B) 2003.
	Measurement	Species			No of stations		Background
	In situ reproduction	A. clausi		22 (2001)	T, Chl-a, microplankton, FA,
		C. typicus	10 (2001) A) Components in 2001	depth-integrated copepod B) Components in 2003
	PC 1	PC 2	PC 3	PC 4	biomass (day) PC 5 PC 1	PC 2	Formatted: Font color: Auto
	C. typicus (27%) (23%)	1 (2003) (16%)	(15%)	(11%)	(80%)	(16%)
	Bioassay reproduction Vertical distribution and biomass EPA:DHA tot T 0.0054 A. tonsa Dominant neritic species F o 4 (2003); chl max and surface 2 (2002): 5 depths, 9 times a day 3 (2003): 11 depths 4 (2005): 11 depths r P r Chl-a, microplankton, FA T, Chl-a, microplankton e 0.339 0.924 e -0.108 0.222 0.514 0.784 Chl -0.519 -0.445 0.561 0.391 0.166 0.557 0.721 S -0.492 0.518 0.482 0.254 -0.183 Flag 0.219 -0.376 -0.151 0.835 -0.111 0.867 0.443 Het dino 0.336 -0.029 0.892 -0.146 -0.017 0.929 0.348 Aut dino -0.218 -0.101 0.912 0.069 0.121 0.970 0.223 Dino 0.080 -0.069 0.988 -0.048 0.054 0.962 0.253 Diat 0.983 -0.011 -0.048 -0.071 -0.005 0.031 0.995 Cil > 50 µm 0.132 0.810 -0.116 -0.003 0.493 Cil < 50 µm -0.130 0.890 -0.265 -0.045 -0.255 Cil tot -0.082 0.939 -0.252 -0.039 -0.108 0.375 0.905 Aut tot 0.973 -0.097 -0.017 0.115 -0.020 0.965 0.248 Het tot 0.137 0.886 0.332 -0.131 -0.115 0.664 0.742 Micropl tot 0.978 0.004 0.020 0.099 -0.033 0.886 0.461 PUFA 0-20 µm 0.628 0.329 0.057 0.562 -0.125 PUFA % 0-20 µm -0.044 0.142 -0.038 0.928 -0.154 PUFA 20-40 µm 0.306 -0.266 -0.080 -0.334 0.666 PUFA % 20-40 µm -0.339 -0.396 0.136 0.641 0.369 PUFA 40-80 µm -0.256 0.021 0.081 -0.206 0.878 EPA:DHA 40-80 µm 0.961 0.008 0.140 -0.145 0.027 FA tot 0.931 0.201 F o r P e e r R e v i e w Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto
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Table III .

 III Average water column temperature and salinity, chl-a concentration, list of dominant algae groups, concentration of heterotrophic and total microplankton biomass, concentration of polyunsaturated fatty acids and the DHA: EPA ratio averaged over the different transects inMarch-September 2001 and August 2003.

													Formatted: Font color: Auto
	Stations		T	S (‰)	Chl-a	Group	Heterotrophs	Microplankton	ALA	DHA	EPA	DHA:EPA	PUFA Formatted: Font color: Auto
			°C		µg L -1		µg C L -1 (%)	µg C L -1	µg L -1	µg L -1	µg L -1		µg L -1 (%) Formatted: Font color: Auto
	In situ												Formatted: Font color: Auto
	March	T3	3.9 ± 1.1	33.7 ± 1.8	138 ± 121	Dia (1)	5.9 (5)	120	0.2 ± 0.02	0.1 ± 0.1	0.2 ± 0.1	1.7 ± 1.8	2.1 ± 0.9 (13.5) Formatted: Font color: Auto
	April	T1	4.7 ± 0.1	34.1 ± 0.5	63 ± 34	-	1 9.6 ± 1.7	-	0.9 ± 0.1	2.4 ± 0.2	1.1 ± 0.3	0.5 ± 0.2	13 ± 0.5 Formatted: Font color: Auto
													(16)
		T3	5.4	34.7	78	-	1 3.7	-	3.1	1.1	3.5	0.3	21 (18) Formatted: Font color: Auto
		T5	6.3 ± 0.1	32.9 ± 0.9	38 ± 16	Dia (3),	40 ± 15 (17 ± 10)	221 ± 96	1.8 ± 1.6	5.8 ± 6.4	7.4 ± 7.2	0.7 ± 0.1	40 ± 28 Formatted: Font color: Auto
						cil (1)							(26)
	May/June: T5	9.7 ± 1.1	33.6 ± 0.5	144 ± 42	Dia (1), flag	26 ± 14 (20 ± 15)	171 ± 202	9.1 ± 14	5.6 ± 1.4	6.6 ± 5.4	1.1 ± 0.5	48 ± 33 Formatted: Font color: Auto
						(1), dino (3)							(36)
	July/August T1 T3 T4 T5	14.0 ± 1.4 11.3 ± 0.7 11.8 ± 1.7 14.4 ± 1.0	33.6 ± 0.3 34.3 ± 0.1 34.8 ± 0.3 F 85 ± 25 102 ± 13 65 ± 19 o 33.4 ± 0.6 91 ± 3.3 r	---Dia (1)	1 4.1 ± 1.8 1 1.2 ± 1.5 1 2.6 12.7 (14)	---76	0.7 ± 0.4 1.1 ± 0.6 2.1 ± 2.0 1.3 ± 1.9	5.5 ± 3.9 3.2 ± 1.4 4.4 ± 3.8 2.1 ± 1.7	4.4 ± 2.8 2.1 ± 0.5 2.4 ± 1.3 2.7 ± 3.4	1.4 ± 0.6 1.5 ± 0.3 0.9 ± 0.4 0.6 ±0.1	17 ± 8 (34) 14 ± 3 (21) 22 ± 18 Formatted: Font color: Auto Formatted: Font color: Auto Formatted: Font color: Auto (17) 12 ± 14 Formatted: Font color: Auto
													(28)
	September T1 T5 Bioassay S M	15.3 ± 2.7 12.2 ± 5.2	--	--P Dia (1), dino (1) Dia (1) 0.3 ± 0.04 Flag + cil (4) 2.4 ± 1.3 (44 ± 13) 13 (20) 4.7 ± 4.0 (13 ± 12) 1.9 ± 0.9 Cil (2), dia (1), dino (1) 21 ± 12 (54 ± 25) e e r	37 ± 4 63 5.2 ± 2.5 67 ± 56	--0.2 ± 0.1 1.1 ± 1.2	--0.04 ± 0.1 0.9 ± 0.8	--0.01 ± 0.02 0.07 0.1 ±	--1.8 ± 2.5 6.2 ± 3.4	-Formatted: Font color: Auto -3.3 ± 3.3 (5) (12) 12 ± 14 Formatted: Font color: Auto
		1 Only ciliates included.		R e v i e w
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Table IV .

 IV Depth-integrated biomass and secondary production of broadcast and sac-spawning copepods, the average egg production used to calculate the secondary production, and the species dominating the biomass of small copepods.

	Station	Broadcast spawners		Sac spawners		Total
		Bm	Ep	Sp	Bm	Ep	Sp	Sp	Dominant species
	2002
	St. 2	6.4	10.0	0.6	75.2	2.1	12.1	12.8	Oithona spp.	Formatted: Font color: Auto
	St. 4	18.7	10.0	1.9	37.8	2.4	6.1	8	Oithona spp., C. typicus	Formatted: Font color: Auto
										Formatted: Font color: Auto
	2003									Formatted: Font color: Auto
	St. 3	97.1	11.0	11.9	23.8	5.0	1.0	12.9	M. pusillus, C. typicus	Formatted: Font color: Auto
	St. 5 St. 8	204.8 165.4	8.1 9.4	17.9 17.6	22.4 19.7	6.0 4.8	1.3 0.9	19.2 18.6	C. typicus, M. pusillus M. pusillus	Formatted: Font color: Auto Formatted: Font color: Auto
	2005 St. 1 St. 3 St. 5	114.3 167.0 39.6	10.2 4.4 13.8	11.7 7.3 4.2	115.5 117.5 134.8 F o r 2.1 1.0 0.5	2.4 1.2 0.7	14.1 8.5 4.9	Oithona spp., T. longicornis T. longicornis, Oithona spp. Oithona spp., Pseudocalanus sp.	Formatted: Font color: Auto Deleted: c
	St. 8	168.0	9.8	16.5	57.7	2.0 P	1.1	17.7	T. longicornis, Paracalanus parvus, Oithona spp.	Deleted: c
						e e r			Deleted: c
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Table V .

 V Average egg production, percentage of females producing eggs and brood size, hatching success and female prosome length of Acartia clausi and Centropages typicus in March -September (in situ reproduction), and Acartia tonsa in August (bioassay).

		Egg production		Hatching	Length
	Eggs f -1 d -1	% prod	Eggs brood -1	%	mm
	A. A. clausi
	March (1)

Table VI .

 VI Multiple regression coefficients and their significance for the models describing the factors influencing A) the egg production and hatching of Acartia clausi and B) the egg production and hatching of Acartia tonsa.

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				Coefficient	SE	F-to-remove F-to-enter	P
	A.	Egg production	Constant	-37.31		32.6	Formatted: Font color: Auto
		N = 22	C1 (body size)	56.18		28.9	0.067	Formatted: Font color: Auto
		R 2 = 0.487	C2 (temperature)	-0.634		0.575	0.284	Formatted: Font color: Auto
		Adj R 2 = 0.434					Formatted: Font color: Auto
							Formatted: Font color: Auto
		Egg production N = 18 R 2 = 0.613	Constant C1 (PUFA % 0-40 µm) C2 (EPA:DHA 40-80 µm) 0.022 0.119 -0.001		0.033 0.0005 8.481 0.008 7.499	0.011* 0.015*	Formatted: Font color: Auto Formatted: Font color: Auto
		Adj R 2 = 0.561	C3 (T)				1.267	0.278	Formatted: Font color: Auto
		August 2003 (1) Egg production	b) 14 ± 17 39 ± 29 Constant C5 (Ciliate bm) F a) 63 ± 36 87 o -39.53 C4 (Chl-a)	b) 19 ± 7 45 ± 26 18.9	--	b) 1.26 ± 0.05 1.20 ± 0.04 1.978 0.180 0.111 0.743	Formatted: Font color: Auto
		C. A. tonsa N = 20 R 2 = 0.68	C1 (body size) C2 (EPA:DHA 40-80 µm) 4.828 45.79 r		20.7 1.67	0.041* 0.010*
		Chl-max (4) Surface (4) Adj R 2 = 0.64 Hatching N = 11 R 2 = 0.577 Adj R 2 = 0.530	a) 4.7 ± 4.2 b) 1.4 ± 1.3 Constant C1 (PUFA % 0-40 µm) C2 (EPA:DHA 40-80 µm) -18.992 P 74.762 e 3.733 5.419 12.284 a) 81 ± 8 (534) a) 86 ± 13 (158) C3 (T) e C4 (Chl-a) r C5 (Ciliate bm)	0.248 0.083 0.373 1.573	0.630 0.007** 0.779 0.555 0.238
	B.	Egg production N = 8 R 2 = 0.447 Adj R 2 = 0.355 Hatching N = 7 R 2 = 0.550 Adj R 2 = 0.460	Constant C1 (EPA: DHA) C2 (Total FA) Constant C1 (EPA: DHA) C2 (Total FA)	2.684 11.396 93.673 -59.720	0.750 R 5.175 4.849 e v 4.285 i 0.118 24.144 6.188 0.501 e w	0.07 0.743 0.056 0.511
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Acknowledgements:

Marja Koski was partly funded through a European Community Marie Curie postdoctoral fellowship and Carlsberg Foundation, Espen Bagøien was funded through a European Community Marie Curie postdoctoral fellowship (MCFI-2002-01148) and the HARVEST project (no. 178477) of the research Council of Norway. The study was additionally financed by LIFECO EU Q5RS-2000-30183 (2001), CONWOY No 2052-01-0034 (2002 and 2003) and No 21-03-0487 to Sigrun Jónasdóttir (2005) from the Danish Research Agency.