Bioenergetics of the copepod Temora longicornis under different nutrient regimes

Bioenergetics of the copepod Temora longicornis under different nutrient regimes Abstract The copepod Temora longicornis depends on constant prey availability, but its performance also depends on how efficiently it utilizes its food sources. Our research goal was to understand copepod energy allocation in relation to diet quality. The working hypothesis was that Temora performs better on the diet whose elemental ratio is closest to its own. Diatoms (Diat) and dinoflagellates (Dino) cultured in nutrient-replete (+) and nitrogen-depleted (−) conditions were fed to the copepods. Ingestion, respiration, excretion and egg and fecal pellet production rates were measured. Carbon (C) and nitrogen (N) budgets were built to investigate differences in dietary C and N partitioning. Copepods fed nitrogen-depleted diatoms (Diat−), which had the most different C:N ratio to that of Temora longicornis, had high metabolic losses and low growth. Copepods fed nitrogen-rich dinoflagellates (Dino+) with a more similar C:N ratio to their own also had high metabolic losses, but displayed the highest investment into somatic growth and egg production. The results indicate that dinoflagellates are a better food source for T. longicornis. Furthermore, consumption of low-quality food leads to higher respiration rates and faster leakage of dissolved organic carbon from copepod fecal pellets; and egestion is a main pathway in copepods for eliminating unabsorbed and non-metabolized carbon. INTRODUCTION Copepods are an important link in the energy transfer between primary producers and higher trophic levels, and contribute to the cycling of organic matter (Juul-Pedersen et al., 2006; Castellani and Altunbas, 2014). The small calanoid copepod Temora longicornis (Müller, 1785) is one of the dominant species in the coastal zooplankton community in the North Atlantic and North Sea, reaching peak densities during the spring and early summer (Hickel, 1975; Castellani and Altunbas, 2014). Off Helgoland, this omnivorous species is abundant all year round and its grazing may have a substantial impact on the phytoplankton standing stock (Gentsch et al., 2009; Maar et al., 2004). Temora longicornis has high metabolic turnover rates, but is unable to accumulate significant amounts of energy reserves (Kreibich et al., 2008, 2011). The species is, thus, dependent on a constant availability of prey and is vulnerable to fluctuations in food supply (Helland et al., 2003; Kreibich et al., 2008, 2011). This can be particularly problematic in systems such as the North Sea, where plankton community composition can change rapidly (Kiørboe and Nielsen, 1994). As T. longicornis inhabits dynamic systems, it must be able to quickly react to changes in trophic conditions (Gentsch et al., 2009; Kreibich et al., 2008), and efficient food utilization is of paramount importance for its survival. Food quantity and quality influence the physiology of copepods, their efficiency in nutrient uptake, and their ability to both convert food into energy and channel stored energy into reproduction (Møller, 2007; Hessen and Anderson, 2008; Jónasdóttir et al., 2009), and changes in these prey characteristics could impact not only copepod populations but also the recruitment of their predators (Boersma et al., 2015). Zooplankton are often limited by food quantity in coastal regions (Hirst and Bunker, 2003), and some food sources are also of lower nutritional value (Sterner and Schulz, 1998). It has been suggested, for example, that protozoans are qualitatively important to copepod diet (Stoecker and Capuzzo, 1990), and that egg hatching success is dependent upon the ingestion of essential fatty acids (Broglio et al., 2003). Elemental composition, digestion resistance and biochemical composition are important factors determining the nutritional value of food particles (Sterner and Schulz, 1998). A vast body of literature is available on the functional responses of calanoids to diet quality (e.g. Dam and Lopes, 2003; Arendt et al., 2005; Jónasdóttir et al., 2009; Nobili et al., 2013), but few address all important vital rates at once (e.g. Abou Debs, 1984). This comprehensive approach would allow for the assessment of energy allocation to different processes in copepods, and is necessary to come to a better understanding of the potential responses of T. longicornis to climate change-induced food regime shifts. The present work investigated metabolism, feeding, growth and reproduction in T. longicornis females in light of different prey elemental compositions/limitations. Based on the concepts of ecological stoichiometry, homeostasis and trophic upgrading (Klein Breteler et al., 1999; Sterner and Elser, 2002; Malzahn et al., 2010), specifically on how prey elemental composition affects consumer performance and energy utilization, the research goal was to investigate the partitioning of dietary carbon (C) and nitrogen (N) in relation to food quality. The working hypothesis was that copepods feeding on a diet with a C:N ratio close to their own body composition would perform best, i.e. would have the highest possible growth and reproduction rates and the lowest possible egestion, respiration and excretion rates. The opposite pattern would thus be observed for copepods feeding on prey with a C:N ratio as different from their own as possible (higher or lower). METHOD Field sampling Zooplankton were collected by horizontal hauls with a 500-µm mesh-size CalCOFI net, which were conducted for 15 minutes at 5 m depth off the German island of Helgoland (54°11′N, 07°54′E), in the southern North Sea. The quantity of females needed for the full experimental design required that the experiment be split into two parts. Samplings were performed as close in time as environmental conditions allowed on 17 (Experiment I) and 30 (Experiment II) May 2016. The samples were taken to the laboratory, where intact and active adult females of T. longicornis were immediately sorted under an Olympus SZX16 stereoscopic microscope. A total of 1150 females were sorted at each date (t0h), 1080 for the experiments, 30 for determination of in situ body C and N contents and another 40 were fixed in 4% formalin buffered with hexamethylenetetramine for measurement of prosome length (PL) under a Leica M205C (Fig. 1). Fig. 1. View largeDownload slide Experimental design used in the present study with females of the copepod Temora longicornis. The two zooplankton sampling dates in May 2016 are indicated, after which Experiments I and II immediately took place. The experimental design was almost equal between these, except that the former was conducted with diet treatments (names in bold) consisting of diatoms cultured in nutrient-rich (Diat+) and N-limited (Diat-) conditions, whereas the latter was conducted with dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. The timing of experimental phases (acclimation period, between t0h and t48h, and incubations, between t48h and t72h) is shown in the left column. Copepods were acclimated and incubated in triplicates. Sampling of copepods for C and N analysis and PL measurement are included in the diagram, as are the estimations of vital rates. Fig. 1. View largeDownload slide Experimental design used in the present study with females of the copepod Temora longicornis. The two zooplankton sampling dates in May 2016 are indicated, after which Experiments I and II immediately took place. The experimental design was almost equal between these, except that the former was conducted with diet treatments (names in bold) consisting of diatoms cultured in nutrient-rich (Diat+) and N-limited (Diat-) conditions, whereas the latter was conducted with dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. The timing of experimental phases (acclimation period, between t0h and t48h, and incubations, between t48h and t72h) is shown in the left column. Copepods were acclimated and incubated in triplicates. Sampling of copepods for C and N analysis and PL measurement are included in the diagram, as are the estimations of vital rates. Prey culture Three prey species were cultured in the laboratory, the cryptophycean Rhodomonas salina (Wislouch) (Hill and Wetherbee, 1989), the heterotrophic dinoflagellate Oxyrrhis marina (Dujardin, 1841) and the diatom Conticribra weissflogii (Grunow) (Stachura-Suchoples and Williams, 2009). Rhodomonas salina was used solely as food for O. marina, whereas the diatoms and dinoflagellates, which are both common prey for T. longicornis (Evans, 1981), were used to feed copepods. A stock solution was maintained for each of the species. New cultures were created daily by diluting part of the stock solution with fresh medium. Two types of medium were used: nutrient-replete f/2 (after Guillard, 1975) and N-depleted (f/2 without the addition of nitrate). Silicate was only added to the medium used for diatoms. Cultures were kept in a temperature-controlled room at 18°C. Rhodomonas salina and C. weissflogii were provided constant light, while O. marina was kept in the dark. Aeration was provided to R. salina, and C. weissflogii and O. marina cultures were stirred twice a day to keep cells suspended. Cultures were grown for 5 days and then used as food suspension for copepods during experiments. Daily feeding of O. marina with R. salina was planned such that prey cells were depleted by the dinoflagellate on Day 5. Cell densities were determined with a BD Accuri C6 Flow Cytometer. Conticribra weissflogii and O. marina cultures were sampled daily for the determination of cell C and N contents by filtering known cell concentrations through Whatman GF/F filters. The remaining volume was then used to feed copepods. Experimental design Experiments were initiated right after field sampling. Figure 1 depicts the distribution of different diet treatments, replication and the sampling scheme. Copepods from Experiment I were fed diatoms cultured in nutrient-replete (Diat+) and N-depleted (Diat−) medium. In Experiment II, the copepods were fed with dinoflagellates cultured with nutrient-replete (Dino+) and N-depleted (Dino−) food. The effects of food quality on predators are more evident when food is abundant than when prey quantity is low (Sterner, 1997). In order to better observe the effects of food quality on the copepods, and to be able to distinguish them from the effects of food quantity, prey were offered to copepods ad libitum (>350 μg C L−1, 8 × 103 diatom and 2 × 103 dinoflagellate cells mL−1) during the entire experiment. The only exceptions were the respiration and excretion incubations, which were performed without any food, as described below. Copepod density varied in the different experimental units for practical reasons, such as the amount of copepods available for performing the experiments and the volumes of the different experimental units themselves. Visual observations of female behavior during the experiments and the results obtained indicate that these differences in density did not affect copepod feeding. Copepods were incubated in groups of 180 females in partially filled 3 L plastic beakers (75 females L−1) fitted with a 300 μm meshed-bottom cylinder to keep the copepods from feeding on eggs and fecal pellets (FP). Although the in situ temperature was different between Experiments I and II (10 and 12°C, respectively), all experiments were conducted in the same temperature, and copepods were kept in a dark temperature-controlled room at 10 ± 0.3°C. The water was gently stirred three times a day for food resuspension. Partial water exchanges (66%) were performed daily in order to remove eggs and FP and to renew copepod food. The copepods were acclimated under these conditions for 48 h. Subsequently, 10 females were sampled from each replicate to determine body C and N contents (samples referred to as t48h). The remaining individuals were distributed among four experimental incubations in order to separately measure grazing, respiration, excretion and egg and FP production rates. Experimental units were kept in the same temperature-controlled room as the beakers. Grazing Ingestion rates were estimated based on changes in prey cell concentrations in the absence and presence of copepods (Frost, 1972). The prey cell concentrations at the beginning and end of the grazing incubation were measured from triplicate 1 L glass bottles containing filtered seawater (FSW) + the respective food suspension (“start bottles”) and FSW + food suspension + 10 copepods (“grazed bottles”), respectively. The start bottles were fixed immediately with 4% buffered formalin. Prey growth during the incubation was accounted for by creating another set of three 1 L glass bottles with FSW + food suspension (“control bottles”). The control and grazed bottles were attached onto a plankton wheel rotating with speed between 0.5 and 1 rpm for 24 h, after which their contents were fixed with formalin as described above. The copepods were retained in 300 μm mesh-sized sieves prior to fixation and sampled to measure body C and N contents. Cell densities of the preserved food suspension were determined within 6 to 8 days of fixation with a BD Accuri C6 Flow Cytometer. These were used to calculate grazing rates as number of prey cells ingested per female and per day. Respiration The sealed chamber method was applied to measure oxygen consumption rates (Harris et al., 2000) with a NTH oxygen microsensor (PreSens GmbH, Regensburg, Germany) connected to a 4-channel oxygen meter (Microx TX3, PreSens GmbH). Calibration of the microsensor was performed with aerated artificial seawater (ASW, salinity 32) as the 100% O2 reference and with a saturated Na2SO3 solution as the 0% O2 reference. In order to avoid complicated corrections for O2 production or consumption by prey (Ikeda, 1976), this incubation was conducted without prey. For each treatment, ASW was added to six 60-mL Winkler bottles. Three of these were replicate control bottles and contained only ASW and the other three were replicates with ASW and 10 copepods each. Females were placed inside the bottles and allowed to acclimate for an hour with the lid open. The O2 saturation of water was then measured, and the lid was closed. After 24 h, it was measured again, and the copepods were sampled to measure body C and N contents. O2 consumption rates (under starvation conditions) were obtained per female and per day. The use of control bottles allowed for correction for potential oxygen consumption or production by microbes during incubation. Air pressure values for Helgoland were obtained from www.wetter.com. Excretion Daily, individual excretion rates were also calculated following the sealed chamber method, and the equation from Miller and Glibert (1998) was used to calculate the total dissolved nitrogen (TDN) and the non-purgeable organic carbon (NPOC) content of filtrate samples. Non-feeding conditions were also employed in this incubation in order to create a low background against which to measure and compare changes in dissolved organic carbon (DOC) and nitrogen (DON). For each treatment, triplicate 100-mL (pre-combusted) glass bottles were prepared with only ASW (controls) and with ASW + ca. 15 copepods. After 24 h of incubation, ASW and copepods were filtered through pre-cleaned (10% HCl) syringes fitted with Whatman GF/F w/GMF (0.7 μm pore size, 25 mm diameter) syringe filters. Filtrates were immediately frozen at −20°C in pre-cleaned high-density polyethylene bottles. DOC and TDN in the filtrate were determined by high temperature catalytic oxidation (HTCO) and subsequent non-dispersive infrared spectroscopy and chemiluminescence detection using a Shimadzu TOC-VCPN analyzer. In the autosampler, the samples (6.5 mL) were acidified with HCl and sparged with oxygen (100 mL min−1) for 5 min to remove inorganic carbon. A 50-μL sample volume was injected directly on the catalyst (heated to 680°C). Final DOC concentrations were averaged values of triplicate measurements. If the standard variation or the coefficient of variation exceeded 0.1 μM or 1%, respectively, up to two additional analyses were performed and outliers were eliminated. After each batch of six samples, one DSR (Deep Sea Water Reference Material, Hansell Research Lab, University of Miami, US), one Milli-Q blank, and one potassium hydrogen phthalate standard were measured. The limit of quantification was 7 μM for DOC and 11 μM for TDN, and the accuracy was ±5%. The copepods were trapped inside the syringe filters and could not be sampled for C and N content analysis. Since both the excretion and the respiration incubations were conducted under similar conditions, the C and N contents of the copepods from the latter were also used for calculating the excretion rates. Egg and FP production For each diet treatment, females were placed individually in triplicate 12-well cell culture plates (12 females per replicate) filled with FSW and food suspension to 4 mL volume. The production of eggs and FP was checked under a binocular 1, 6, 12, 18 and 24 h after the beginning of the experiment. After eggs and feces were counted, they were carefully pipetted out of the well, at times being collected in filters for determination of their C and N contents. Due to the high amount of eggs and feces required for C and N analysis, only one pooled sample was taken from each of the different treatments, with a minimum of 400 eggs and 379 FP. Due to time limitations, eggs and FP were not washed in distilled water before sampling, but the presence of algal cells in the samples was unlikely, since most of the food had been eaten by copepods at the time of collection. Food was immediately replenished until the next count, and plates were left undisturbed. Copepods were sampled after 24 h for determination of body C and N contents. Individual egg production rates (EPRs) were calculated as a daily estimate based on the production observed over 24 h. FP production rates (FPRs) were also observed during 24 h, but due to problems during the last 6 h period, only the data from the first 18 h of incubation were used to estimate daily rates. C and N content analysis Copepods, prey cultures, eggs and FP were sampled for the determination of their C and N contents. Copepods were gently washed in distilled water, placed into pre-weighed tin cartridges and stored at −80°C until further analysis. In situ water samples and prey cultures were filtered onto pre-combusted (500°C for 24 h) Whatman GF/F filters (0.7 μm pore size, 25 mm diameter). Eggs and FP were counted and pipetted onto pre-combusted Whatman GF/F filters. Both tin cartridges and filters with samples were dried at 60°C for 48 h, folded (filters inside aluminum foil), and stored in a desiccator. The C and N contents of all samples were later measured with an elemental analyzer (detection limit: 2 μg C / 0.5 μg N; maximum error: ± 3%, Euro EA 3000, EuroVector S.P.A., Milan, Italy) using acetanilide as a standard. C and N budgets The amount of energy ingested by an organism should equal the sum of the amounts of energy egested and used for growth and metabolism. In that sense, the vital rates of an individual can also be expressed as a balanced equation, such that I = G + R + U + E + F, where I is ingestion, G is somatic growth, R is respiration, U is excretion, E is egg production and F is FP production. In order to do so, the vital rates were converted to C and N units. The number of prey cells ingested and of eggs and FP produced were multiplied by their respective C and N contents. Respiratory quotients (RQs), which ranged from 0.74 to 0.76, were estimated for each replicate (as described by McConnaughey, 1978) and used to convert the O2 consumption into C-equivalent respiration (Harris et al., 2000). The excretion incubations did not allow for the differentiation between C properly excreted (in the form of urea and amino acids, for example) and C leaked from the FP before absorption by the copepods. The majority of the DOC measured was likely leaked from FP, and thus added to the calculation of F. The G term was obtained by the formula G = (Xt48h – Xt0h) / (N * Δt), where Xt48h and Xt0h are the C or the N content of the copepod samples from t48h and t0h, respectively, N is the number of females in the sample and Δt is the time in days between t48h and t0h. Values were compared in the format μg C or N female−1 day−1. Some organisms have a limited ability to store C relative to other elements, which is especially true for many copepod species (Meunier et al., 2014), and will be in excess of this element when they are supplied a diet with more C than they can absorb and/or utilize (Hessen and Anderson, 2008). Therefore, the discussion on the C budget also approaches the topic of excess C and the pathways for returning this excess C to the environment (i.e. eliminating it). Turnover rates The budget data were further standardized to daily C and N turnover rates (% body C or N day−1). This was achieved by dividing the budget values by the median C or N contents of the copepods sampled at t48h and at the end of each incubation (to account for possible weight losses during the incubations). Somatic growth was derived from weight differences between t0 h and t48h, so the budget values for this specific turnover rate were divided by the median C or N contents of the copepods sampled at t0h and t48h (to account for possible weight gain during the acclimation). An arcsine square root transformation was then applied to the calculated percentage values. Given that there were negative values in the N growth term, and that the transformation is not possible for negative numbers, a fixed, minimum value was added to all treatments in that category to ensure values above zero. Efficiencies Different physiological efficiencies were calculated to evaluate copepod performance in relation to food quality. Absorption efficiency (AE), the percentage of ingested carbon absorbed in the gut of copepods, was calculated as AE = 1 – (F/I), where F is the egestion and I is the ingestion (Harris et al., 2000). The DOC excreted by copepods was added to the F term in the carbon AE to account for leakage from FP. The net somatic growth efficiency (NSGE) and the net egg production efficiency (NEPE) indicated the percentage of absorbed food converted into somatic growth and egg production, respectively. The formulas were adapted from Kiørboe et al. (1985) and Wendt and Thor (2015) as NSGE = G/(G + E + R + U) and NEPE = E/(G + E + R + U), where G is the somatic growth, E is the egg production, R is the respiration and U is the excretion. Statistical analysis Differences in seston and in copepod in situ elemental composition and PL were verified with t-tests. Differences in vital rates, turnover rates and efficiency calculations were tested with one-way analysis of variance (ANOVA). Differences in copepod elemental composition during the experiments were tested with a two-way ANOVA, in order to account for interactions between diet treatment and sampling time. When these results were significant, the Tukey HSD post hoc test was used at 95% confidence limits to further identify origin of differences. Prior to the ANOVA, the data were tested for normality and homogeneity of variances with Shapiro–Wilk and Bartlett tests, respectively. If the data were non-normal and/or heteroscedastic, they were analyzed with a Kruskal–Wallis test and with the post hoc Nemenyi test (with P-value being determined with the Tukey method). Analyses were performed using R ver. 3.2.5 (Ihaka and Gentleman, 1993). RESULTS C and N contents Prey culture The C and N contents and molar C:N ratio of prey cultures are presented in Table I. The average C content was always above 500 μg C L−1, confirming the ad libitum feeding condition. The average N content was always above 100 μg N L−1, with the exception of the Diat− treatment, which was clearly N-limited and contained less than half this amount. The molar C:N ratio was also similar between all treatments with the exception of the Diat−, such that the approximate values were 6 and 19 for the former and the latter, respectively. Cultures were thus significantly different from one another (Supplementary Tables S1 and S2). Table I: C and N contents and molar C:N ratio of the prey cultures (nutrient-replete and N-depleted C. weissflogii, Diat+ and Diat−, and O. marina, Dino+ and Dino−, respectively). Values are mean ± standard deviation from the three batches (temporal replicates) used for feeding in each experiment Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Table I: C and N contents and molar C:N ratio of the prey cultures (nutrient-replete and N-depleted C. weissflogii, Diat+ and Diat−, and O. marina, Dino+ and Dino−, respectively). Values are mean ± standard deviation from the three batches (temporal replicates) used for feeding in each experiment Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Copepods The PL of in situ females was significantly greater in Experiment II (Table II, Supplementary Table S1), but their elemental compositions were not statistically different. Table II: C and N contents (in μg C copepod−1 and μg N copepod−1), and molar C:N ratio of T. longicornis. Values presented for samples obtained at t0h (in situ condition), t48h (after 48h of acclimation) and after the experimental incubations on Day 3 (grazing = G; respiration = R; and egg and FP production = EF). PL (in mm) are shown for t0h copepods. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Values presented are mean ± standard deviation of triplicate samples containing between 10 and 12 copepods each (except for PLs, which had 40 replicates each) Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Table II: C and N contents (in μg C copepod−1 and μg N copepod−1), and molar C:N ratio of T. longicornis. Values presented for samples obtained at t0h (in situ condition), t48h (after 48h of acclimation) and after the experimental incubations on Day 3 (grazing = G; respiration = R; and egg and FP production = EF). PL (in mm) are shown for t0h copepods. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Values presented are mean ± standard deviation of triplicate samples containing between 10 and 12 copepods each (except for PLs, which had 40 replicates each) Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Copepod C and N contents varied between 13 and 21 μg C copepod−1 and between 3 and 5 μg N copepod−1, and molar C:N ratios ranged from 4.0 to 5.6 (Table II). Although no significant differences were found between the t0h samples from Experiments I and II, the C and N contents and the molar C:N ratio were significantly different when comparing t0h with t48h and t72h, both within and between experiments (Supplementary Tables 1 and 2). A significant increase in C and N contents was observed between t0h and t48h, except in the Diat− treatment, for which the N content remained the same throughout the experiment. C and N contents of copepods sampled between t48h and the end of the incubations remained constant in the grazing incubation (Fig. 2, Table II, Supplementary Table S2), for which surplus conditions were maintained. Some level of food limitation was present in the egg and FP production rate incubations, given the small volume of the experimental unit, and copepods in the respiration incubation were food-deprived. In both incubations, the C and N contents of copepods decreased (Table II, Supplementary Table S2). The highest increase in molar C:N ratio was observed for the Diat− treatment (from 4.7 at t0h to 5.6 at the end of the grazing incubation), which was always significantly different from all other treatments (Fig. 2, Table II, Supplementary Table S2). Fig. 2. View largeDownload slide C and N contents and molar C:N ratio of T. longicornis females during Experiments I and II. Experiment I tested diet treatments consisting of diatoms cultured in nutrient-rich (Diat+, gray squares and dotted lines) and N-depleted (Diat−, black squares and full lines) conditions, whereas Experiment II tested diets consisting of dinoflagellates fed with nutrient-rich (Dino+, gray triangle and dotted lines) and N-limited (Dino−, black triangle and dotted lines) prey. Samples were taken of in situ conditions (t0h, empty symbols), after 48h of acclimation (t48h), and after 24h of incubations (t72h). Bars represent mean values ± one standard deviation. Fig. 2. View largeDownload slide C and N contents and molar C:N ratio of T. longicornis females during Experiments I and II. Experiment I tested diet treatments consisting of diatoms cultured in nutrient-rich (Diat+, gray squares and dotted lines) and N-depleted (Diat−, black squares and full lines) conditions, whereas Experiment II tested diets consisting of dinoflagellates fed with nutrient-rich (Dino+, gray triangle and dotted lines) and N-limited (Dino−, black triangle and dotted lines) prey. Samples were taken of in situ conditions (t0h, empty symbols), after 48h of acclimation (t48h), and after 24h of incubations (t72h). Bars represent mean values ± one standard deviation. Eggs and FP Diatom-fed copepods produced eggs with the highest C content and the highest and the lowest N content (72 and 69 ng C egg−1 and 13 and 9 ng N egg−1 for nutrient-replete and N-depleted treatments, respectively). Dinoflagellate-fed copepods produced eggs with similar C and N contents (60 ng C egg−1 and 12 and 11 ng N egg−1 for Dino+ and Dino−, respectively) The molar C:N ratio of eggs was similar between the Diat+ (6.5) and Dino− (6.4) diets, and lower and higher in the Dino+ (5.8) and Diat− (8.9) treatments, respectively. The FP from diatom-fed copepods had the lowest C and N contents (26 and 20 ng C FP−1 and 4 and 2 ng N FP−1 for nutrient-replete and N-depleted treatments, respectively). The C and N contents of the FP from dinoflagellate-fed copepods were almost twice as high (52 and 48 ng C FP−1 and 9 and 7 ng N FP−1 for Dino+ and Dino−, respectively). The molar C:N ratio of FP was lower in the Dino+ (6.7), Diat+ (7.6) and Dino− (8.0) diets than in the Diat− treatment (11.7). It was not possible to measure FP sizes, however, visual observations indicate that FPs from copepods fed with diatoms were slightly longer and thicker than those produced by copepods fed with dinoflagellates. Vital rates and turnover rates Diatom-fed copepods had significantly higher average ingestion rates (8.6 ± 0.5 × 104 and 10.1 ± 1.2 × 104 cells female−1 day−1 for Diat+ and Diat−, respectively) than the dinoflagellate-fed copepods (2.4 ± 0.8 × 104 and 1.6 ± 0.5 × 104 cells female−1 day−1 for Dino+ and Dino−, respectively) (Supplementary Tables S1 and S3). C ingestion, which ranged from 17 to 86% body C day−1, was significantly higher in the Dino+ treatment than in the Dino−, whereas N ingestion, which ranged from 12 to 66% body N day−1 (Table III), was significantly higher in the Dino+ diet than in the N-depleted treatments (Supplementary Tables S1 and S3). Table III: C and N turnover rates and AE, NSGE and NEPE efficiencies from experimental incubations with T. longicornis. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Mass-specific values for ingestion (I), growth (G), carbon-equivalent respiration (R), excretion (U), egg production (E) and FP production (F) turnover rates are expressed as % body C or N day−1, and efficiencies as %. Values presented are mean ± standard deviation. Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Table III: C and N turnover rates and AE, NSGE and NEPE efficiencies from experimental incubations with T. longicornis. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Mass-specific values for ingestion (I), growth (G), carbon-equivalent respiration (R), excretion (U), egg production (E) and FP production (F) turnover rates are expressed as % body C or N day−1, and efficiencies as %. Values presented are mean ± standard deviation. Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 The average oxygen consumption rates were lower in the diatom treatments (3.4 ± 0.3 and 4.3 ± 0.1 mL O2 female−1 day−1 for Diat+ and Diat−, respectively) than in the dinoflagellate treatments (6.0 ± 1.3 and 4.2 ± 0.4 mL O2 female−1 day−1 for Dino+ and Dino−, respectively). They corresponded to a C-equivalent respiration of 6–18% body C day−1 (Table III). A significant difference in O2 consumption and turnover of respired C was only found between the nutrient-replete treatments, with higher values in the Dino+ diet (Supplementary Tables S1 and S3 and Table III). Surprisingly, the average N excretion values were higher in the N-depleted treatments (1.0 ± 0.3 and 0.6 ± 0.1 μg N female−1 day−1 for the Diat− and Dino−, respectively) than in the nutrient-replete diets (0.2 ± 0.2 and 0.3 ± 0.1 μg N female−1 day−1 for the Diat+ and Dino+, respectively). They corresponded to 5–32% body N day−1 (Table III) and were significantly lower in the Diat− treatment (Supplementary Tables S1 and S3). The average FP production rates were not significantly different between the Diat+ (89 ± 4 pellets female−1 day−1) and Diat− (86 ± 8 pellets female−1 day−1) treatments. FP production rates recorded for copepods fed with dinoflagellates were lower, 72 ± 5 and 49 ± 1 pellets female−1 day−1 for the Dino+ and Dino− treatments, respectively (Supplementary Tables S1 and S3). The average amount of DOC leaked from FP, which was significantly different between all treatments (Supplementary Table S1), was higher in the N-depleted diets (1.6 ± 0.2 and 1.1 ± 0.1 μg C female−1 day−1 for Diat− and Dino−, respectively) than in the nutrient-replete treatments (0.2 ± 0.1 and 0.7 ± 0.1 μg C female−1 day−1 for Diat+ and Dino+, respectively). The C and N turnover rates for FP production, which varied between 12 and 24% body C day−1 and between 4 and 14% body N day−1 (Table III), were significantly different between almost all treatment pairs (Supplementary Tables S1 and S3). Somatic growth was significantly different between treatments in terms of both C and N (Supplementary Table S1). It ranged from 4 to 14% body C day−1 and from 0 to 11% body N day−1 (Table III), with the lowest values recorded for the N-limited diatom treatment (Supplementary Table S3). The average EPRs were significantly higher in the Dino+ treatment (61 ± 4 eggs female−1 day−1) than for copepods fed with the other diets (39 ± 9, 36 ± 4, and 41 ± 5 eggs female−1 day−1 for Diat+, Diat− and Dino−, respectively) (Supplementary Tables S1 and S3). The egg C turnover (13–21% body C day−1) was similar between treatments (Table III), but the N turnover (7–16% body N day−1) was significantly different between the Dino+ and Diat− treatments (Supplementary Tables S1 and S3). C and N budgets The C and N budgets obtained for each of the treatments are represented in Fig. 3. They were mostly unbalanced, which is represented in the figure by the equation symbols “−” and “+”. The “I” values are represented on the left side of the copepod antenna, whereas the right side represents the sum of “G + R + U + E + F”. When the gain of energy via feeding was higher than growth and energy expenditure (e.g. Fig 3B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (e.g. Fig 3D and F), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (Fig. 3C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The budgets recorded for the Dino+ treatment seem to have the best estimates (percentages close to 100%). The budgets for the other treatments mostly point to higher growth and energy expenditure. The Dino− treatment had the most deviating results, with expenses amounting to twice as much the recorded energy ingested, a pattern visually represented by the use of double equation symbols (Fig. 3D and H). The Diat+ budgets also show higher expenses than energy gain (Fig. 3A and E), whereas the Diat− budgets show a mixed response, with an accurate estimate for the C budget (Fig. 3B) but an inaccurate estimate for the N budget (Fig. 3F). The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows in Fig. 3. Fig. 3. View largeDownload slide Carbon (A–D) and nitrogen (E–H) budgets obtained for T. longicornis females feeding on diatoms cultured in nutrient-rich (Diat+) and N-depleted (Diat−) conditions and on dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. Values given represent daily ingestion, egestion, respiration, excretion, reproduction and somatic growth rates for one individual copepod, and are visually represented by proportionately sized arrows. The comparison between the amount of C and N ingested by copepods (“I” values on the right side of the copepod antenna) and the amount used by them (the sum of “G + R + U + E + F” values on the left side of the copepod antenna) is represented in the figure by the equation symbols “+”, “−” and “=”. When the gain of energy via feeding was higher than growth and energy expenditure (B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (A, D, E, F and H), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The closer to 100%, the more balanced the measurements; values below and above 100% indicated higher energy gain from feeding than energy expenditure and higher energy expenditure than energy gain from ingestion, respectively. The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows. Fig. 3. View largeDownload slide Carbon (A–D) and nitrogen (E–H) budgets obtained for T. longicornis females feeding on diatoms cultured in nutrient-rich (Diat+) and N-depleted (Diat−) conditions and on dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. Values given represent daily ingestion, egestion, respiration, excretion, reproduction and somatic growth rates for one individual copepod, and are visually represented by proportionately sized arrows. The comparison between the amount of C and N ingested by copepods (“I” values on the right side of the copepod antenna) and the amount used by them (the sum of “G + R + U + E + F” values on the left side of the copepod antenna) is represented in the figure by the equation symbols “+”, “−” and “=”. When the gain of energy via feeding was higher than growth and energy expenditure (B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (A, D, E, F and H), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The closer to 100%, the more balanced the measurements; values below and above 100% indicated higher energy gain from feeding than energy expenditure and higher energy expenditure than energy gain from ingestion, respectively. The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows. Efficiencies The AEs varied between 33% and 75% and between 46% and 81% of C and N ingested, respectively (Table III), with the carbon AE for Dino− being significantly lower than that of Diat− and Dino+ (Supplementary Tables S1 and S3). The carbon NSGEs varied between 15% and 36%, and were significantly different between the diatom treatments, while the nitrogen NSGEs varied from −4% to 48% and were significantly lower in Diat− (Table III, Supplementary Tables S1 and S3). No significant differences were found between the carbon NEPE, which ranged from 39% to 50%, whereas the nitrogen NEPEs, which varied from 19% to 56% (Table III), were significantly higher in the nutrient-replete treatments (Supplementary Tables S1 and S3). DISCUSSION To our knowledge, this is the first study of energy budgets of T. longicornis females feeding on prey items of different qualities, which enabled the comparison of copepod performance under different food regimes. Our working hypothesis was that the highest growth and reproduction rates and the lowest catabolic rates would be recorded for copepods feeding on a diet with a C:N ratio as close as possible to their own body composition. Our study was able to partially confirm the working hypothesis. The highest investment in somatic and reproductive growth was indeed shown by copepods fed with a food source of C:N ratio close to their own (Dino+), but so were the highest expenditures with egestion and catabolism, contrary to what had been postulated. Performance entails the amount of energy ingested and used by an individual for different vital activities, but is ultimately defined by what is invested in reproduction and how successful this process is. Thus, the major finding of this study is that dinoflagellates are a food source for copepods of superior or similar quality to diatoms under nutrient-replete or N-depleted conditions, respectively. This is, however, only valid for the temperatures investigated in the present study, as individual metabolism and C requirements are affected by temperature (e.g. Boersma et al., 2016). Results also revealed that egestion is a major pathway for T. longicornis females to eliminate the excess C; and that low food quality can influence copepod respiration (regardless of its C-to-nutrient ratio) and the intensity and speed with which DOC leaks from FP. The fact that results only partially agreed with predictions from stoichiometric theory indicates the need for further investigations into copepod ecophysiology and adaptive capacity to shifting food regimes. The level of N-limitation achieved in the Diat− treatment was not expected for the Dino− diet, as it has been shown that O. marina can regulate its body composition to incorporate the stoichiometric imbalances of its prey in an attenuated form (Malzahn et al., 2010; Meunier et al., 2014). Although the elemental composition of the Dino− treatment was similar to that of the Dino+, its fatty acid profile was not (unpublished data). This could explain why performance was different for copepods fed with these two diets, and why the former constituted a food source of lower quality. Copepod feeding The C ingestion rates reported by Arendt et al. (2005) and Jónasdóttir et al. (2009) for T. longicornis feeding on C. weissflogii are twice as high as those reported herein, despite their use of lower prey concentrations (Table IV). The values from our study might be underestimated, as indicated by the comparison of the amount of energy ingested and used by copepods (symbols on the top left and right sides of the copepod antenna in Fig. 3, respectively). Despite measurements being recorded in the same fashion for all treatments, some show a nearly balanced budget (e.g. Fig. 3C) while others reveal unbalanced budgets (e.g. Fig. 3F). We speculate that the ingestion rates are underestimated and that this might be due to copepods feeding on food sources that were unaccounted for, which were ingested in quantities inversely proportional to the quality of the diet treatments. Coprophagy and filial cannibalism are known to occur among copepods, regardless of availability of alternative food, and can reach values of up to 50% of produced FP and 60 eggs female−1 day−1 (e.g. Lampitt et al., 1990; Noji et al., 1991; Dam and Lopes, 2003; Boersma et al., 2014) in starved or food-limited conditions. The glass bottles used for the grazing incubation in the present study did not allow for the separation of copepods from eggs and FP. The eggs had a similar or higher nutritional quality (in terms of elemental composition/molar C:N ratio) than the prey items, and the microbiota associated with the peritrophic membrane of the FPs might be a valuable food source for copepods (as suggested by Lampitt et al., 1990), so by feeding on its own eggs and FPs, copepods could have complemented nutritionally inadequate diets. This response would be a form of compensatory behavior for this copepod, and has been identified in other studies (Augustin and Boersma, 2006; Siuda and Dam, 2010) as a mechanism for other small calanoids to overcome the elemental limitation of their prey. Table IV: Comparative values between prey species and quantity used in laboratory experiments and the ingestion and EPR of T. longicornis recorded for that prey in the present study and elsewhere in the literature Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study aUsing a carbon:chlorophyll α ratio of 50, according to Dam and Peterson (1991). Table IV: Comparative values between prey species and quantity used in laboratory experiments and the ingestion and EPR of T. longicornis recorded for that prey in the present study and elsewhere in the literature Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study aUsing a carbon:chlorophyll α ratio of 50, according to Dam and Peterson (1991). Copepod waste production (or catabolic activities) Few studies are available on the metabolic activities of small copepods such as T. longicornis (e.g. Berner, 1962; Dam and Peterson, 1993; Nobili et al., 2013; Castellani and Altunbas, 2014), all reporting lower O2 consumption and C-equivalent respiration than the ones described herein (1–2 μL O2 ind−1 day−1 and 1% body C day−1). Our respiration and excretion incubations were performed without feeding the copepods, and probably underestimate the rates of fed copepods (Miller and Glibert, 1998; Thor, 2002a, 2002b; Nobili et al., 2013). High respiration rates have been suggested as a mechanism for removal of excess C in unbalanced nutrient conditions (Anderson et al., 2005; Hessen and Anderson, 2008), even though the mechanism itself is yet to be described (Malzahn et al., 2010). The results of the present study partially agree with this assumption. The second highest respiration rates were reported for copepods fed with the N-poor diets, but one of them was enriched in C in relation to N (Diat−) and the other had a C:N ratio close to that of the copepods (Dino−). Furthermore, the highest rates were measured for another one of the diets whose C:N more closely resembled that of the copepods (Dino+). The type of substrate catabolized by starved copepods in order to obtain energy influences respiration (Castellani and Altunbas, 2014) and excretion (Anderson, 1992) rates. Proteins are the main substrates catabolized in small calanoids (Thor, 2002). The RQ of proteins is higher than that of lipids (Gnaiger, 1983) and would, thus, generate higher C-equivalent respiration rates in starved individuals that were previously fed N-rich diets, as observed for O. marina by Meunier et al. (2012). Our results thus suggest that O2 consumption increases with decreasing food quality, regardless of an excess of C or not. If prey is of high nutritional quality, then O2 consumption seems to be modulated by the amount of food ingested. The preferential utilization of proteins by copepods previously fed with a N-rich diet should also result in higher ammonia excretion rates than those of copepods previously fed N-poor diets, but the opposite was recorded. It is possible that the high food C:N ratio of 19 in the Diat− treatment led to an increase in ingestion rates and a decrease in gut passage time. This, in turn, reduced the efficiency with which nutrients were absorbed and resulted in higher excretion rates (Plath and Boersma, 2001). FP production by copepods is an important source of POM and DOC in the epipelagic (Møller et al., 2003; Thor et al., 2003), contributing to microbial production with recycled nutrients (Smetacek, 1980; Strom et al., 1997). Although it has been claimed that undisturbed FP do not release DOC (Strom et al., 1997), it has been shown that 50% of the total C content of FP is released as DOC at the onset of defecation (Thor et al., 2003). DOC can be generated by copepods before absorption, as leakage from egested FP, and after absorption, as excretion in the form of nitrogenous organic compounds (Frangoulis et al., 2004). The methodology did not allow for the separation of both components. Even though some DOC may have been excreted, it was considered that the majority of DOC measured was leaked from FP, which is why DOC values were added to the C content of FP to calculate the egestion term (“F”) of the C budget. In our study, the percentage of total C egested (“F”) that corresponded to DOC varied widely between treatments, going from approximately 8% in Diat+ to 16% in Dino+, 31% in Dino− and 50% in Diat−. It would seem, thus, that the level of DOC leakage from FP is directly or indirectly influenced by food quality. Small calanoids such as T. longicornis are unable to store significant amounts of energy reserves, and must return to the environment any C consumed in excess of its needs and ability to absorb. Our results suggest that the primary pathway for T. longicornis to eliminate excess C was egestion, which removed twice as much C as that burnt via respiration (Fig. 3A–D). Furthermore, if all C had been assimilated and metabolized and no excess was left, assuming that all N was also assimilated, the molar C:N ratio of FP would not have differed from that of the diet treatments (Checkley, 1980). The fact that it was higher in the FP (except for Diat−) indicates that N was used constantly and efficiently for production and that the excess C was egested (Checkley, 1980). This pattern is further supported by the statistically similar nitrogen AE in the treatments, contrary to what stoichiometric theory would have predicted. The carbon AE, on the other hand, was significantly lower in the Dino− treatment. It could be that the resulting biochemical composition of the Dino− diet indirectly caused an inefficient absorption of C in copepods and led to an egestion similar to that of the other treatments despite a lower ingestion rate. Our estimates for the carbon AE were greater than the assimilation efficiency estimates for another small copepod (A. tonsa) feeding on the same prey species (Besiktepe and Dam, 2002). Copepod growth and reproduction (or anabolic activities) Most studies assume negligible increases in body weight in adult copepods which do not store energy reserves and only investigate reproductive investment as a measure of growth (Hirst and McKinnon, 2001). The C and N somatic growth rates observed in the present study, though lower than the EPRs, contest this assumption, as do other studies (Hirst and McKinnon, 2001; Dam and Lopes, 2003). A comparison between the net efficiencies for somatic growth (NSGE) and egg production (NEPE) emphasizes how the majority of assimilated energy is used in reproduction, but also that the portion destined to somatic growth is not negligible. The only absence of somatic growth was observed in terms of N for the Diat− treatment, as expected. The EPR recorded for T. longicornis in this study were within the range of those obtained when this copepod was fed with natural plankton (Peterson and Dam, 1996), similar prey species (Niehoff et al., 2015) and with water from the spring phytoplankton bloom (Castellani and Altunbas, 2014; Peterson and Kimmerer, 1994), but higher than when it was fed water from a dinoflagellate bloom (Jansen et al., 2006) (Table IV). The highest EPR in our study was obtained with the nutrient-rich dinoflagellate diet, as opposed to results from other studies (Turner et al., 2001; Dam and Lopes, 2003; Jónasdóttir et al., 2009). It must be noted that great controversy involves the topic of diatoms being good or bad quality diet items (for reviews, see Jónasdóttir et al., 1998; Paffenhöfer et al., 2005), with many suggesting its suitability for egg production but reduced success for hatching (Miralto et al., 1999; Ban et al., 1997). The quantity of body C invested into reproduction was similar across treatments, but body N investment was significantly different between the Diat− and the Dino+ treatments. This indicates N-limitation in egg production, as also suggested by Nobili et al. (2013) for T. longicornis. The nitrogen NEPE observed in our study further indicates a clear limitation of this element in copepods fed with the N-depleted diets. This contrasts the assumption that nutrients in limited amounts should be used with higher efficiency. Other studies have reported similar trends, with lower egg production efficiencies associated with higher food C:N ratios and with P-limitation (Anderson et al., 2005; Nobili et al., 2013). This pattern might be due to increased maintenance requirements for copepods fed with nutrient-limited diets (Anderson et al., 2005; Wendt and Thor, 2015). Even so, the NEPE values reported herein seem to be mostly higher than those obtained from the literature as either egg production efficiency or gross growth efficiency (e.g. Dam and Lopes, 2003; Thor et al., 2007; Nobili et al., 2013; Wendt and Thor, 2015). Few studies have approached the effect of N-limitation on EPR, which has been said to be both deleterious (Koski et al., 2006; Nobili et al., 2013) and advantageous (Augustin and Boersma, 2006). EPR does not provide information on egg viability (hatching success), so it would be necessary to conduct further investigations in order to understand the importance of diet quality for secondary production by T. longicornis. Climate change and marine C and N cycles Long-term monitoring data from Helgoland have shown a drastic decrease in calanoid copepod densities since 1985, a decline most likely caused by the decrease in nutrient (N and P) loading coupled to the increase in light penetration in the region (Boersma et al., 2015). These would have resulted in phytoplankton with higher C:nutrient ratios and, thus, of lower nutritional value for zooplankton (Boersma et al., 2015). Recent studies show controversial results regarding the relative dominance in recent years of diatoms and dinoflagellates in the southern North Sea (Wiltshire et al., 2010; Alvarez-Fernandes et al. 2012; Hinder et al., 2012; Boersma et al., 2015). Changes in the abundance of these prey species can affect secondary production, and a further increase in their C:N ratio could have major consequences for food web processes (Jones and Flynn, 2005; Malzahn and Boersma, 2011; Nobili et al., 2013; Boersma et al., 2015). On the other hand, increasing local temperatures (Wiltshire et al., 2010) could result in higher metabolic costs for copepods (Castellani and Altunbas, 2014), which can only be met by an increased C consumption (Boersma et al., 2016; Malzahn et al., 2016). In that scenario, increasing temperatures would dampen the effects of prey with higher C:nutrient ratios (Boersma et al., 2016; Malzahn et al., 2016). In N-limitation conditions (e.g. late spring bloom), dinoflagellates are still of better nutritional value than diatoms (Jones and Flynn, 2005; this study), but at higher temperatures, copepods might need to preferentially consume autotrophs to supply metabolic demand, as observed for T. longicornis (Boersma et al., 2016) and A. tonsa (Malzahn et al., 2016). All of these variables make it difficult to say at this point how T. longicornis (and possibly other calanoid copepods) will react to future changes in prey composition, even though its omnivorous strategy and related adaptations allow it to switch food sources depending on their availability (Daan et al., 1988; Gentsch et al., 2009). CONCLUSIONS The results obtained in the present study indicate that, under nutrient-replete conditions, dinoflagellates such as O. marina are a better food source for T. longicornis than diatoms. This major finding is, however, dependent upon environmental conditions. Furthermore, low-quality food also leads to higher respiration rates, regardless of its C-to-nutrient ratio, and to faster leakage of DOC from copepod FP. In addition, egestion seems to be the main pathway for eliminating excess C, contrary to the common belief of excess C being mostly respired. The vital rates measured herein for T. longicornis provide important information on this species’ food utilization efficiency and maintenance costs in relation to diet quality. The budget approach used herein is not free of flaws, and future research in the field should investigate more complex scenarios (mixed diets, different temperatures, varying food concentrations, include all life stages). SUPPLEMENTARY DATA Supplementary data are available at Journal of Plankton Research online. ACKNOWLEDGEMENTS We kindly thank the crew of the Aade research vessel (AWI-BAH) for providing us with zooplankton samples and the technicians at the AWI, BAH and Claudia Burau for the help with sample analyses. Funding R.M.F.-S. was supported by a doctoral research grant from the Doctoral Programme on Marine Ecosystem Health and Conservation (MARES, Framework Agreement Number 2011-0016). The work was further supported by Ghent University (BOF-GOA project 01G02617). C.L.M. was supported by the Bundesministerium für Bildung und Forschung (BMBF grant no. 01LN1702A). Data archiving The laboratory experimental raw data and metadata for this study are available through the PANGAEA repository under https://doi.pangaea.de/10.1594/PANGAEA.886050. Ethical standards The experiments described in the present study comply with the current laws of the country in which they were performed. Conflict of interest The authors declare that they have no conflict of interest. References Abou Debs , C. ( 1984 ) Carbon and nitrogen budget of the calanoid copepod Temora stylifera: effect of concentration and composition of food . Mar. Ecol. Prog. Ser. , 15 , 213 – 223 . 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H. , Kraberg , A. , Bartsch , I. , Boersma , M. , Franke , H.-D. , Freund , J. , Gebühr , C. , Gerdts , G. et al. ( 2010 ) Helgoland Roads, North sea: 45 years of change . Estuar. Coast. , 33 , 295 – 310 . Google Scholar CrossRef Search ADS Author notes Corresponding Editor: Xabier Irigoien © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Plankton Research Oxford University Press

Bioenergetics of the copepod Temora longicornis under different nutrient regimes

Journal of Plankton Research , Volume Advance Article (4) – May 21, 2018

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Abstract

Abstract The copepod Temora longicornis depends on constant prey availability, but its performance also depends on how efficiently it utilizes its food sources. Our research goal was to understand copepod energy allocation in relation to diet quality. The working hypothesis was that Temora performs better on the diet whose elemental ratio is closest to its own. Diatoms (Diat) and dinoflagellates (Dino) cultured in nutrient-replete (+) and nitrogen-depleted (−) conditions were fed to the copepods. Ingestion, respiration, excretion and egg and fecal pellet production rates were measured. Carbon (C) and nitrogen (N) budgets were built to investigate differences in dietary C and N partitioning. Copepods fed nitrogen-depleted diatoms (Diat−), which had the most different C:N ratio to that of Temora longicornis, had high metabolic losses and low growth. Copepods fed nitrogen-rich dinoflagellates (Dino+) with a more similar C:N ratio to their own also had high metabolic losses, but displayed the highest investment into somatic growth and egg production. The results indicate that dinoflagellates are a better food source for T. longicornis. Furthermore, consumption of low-quality food leads to higher respiration rates and faster leakage of dissolved organic carbon from copepod fecal pellets; and egestion is a main pathway in copepods for eliminating unabsorbed and non-metabolized carbon. INTRODUCTION Copepods are an important link in the energy transfer between primary producers and higher trophic levels, and contribute to the cycling of organic matter (Juul-Pedersen et al., 2006; Castellani and Altunbas, 2014). The small calanoid copepod Temora longicornis (Müller, 1785) is one of the dominant species in the coastal zooplankton community in the North Atlantic and North Sea, reaching peak densities during the spring and early summer (Hickel, 1975; Castellani and Altunbas, 2014). Off Helgoland, this omnivorous species is abundant all year round and its grazing may have a substantial impact on the phytoplankton standing stock (Gentsch et al., 2009; Maar et al., 2004). Temora longicornis has high metabolic turnover rates, but is unable to accumulate significant amounts of energy reserves (Kreibich et al., 2008, 2011). The species is, thus, dependent on a constant availability of prey and is vulnerable to fluctuations in food supply (Helland et al., 2003; Kreibich et al., 2008, 2011). This can be particularly problematic in systems such as the North Sea, where plankton community composition can change rapidly (Kiørboe and Nielsen, 1994). As T. longicornis inhabits dynamic systems, it must be able to quickly react to changes in trophic conditions (Gentsch et al., 2009; Kreibich et al., 2008), and efficient food utilization is of paramount importance for its survival. Food quantity and quality influence the physiology of copepods, their efficiency in nutrient uptake, and their ability to both convert food into energy and channel stored energy into reproduction (Møller, 2007; Hessen and Anderson, 2008; Jónasdóttir et al., 2009), and changes in these prey characteristics could impact not only copepod populations but also the recruitment of their predators (Boersma et al., 2015). Zooplankton are often limited by food quantity in coastal regions (Hirst and Bunker, 2003), and some food sources are also of lower nutritional value (Sterner and Schulz, 1998). It has been suggested, for example, that protozoans are qualitatively important to copepod diet (Stoecker and Capuzzo, 1990), and that egg hatching success is dependent upon the ingestion of essential fatty acids (Broglio et al., 2003). Elemental composition, digestion resistance and biochemical composition are important factors determining the nutritional value of food particles (Sterner and Schulz, 1998). A vast body of literature is available on the functional responses of calanoids to diet quality (e.g. Dam and Lopes, 2003; Arendt et al., 2005; Jónasdóttir et al., 2009; Nobili et al., 2013), but few address all important vital rates at once (e.g. Abou Debs, 1984). This comprehensive approach would allow for the assessment of energy allocation to different processes in copepods, and is necessary to come to a better understanding of the potential responses of T. longicornis to climate change-induced food regime shifts. The present work investigated metabolism, feeding, growth and reproduction in T. longicornis females in light of different prey elemental compositions/limitations. Based on the concepts of ecological stoichiometry, homeostasis and trophic upgrading (Klein Breteler et al., 1999; Sterner and Elser, 2002; Malzahn et al., 2010), specifically on how prey elemental composition affects consumer performance and energy utilization, the research goal was to investigate the partitioning of dietary carbon (C) and nitrogen (N) in relation to food quality. The working hypothesis was that copepods feeding on a diet with a C:N ratio close to their own body composition would perform best, i.e. would have the highest possible growth and reproduction rates and the lowest possible egestion, respiration and excretion rates. The opposite pattern would thus be observed for copepods feeding on prey with a C:N ratio as different from their own as possible (higher or lower). METHOD Field sampling Zooplankton were collected by horizontal hauls with a 500-µm mesh-size CalCOFI net, which were conducted for 15 minutes at 5 m depth off the German island of Helgoland (54°11′N, 07°54′E), in the southern North Sea. The quantity of females needed for the full experimental design required that the experiment be split into two parts. Samplings were performed as close in time as environmental conditions allowed on 17 (Experiment I) and 30 (Experiment II) May 2016. The samples were taken to the laboratory, where intact and active adult females of T. longicornis were immediately sorted under an Olympus SZX16 stereoscopic microscope. A total of 1150 females were sorted at each date (t0h), 1080 for the experiments, 30 for determination of in situ body C and N contents and another 40 were fixed in 4% formalin buffered with hexamethylenetetramine for measurement of prosome length (PL) under a Leica M205C (Fig. 1). Fig. 1. View largeDownload slide Experimental design used in the present study with females of the copepod Temora longicornis. The two zooplankton sampling dates in May 2016 are indicated, after which Experiments I and II immediately took place. The experimental design was almost equal between these, except that the former was conducted with diet treatments (names in bold) consisting of diatoms cultured in nutrient-rich (Diat+) and N-limited (Diat-) conditions, whereas the latter was conducted with dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. The timing of experimental phases (acclimation period, between t0h and t48h, and incubations, between t48h and t72h) is shown in the left column. Copepods were acclimated and incubated in triplicates. Sampling of copepods for C and N analysis and PL measurement are included in the diagram, as are the estimations of vital rates. Fig. 1. View largeDownload slide Experimental design used in the present study with females of the copepod Temora longicornis. The two zooplankton sampling dates in May 2016 are indicated, after which Experiments I and II immediately took place. The experimental design was almost equal between these, except that the former was conducted with diet treatments (names in bold) consisting of diatoms cultured in nutrient-rich (Diat+) and N-limited (Diat-) conditions, whereas the latter was conducted with dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. The timing of experimental phases (acclimation period, between t0h and t48h, and incubations, between t48h and t72h) is shown in the left column. Copepods were acclimated and incubated in triplicates. Sampling of copepods for C and N analysis and PL measurement are included in the diagram, as are the estimations of vital rates. Prey culture Three prey species were cultured in the laboratory, the cryptophycean Rhodomonas salina (Wislouch) (Hill and Wetherbee, 1989), the heterotrophic dinoflagellate Oxyrrhis marina (Dujardin, 1841) and the diatom Conticribra weissflogii (Grunow) (Stachura-Suchoples and Williams, 2009). Rhodomonas salina was used solely as food for O. marina, whereas the diatoms and dinoflagellates, which are both common prey for T. longicornis (Evans, 1981), were used to feed copepods. A stock solution was maintained for each of the species. New cultures were created daily by diluting part of the stock solution with fresh medium. Two types of medium were used: nutrient-replete f/2 (after Guillard, 1975) and N-depleted (f/2 without the addition of nitrate). Silicate was only added to the medium used for diatoms. Cultures were kept in a temperature-controlled room at 18°C. Rhodomonas salina and C. weissflogii were provided constant light, while O. marina was kept in the dark. Aeration was provided to R. salina, and C. weissflogii and O. marina cultures were stirred twice a day to keep cells suspended. Cultures were grown for 5 days and then used as food suspension for copepods during experiments. Daily feeding of O. marina with R. salina was planned such that prey cells were depleted by the dinoflagellate on Day 5. Cell densities were determined with a BD Accuri C6 Flow Cytometer. Conticribra weissflogii and O. marina cultures were sampled daily for the determination of cell C and N contents by filtering known cell concentrations through Whatman GF/F filters. The remaining volume was then used to feed copepods. Experimental design Experiments were initiated right after field sampling. Figure 1 depicts the distribution of different diet treatments, replication and the sampling scheme. Copepods from Experiment I were fed diatoms cultured in nutrient-replete (Diat+) and N-depleted (Diat−) medium. In Experiment II, the copepods were fed with dinoflagellates cultured with nutrient-replete (Dino+) and N-depleted (Dino−) food. The effects of food quality on predators are more evident when food is abundant than when prey quantity is low (Sterner, 1997). In order to better observe the effects of food quality on the copepods, and to be able to distinguish them from the effects of food quantity, prey were offered to copepods ad libitum (>350 μg C L−1, 8 × 103 diatom and 2 × 103 dinoflagellate cells mL−1) during the entire experiment. The only exceptions were the respiration and excretion incubations, which were performed without any food, as described below. Copepod density varied in the different experimental units for practical reasons, such as the amount of copepods available for performing the experiments and the volumes of the different experimental units themselves. Visual observations of female behavior during the experiments and the results obtained indicate that these differences in density did not affect copepod feeding. Copepods were incubated in groups of 180 females in partially filled 3 L plastic beakers (75 females L−1) fitted with a 300 μm meshed-bottom cylinder to keep the copepods from feeding on eggs and fecal pellets (FP). Although the in situ temperature was different between Experiments I and II (10 and 12°C, respectively), all experiments were conducted in the same temperature, and copepods were kept in a dark temperature-controlled room at 10 ± 0.3°C. The water was gently stirred three times a day for food resuspension. Partial water exchanges (66%) were performed daily in order to remove eggs and FP and to renew copepod food. The copepods were acclimated under these conditions for 48 h. Subsequently, 10 females were sampled from each replicate to determine body C and N contents (samples referred to as t48h). The remaining individuals were distributed among four experimental incubations in order to separately measure grazing, respiration, excretion and egg and FP production rates. Experimental units were kept in the same temperature-controlled room as the beakers. Grazing Ingestion rates were estimated based on changes in prey cell concentrations in the absence and presence of copepods (Frost, 1972). The prey cell concentrations at the beginning and end of the grazing incubation were measured from triplicate 1 L glass bottles containing filtered seawater (FSW) + the respective food suspension (“start bottles”) and FSW + food suspension + 10 copepods (“grazed bottles”), respectively. The start bottles were fixed immediately with 4% buffered formalin. Prey growth during the incubation was accounted for by creating another set of three 1 L glass bottles with FSW + food suspension (“control bottles”). The control and grazed bottles were attached onto a plankton wheel rotating with speed between 0.5 and 1 rpm for 24 h, after which their contents were fixed with formalin as described above. The copepods were retained in 300 μm mesh-sized sieves prior to fixation and sampled to measure body C and N contents. Cell densities of the preserved food suspension were determined within 6 to 8 days of fixation with a BD Accuri C6 Flow Cytometer. These were used to calculate grazing rates as number of prey cells ingested per female and per day. Respiration The sealed chamber method was applied to measure oxygen consumption rates (Harris et al., 2000) with a NTH oxygen microsensor (PreSens GmbH, Regensburg, Germany) connected to a 4-channel oxygen meter (Microx TX3, PreSens GmbH). Calibration of the microsensor was performed with aerated artificial seawater (ASW, salinity 32) as the 100% O2 reference and with a saturated Na2SO3 solution as the 0% O2 reference. In order to avoid complicated corrections for O2 production or consumption by prey (Ikeda, 1976), this incubation was conducted without prey. For each treatment, ASW was added to six 60-mL Winkler bottles. Three of these were replicate control bottles and contained only ASW and the other three were replicates with ASW and 10 copepods each. Females were placed inside the bottles and allowed to acclimate for an hour with the lid open. The O2 saturation of water was then measured, and the lid was closed. After 24 h, it was measured again, and the copepods were sampled to measure body C and N contents. O2 consumption rates (under starvation conditions) were obtained per female and per day. The use of control bottles allowed for correction for potential oxygen consumption or production by microbes during incubation. Air pressure values for Helgoland were obtained from www.wetter.com. Excretion Daily, individual excretion rates were also calculated following the sealed chamber method, and the equation from Miller and Glibert (1998) was used to calculate the total dissolved nitrogen (TDN) and the non-purgeable organic carbon (NPOC) content of filtrate samples. Non-feeding conditions were also employed in this incubation in order to create a low background against which to measure and compare changes in dissolved organic carbon (DOC) and nitrogen (DON). For each treatment, triplicate 100-mL (pre-combusted) glass bottles were prepared with only ASW (controls) and with ASW + ca. 15 copepods. After 24 h of incubation, ASW and copepods were filtered through pre-cleaned (10% HCl) syringes fitted with Whatman GF/F w/GMF (0.7 μm pore size, 25 mm diameter) syringe filters. Filtrates were immediately frozen at −20°C in pre-cleaned high-density polyethylene bottles. DOC and TDN in the filtrate were determined by high temperature catalytic oxidation (HTCO) and subsequent non-dispersive infrared spectroscopy and chemiluminescence detection using a Shimadzu TOC-VCPN analyzer. In the autosampler, the samples (6.5 mL) were acidified with HCl and sparged with oxygen (100 mL min−1) for 5 min to remove inorganic carbon. A 50-μL sample volume was injected directly on the catalyst (heated to 680°C). Final DOC concentrations were averaged values of triplicate measurements. If the standard variation or the coefficient of variation exceeded 0.1 μM or 1%, respectively, up to two additional analyses were performed and outliers were eliminated. After each batch of six samples, one DSR (Deep Sea Water Reference Material, Hansell Research Lab, University of Miami, US), one Milli-Q blank, and one potassium hydrogen phthalate standard were measured. The limit of quantification was 7 μM for DOC and 11 μM for TDN, and the accuracy was ±5%. The copepods were trapped inside the syringe filters and could not be sampled for C and N content analysis. Since both the excretion and the respiration incubations were conducted under similar conditions, the C and N contents of the copepods from the latter were also used for calculating the excretion rates. Egg and FP production For each diet treatment, females were placed individually in triplicate 12-well cell culture plates (12 females per replicate) filled with FSW and food suspension to 4 mL volume. The production of eggs and FP was checked under a binocular 1, 6, 12, 18 and 24 h after the beginning of the experiment. After eggs and feces were counted, they were carefully pipetted out of the well, at times being collected in filters for determination of their C and N contents. Due to the high amount of eggs and feces required for C and N analysis, only one pooled sample was taken from each of the different treatments, with a minimum of 400 eggs and 379 FP. Due to time limitations, eggs and FP were not washed in distilled water before sampling, but the presence of algal cells in the samples was unlikely, since most of the food had been eaten by copepods at the time of collection. Food was immediately replenished until the next count, and plates were left undisturbed. Copepods were sampled after 24 h for determination of body C and N contents. Individual egg production rates (EPRs) were calculated as a daily estimate based on the production observed over 24 h. FP production rates (FPRs) were also observed during 24 h, but due to problems during the last 6 h period, only the data from the first 18 h of incubation were used to estimate daily rates. C and N content analysis Copepods, prey cultures, eggs and FP were sampled for the determination of their C and N contents. Copepods were gently washed in distilled water, placed into pre-weighed tin cartridges and stored at −80°C until further analysis. In situ water samples and prey cultures were filtered onto pre-combusted (500°C for 24 h) Whatman GF/F filters (0.7 μm pore size, 25 mm diameter). Eggs and FP were counted and pipetted onto pre-combusted Whatman GF/F filters. Both tin cartridges and filters with samples were dried at 60°C for 48 h, folded (filters inside aluminum foil), and stored in a desiccator. The C and N contents of all samples were later measured with an elemental analyzer (detection limit: 2 μg C / 0.5 μg N; maximum error: ± 3%, Euro EA 3000, EuroVector S.P.A., Milan, Italy) using acetanilide as a standard. C and N budgets The amount of energy ingested by an organism should equal the sum of the amounts of energy egested and used for growth and metabolism. In that sense, the vital rates of an individual can also be expressed as a balanced equation, such that I = G + R + U + E + F, where I is ingestion, G is somatic growth, R is respiration, U is excretion, E is egg production and F is FP production. In order to do so, the vital rates were converted to C and N units. The number of prey cells ingested and of eggs and FP produced were multiplied by their respective C and N contents. Respiratory quotients (RQs), which ranged from 0.74 to 0.76, were estimated for each replicate (as described by McConnaughey, 1978) and used to convert the O2 consumption into C-equivalent respiration (Harris et al., 2000). The excretion incubations did not allow for the differentiation between C properly excreted (in the form of urea and amino acids, for example) and C leaked from the FP before absorption by the copepods. The majority of the DOC measured was likely leaked from FP, and thus added to the calculation of F. The G term was obtained by the formula G = (Xt48h – Xt0h) / (N * Δt), where Xt48h and Xt0h are the C or the N content of the copepod samples from t48h and t0h, respectively, N is the number of females in the sample and Δt is the time in days between t48h and t0h. Values were compared in the format μg C or N female−1 day−1. Some organisms have a limited ability to store C relative to other elements, which is especially true for many copepod species (Meunier et al., 2014), and will be in excess of this element when they are supplied a diet with more C than they can absorb and/or utilize (Hessen and Anderson, 2008). Therefore, the discussion on the C budget also approaches the topic of excess C and the pathways for returning this excess C to the environment (i.e. eliminating it). Turnover rates The budget data were further standardized to daily C and N turnover rates (% body C or N day−1). This was achieved by dividing the budget values by the median C or N contents of the copepods sampled at t48h and at the end of each incubation (to account for possible weight losses during the incubations). Somatic growth was derived from weight differences between t0 h and t48h, so the budget values for this specific turnover rate were divided by the median C or N contents of the copepods sampled at t0h and t48h (to account for possible weight gain during the acclimation). An arcsine square root transformation was then applied to the calculated percentage values. Given that there were negative values in the N growth term, and that the transformation is not possible for negative numbers, a fixed, minimum value was added to all treatments in that category to ensure values above zero. Efficiencies Different physiological efficiencies were calculated to evaluate copepod performance in relation to food quality. Absorption efficiency (AE), the percentage of ingested carbon absorbed in the gut of copepods, was calculated as AE = 1 – (F/I), where F is the egestion and I is the ingestion (Harris et al., 2000). The DOC excreted by copepods was added to the F term in the carbon AE to account for leakage from FP. The net somatic growth efficiency (NSGE) and the net egg production efficiency (NEPE) indicated the percentage of absorbed food converted into somatic growth and egg production, respectively. The formulas were adapted from Kiørboe et al. (1985) and Wendt and Thor (2015) as NSGE = G/(G + E + R + U) and NEPE = E/(G + E + R + U), where G is the somatic growth, E is the egg production, R is the respiration and U is the excretion. Statistical analysis Differences in seston and in copepod in situ elemental composition and PL were verified with t-tests. Differences in vital rates, turnover rates and efficiency calculations were tested with one-way analysis of variance (ANOVA). Differences in copepod elemental composition during the experiments were tested with a two-way ANOVA, in order to account for interactions between diet treatment and sampling time. When these results were significant, the Tukey HSD post hoc test was used at 95% confidence limits to further identify origin of differences. Prior to the ANOVA, the data were tested for normality and homogeneity of variances with Shapiro–Wilk and Bartlett tests, respectively. If the data were non-normal and/or heteroscedastic, they were analyzed with a Kruskal–Wallis test and with the post hoc Nemenyi test (with P-value being determined with the Tukey method). Analyses were performed using R ver. 3.2.5 (Ihaka and Gentleman, 1993). RESULTS C and N contents Prey culture The C and N contents and molar C:N ratio of prey cultures are presented in Table I. The average C content was always above 500 μg C L−1, confirming the ad libitum feeding condition. The average N content was always above 100 μg N L−1, with the exception of the Diat− treatment, which was clearly N-limited and contained less than half this amount. The molar C:N ratio was also similar between all treatments with the exception of the Diat−, such that the approximate values were 6 and 19 for the former and the latter, respectively. Cultures were thus significantly different from one another (Supplementary Tables S1 and S2). Table I: C and N contents and molar C:N ratio of the prey cultures (nutrient-replete and N-depleted C. weissflogii, Diat+ and Diat−, and O. marina, Dino+ and Dino−, respectively). Values are mean ± standard deviation from the three batches (temporal replicates) used for feeding in each experiment Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Table I: C and N contents and molar C:N ratio of the prey cultures (nutrient-replete and N-depleted C. weissflogii, Diat+ and Diat−, and O. marina, Dino+ and Dino−, respectively). Values are mean ± standard deviation from the three batches (temporal replicates) used for feeding in each experiment Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Experiment Treatment Prey culture μg C L−1 pg C cell−1 μg N L−1 pg N cell−1 C:N ratio I Diat+ 572 ± 55 72 ± 7 105 ± 18 13 ± 2 6.4 ± 0.5 Diat− 696 ± 46 87 ± 6 43 ± 4 5 ± 1 19.1 ± 2.2 II Dino+ 906 ± 179 453 ± 90 179 ± 26 89 ± 13 5.9 ± 0.3 Dino− 534 ± 44 267 ± 22 108 ± 4 54 ± 2 5.8 ± 0.4 Copepods The PL of in situ females was significantly greater in Experiment II (Table II, Supplementary Table S1), but their elemental compositions were not statistically different. Table II: C and N contents (in μg C copepod−1 and μg N copepod−1), and molar C:N ratio of T. longicornis. Values presented for samples obtained at t0h (in situ condition), t48h (after 48h of acclimation) and after the experimental incubations on Day 3 (grazing = G; respiration = R; and egg and FP production = EF). PL (in mm) are shown for t0h copepods. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Values presented are mean ± standard deviation of triplicate samples containing between 10 and 12 copepods each (except for PLs, which had 40 replicates each) Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Table II: C and N contents (in μg C copepod−1 and μg N copepod−1), and molar C:N ratio of T. longicornis. Values presented for samples obtained at t0h (in situ condition), t48h (after 48h of acclimation) and after the experimental incubations on Day 3 (grazing = G; respiration = R; and egg and FP production = EF). PL (in mm) are shown for t0h copepods. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Values presented are mean ± standard deviation of triplicate samples containing between 10 and 12 copepods each (except for PLs, which had 40 replicates each) Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Treatment Carbon Nitrogen C:N PL t0h t48h G R EF t0h t48h G R EF t0h t48h G R EF t0h Diat+ 16 ± 1 20 ± 0 20 ± 1 18 ± 1 18 ± 1 4 ± 0 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.7 ± 0.2 4.9 ± 0.1 5.0 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 0.99 ± 0.1 Diat− 19 ± 1 20 ± 0 17 ± 1 17 ± 2 4 ± 0 4 ± 0 4 ± 0 4 ± 0 5.4 ± 0.2 5.6 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 Dino+ 15 ± 0 20 ± 0 20 ± 1 15 ± 2 18 ± 2 4 ± 0 5 ± 0 5 ± 0 4 ± 1 4 ± 0 4.4 ± 0.1 4.8 ± 0.0 4.8 ± 0.0 4.6 ± 0.0 4.9 ± 0.0 1.05 ± 0.1 Dino− 18 ± 1 18 ± 1 15 ± 1 15 ± 1 5 ± 0 5 ± 0 4 ± 0 4 ± 0 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1 4.6 ± 0.0 Copepod C and N contents varied between 13 and 21 μg C copepod−1 and between 3 and 5 μg N copepod−1, and molar C:N ratios ranged from 4.0 to 5.6 (Table II). Although no significant differences were found between the t0h samples from Experiments I and II, the C and N contents and the molar C:N ratio were significantly different when comparing t0h with t48h and t72h, both within and between experiments (Supplementary Tables 1 and 2). A significant increase in C and N contents was observed between t0h and t48h, except in the Diat− treatment, for which the N content remained the same throughout the experiment. C and N contents of copepods sampled between t48h and the end of the incubations remained constant in the grazing incubation (Fig. 2, Table II, Supplementary Table S2), for which surplus conditions were maintained. Some level of food limitation was present in the egg and FP production rate incubations, given the small volume of the experimental unit, and copepods in the respiration incubation were food-deprived. In both incubations, the C and N contents of copepods decreased (Table II, Supplementary Table S2). The highest increase in molar C:N ratio was observed for the Diat− treatment (from 4.7 at t0h to 5.6 at the end of the grazing incubation), which was always significantly different from all other treatments (Fig. 2, Table II, Supplementary Table S2). Fig. 2. View largeDownload slide C and N contents and molar C:N ratio of T. longicornis females during Experiments I and II. Experiment I tested diet treatments consisting of diatoms cultured in nutrient-rich (Diat+, gray squares and dotted lines) and N-depleted (Diat−, black squares and full lines) conditions, whereas Experiment II tested diets consisting of dinoflagellates fed with nutrient-rich (Dino+, gray triangle and dotted lines) and N-limited (Dino−, black triangle and dotted lines) prey. Samples were taken of in situ conditions (t0h, empty symbols), after 48h of acclimation (t48h), and after 24h of incubations (t72h). Bars represent mean values ± one standard deviation. Fig. 2. View largeDownload slide C and N contents and molar C:N ratio of T. longicornis females during Experiments I and II. Experiment I tested diet treatments consisting of diatoms cultured in nutrient-rich (Diat+, gray squares and dotted lines) and N-depleted (Diat−, black squares and full lines) conditions, whereas Experiment II tested diets consisting of dinoflagellates fed with nutrient-rich (Dino+, gray triangle and dotted lines) and N-limited (Dino−, black triangle and dotted lines) prey. Samples were taken of in situ conditions (t0h, empty symbols), after 48h of acclimation (t48h), and after 24h of incubations (t72h). Bars represent mean values ± one standard deviation. Eggs and FP Diatom-fed copepods produced eggs with the highest C content and the highest and the lowest N content (72 and 69 ng C egg−1 and 13 and 9 ng N egg−1 for nutrient-replete and N-depleted treatments, respectively). Dinoflagellate-fed copepods produced eggs with similar C and N contents (60 ng C egg−1 and 12 and 11 ng N egg−1 for Dino+ and Dino−, respectively) The molar C:N ratio of eggs was similar between the Diat+ (6.5) and Dino− (6.4) diets, and lower and higher in the Dino+ (5.8) and Diat− (8.9) treatments, respectively. The FP from diatom-fed copepods had the lowest C and N contents (26 and 20 ng C FP−1 and 4 and 2 ng N FP−1 for nutrient-replete and N-depleted treatments, respectively). The C and N contents of the FP from dinoflagellate-fed copepods were almost twice as high (52 and 48 ng C FP−1 and 9 and 7 ng N FP−1 for Dino+ and Dino−, respectively). The molar C:N ratio of FP was lower in the Dino+ (6.7), Diat+ (7.6) and Dino− (8.0) diets than in the Diat− treatment (11.7). It was not possible to measure FP sizes, however, visual observations indicate that FPs from copepods fed with diatoms were slightly longer and thicker than those produced by copepods fed with dinoflagellates. Vital rates and turnover rates Diatom-fed copepods had significantly higher average ingestion rates (8.6 ± 0.5 × 104 and 10.1 ± 1.2 × 104 cells female−1 day−1 for Diat+ and Diat−, respectively) than the dinoflagellate-fed copepods (2.4 ± 0.8 × 104 and 1.6 ± 0.5 × 104 cells female−1 day−1 for Dino+ and Dino−, respectively) (Supplementary Tables S1 and S3). C ingestion, which ranged from 17 to 86% body C day−1, was significantly higher in the Dino+ treatment than in the Dino−, whereas N ingestion, which ranged from 12 to 66% body N day−1 (Table III), was significantly higher in the Dino+ diet than in the N-depleted treatments (Supplementary Tables S1 and S3). Table III: C and N turnover rates and AE, NSGE and NEPE efficiencies from experimental incubations with T. longicornis. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Mass-specific values for ingestion (I), growth (G), carbon-equivalent respiration (R), excretion (U), egg production (E) and FP production (F) turnover rates are expressed as % body C or N day−1, and efficiencies as %. Values presented are mean ± standard deviation. Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Table III: C and N turnover rates and AE, NSGE and NEPE efficiencies from experimental incubations with T. longicornis. Diet treatments: single cultures of C. weissflogii (Diat) and O. marina (Dino) in nutrient-replete (+) and N-depleted (−) conditions. Mass-specific values for ingestion (I), growth (G), carbon-equivalent respiration (R), excretion (U), egg production (E) and FP production (F) turnover rates are expressed as % body C or N day−1, and efficiencies as %. Values presented are mean ± standard deviation. Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 Treatment Carbon Nitrogen I G R E F AE NSGE NEPE I G U E F AE NSGE NEPE Diat+ 32 ± 1 11 ± 1 7 ± 1 15 ± 4 13 ± 1 60 ± 3 33 ± 4 45 ± 5 24 ± 1 9 ± 1 5 ± 9 11 ± 3 8 ± 0 68 ± 1 36 ± 11 48 ± 6 Diat− 49 ± 6 6 ± 2 10 ± 0 14 ± 1 19 ± 2 64 ± 7 20 ± 5 47 ± 2 14 ± 2 0 ± 1 27 ± 5 8 ± 1 5 ± 0 67 ± 7 −1 ± 4 23 ± 4 Dino+ 65 ± 23 13 ± 1 14 ± 3 19 ± 1 23 ± 1 64 ± 12 27 ± 2 44 ± 4 50 ± 17 10 ± 1 8 ± 4 15 ± 1 13 ± 1 73 ± 9 29 ± 3 48 ± 7 Dino− 27 ± 10 10 ± 2 10 ± 1 14 ± 1 20 ± 1 36 ± 5 29 ± 5 42 ± 2 21 ± 8 9 ± 2 15 ± 3 11 ± 1 8 ± 0 60 ± 12 25 ± 6 32 ± 3 The average oxygen consumption rates were lower in the diatom treatments (3.4 ± 0.3 and 4.3 ± 0.1 mL O2 female−1 day−1 for Diat+ and Diat−, respectively) than in the dinoflagellate treatments (6.0 ± 1.3 and 4.2 ± 0.4 mL O2 female−1 day−1 for Dino+ and Dino−, respectively). They corresponded to a C-equivalent respiration of 6–18% body C day−1 (Table III). A significant difference in O2 consumption and turnover of respired C was only found between the nutrient-replete treatments, with higher values in the Dino+ diet (Supplementary Tables S1 and S3 and Table III). Surprisingly, the average N excretion values were higher in the N-depleted treatments (1.0 ± 0.3 and 0.6 ± 0.1 μg N female−1 day−1 for the Diat− and Dino−, respectively) than in the nutrient-replete diets (0.2 ± 0.2 and 0.3 ± 0.1 μg N female−1 day−1 for the Diat+ and Dino+, respectively). They corresponded to 5–32% body N day−1 (Table III) and were significantly lower in the Diat− treatment (Supplementary Tables S1 and S3). The average FP production rates were not significantly different between the Diat+ (89 ± 4 pellets female−1 day−1) and Diat− (86 ± 8 pellets female−1 day−1) treatments. FP production rates recorded for copepods fed with dinoflagellates were lower, 72 ± 5 and 49 ± 1 pellets female−1 day−1 for the Dino+ and Dino− treatments, respectively (Supplementary Tables S1 and S3). The average amount of DOC leaked from FP, which was significantly different between all treatments (Supplementary Table S1), was higher in the N-depleted diets (1.6 ± 0.2 and 1.1 ± 0.1 μg C female−1 day−1 for Diat− and Dino−, respectively) than in the nutrient-replete treatments (0.2 ± 0.1 and 0.7 ± 0.1 μg C female−1 day−1 for Diat+ and Dino+, respectively). The C and N turnover rates for FP production, which varied between 12 and 24% body C day−1 and between 4 and 14% body N day−1 (Table III), were significantly different between almost all treatment pairs (Supplementary Tables S1 and S3). Somatic growth was significantly different between treatments in terms of both C and N (Supplementary Table S1). It ranged from 4 to 14% body C day−1 and from 0 to 11% body N day−1 (Table III), with the lowest values recorded for the N-limited diatom treatment (Supplementary Table S3). The average EPRs were significantly higher in the Dino+ treatment (61 ± 4 eggs female−1 day−1) than for copepods fed with the other diets (39 ± 9, 36 ± 4, and 41 ± 5 eggs female−1 day−1 for Diat+, Diat− and Dino−, respectively) (Supplementary Tables S1 and S3). The egg C turnover (13–21% body C day−1) was similar between treatments (Table III), but the N turnover (7–16% body N day−1) was significantly different between the Dino+ and Diat− treatments (Supplementary Tables S1 and S3). C and N budgets The C and N budgets obtained for each of the treatments are represented in Fig. 3. They were mostly unbalanced, which is represented in the figure by the equation symbols “−” and “+”. The “I” values are represented on the left side of the copepod antenna, whereas the right side represents the sum of “G + R + U + E + F”. When the gain of energy via feeding was higher than growth and energy expenditure (e.g. Fig 3B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (e.g. Fig 3D and F), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (Fig. 3C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The budgets recorded for the Dino+ treatment seem to have the best estimates (percentages close to 100%). The budgets for the other treatments mostly point to higher growth and energy expenditure. The Dino− treatment had the most deviating results, with expenses amounting to twice as much the recorded energy ingested, a pattern visually represented by the use of double equation symbols (Fig. 3D and H). The Diat+ budgets also show higher expenses than energy gain (Fig. 3A and E), whereas the Diat− budgets show a mixed response, with an accurate estimate for the C budget (Fig. 3B) but an inaccurate estimate for the N budget (Fig. 3F). The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows in Fig. 3. Fig. 3. View largeDownload slide Carbon (A–D) and nitrogen (E–H) budgets obtained for T. longicornis females feeding on diatoms cultured in nutrient-rich (Diat+) and N-depleted (Diat−) conditions and on dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. Values given represent daily ingestion, egestion, respiration, excretion, reproduction and somatic growth rates for one individual copepod, and are visually represented by proportionately sized arrows. The comparison between the amount of C and N ingested by copepods (“I” values on the right side of the copepod antenna) and the amount used by them (the sum of “G + R + U + E + F” values on the left side of the copepod antenna) is represented in the figure by the equation symbols “+”, “−” and “=”. When the gain of energy via feeding was higher than growth and energy expenditure (B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (A, D, E, F and H), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The closer to 100%, the more balanced the measurements; values below and above 100% indicated higher energy gain from feeding than energy expenditure and higher energy expenditure than energy gain from ingestion, respectively. The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows. Fig. 3. View largeDownload slide Carbon (A–D) and nitrogen (E–H) budgets obtained for T. longicornis females feeding on diatoms cultured in nutrient-rich (Diat+) and N-depleted (Diat−) conditions and on dinoflagellates fed with nutrient-rich (Dino+) and N-limited (Dino−) prey. Values given represent daily ingestion, egestion, respiration, excretion, reproduction and somatic growth rates for one individual copepod, and are visually represented by proportionately sized arrows. The comparison between the amount of C and N ingested by copepods (“I” values on the right side of the copepod antenna) and the amount used by them (the sum of “G + R + U + E + F” values on the left side of the copepod antenna) is represented in the figure by the equation symbols “+”, “−” and “=”. When the gain of energy via feeding was higher than growth and energy expenditure (B and G), the “+” and “−” signs are placed on the left and right sides of the antenna, respectively. When the gain of energy was lower than growth and energy expenditure (A, D, E, F and H), the “+” and “−” signs are placed on the right and left sides of the antenna, respectively. The symbol “=” was used for the C budget which was nearly balanced (C). To the right of these symbols is a percentage, which indicates the percentage of energy gained via feeding that was recorded as being used. The closer to 100%, the more balanced the measurements; values below and above 100% indicated higher energy gain from feeding than energy expenditure and higher energy expenditure than energy gain from ingestion, respectively. The DOC and FP components of the egestion term of the C budget (percentage of total “F”) are indicated in gray and white colors (respectively) in the corresponding arrows. Efficiencies The AEs varied between 33% and 75% and between 46% and 81% of C and N ingested, respectively (Table III), with the carbon AE for Dino− being significantly lower than that of Diat− and Dino+ (Supplementary Tables S1 and S3). The carbon NSGEs varied between 15% and 36%, and were significantly different between the diatom treatments, while the nitrogen NSGEs varied from −4% to 48% and were significantly lower in Diat− (Table III, Supplementary Tables S1 and S3). No significant differences were found between the carbon NEPE, which ranged from 39% to 50%, whereas the nitrogen NEPEs, which varied from 19% to 56% (Table III), were significantly higher in the nutrient-replete treatments (Supplementary Tables S1 and S3). DISCUSSION To our knowledge, this is the first study of energy budgets of T. longicornis females feeding on prey items of different qualities, which enabled the comparison of copepod performance under different food regimes. Our working hypothesis was that the highest growth and reproduction rates and the lowest catabolic rates would be recorded for copepods feeding on a diet with a C:N ratio as close as possible to their own body composition. Our study was able to partially confirm the working hypothesis. The highest investment in somatic and reproductive growth was indeed shown by copepods fed with a food source of C:N ratio close to their own (Dino+), but so were the highest expenditures with egestion and catabolism, contrary to what had been postulated. Performance entails the amount of energy ingested and used by an individual for different vital activities, but is ultimately defined by what is invested in reproduction and how successful this process is. Thus, the major finding of this study is that dinoflagellates are a food source for copepods of superior or similar quality to diatoms under nutrient-replete or N-depleted conditions, respectively. This is, however, only valid for the temperatures investigated in the present study, as individual metabolism and C requirements are affected by temperature (e.g. Boersma et al., 2016). Results also revealed that egestion is a major pathway for T. longicornis females to eliminate the excess C; and that low food quality can influence copepod respiration (regardless of its C-to-nutrient ratio) and the intensity and speed with which DOC leaks from FP. The fact that results only partially agreed with predictions from stoichiometric theory indicates the need for further investigations into copepod ecophysiology and adaptive capacity to shifting food regimes. The level of N-limitation achieved in the Diat− treatment was not expected for the Dino− diet, as it has been shown that O. marina can regulate its body composition to incorporate the stoichiometric imbalances of its prey in an attenuated form (Malzahn et al., 2010; Meunier et al., 2014). Although the elemental composition of the Dino− treatment was similar to that of the Dino+, its fatty acid profile was not (unpublished data). This could explain why performance was different for copepods fed with these two diets, and why the former constituted a food source of lower quality. Copepod feeding The C ingestion rates reported by Arendt et al. (2005) and Jónasdóttir et al. (2009) for T. longicornis feeding on C. weissflogii are twice as high as those reported herein, despite their use of lower prey concentrations (Table IV). The values from our study might be underestimated, as indicated by the comparison of the amount of energy ingested and used by copepods (symbols on the top left and right sides of the copepod antenna in Fig. 3, respectively). Despite measurements being recorded in the same fashion for all treatments, some show a nearly balanced budget (e.g. Fig. 3C) while others reveal unbalanced budgets (e.g. Fig. 3F). We speculate that the ingestion rates are underestimated and that this might be due to copepods feeding on food sources that were unaccounted for, which were ingested in quantities inversely proportional to the quality of the diet treatments. Coprophagy and filial cannibalism are known to occur among copepods, regardless of availability of alternative food, and can reach values of up to 50% of produced FP and 60 eggs female−1 day−1 (e.g. Lampitt et al., 1990; Noji et al., 1991; Dam and Lopes, 2003; Boersma et al., 2014) in starved or food-limited conditions. The glass bottles used for the grazing incubation in the present study did not allow for the separation of copepods from eggs and FP. The eggs had a similar or higher nutritional quality (in terms of elemental composition/molar C:N ratio) than the prey items, and the microbiota associated with the peritrophic membrane of the FPs might be a valuable food source for copepods (as suggested by Lampitt et al., 1990), so by feeding on its own eggs and FPs, copepods could have complemented nutritionally inadequate diets. This response would be a form of compensatory behavior for this copepod, and has been identified in other studies (Augustin and Boersma, 2006; Siuda and Dam, 2010) as a mechanism for other small calanoids to overcome the elemental limitation of their prey. Table IV: Comparative values between prey species and quantity used in laboratory experiments and the ingestion and EPR of T. longicornis recorded for that prey in the present study and elsewhere in the literature Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study aUsing a carbon:chlorophyll α ratio of 50, according to Dam and Peterson (1991). Table IV: Comparative values between prey species and quantity used in laboratory experiments and the ingestion and EPR of T. longicornis recorded for that prey in the present study and elsewhere in the literature Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study Copepod Prey Ingestion EPR Reference Type μg C L−1 Cells ind−1 day−1 μg C ind−1 day−1 % body C day−1 Eggs ind−1 day−1 Temora longicornis Natural prey assemblage 200–800a 1–4a 2–55 Dam and Peterson (1991) and Peterson and Dam (1996) Phytoplankton 50 Castellani and Altunbas (2014) Jansen et al. (2006) Dinoflagellates 3 Jansen et al. (2006) Conticribra weissflogii 349 204 000 ± 70 000 15.6 ± 4.8 124 27.8 ± 7.3 Arendt et al. (2005) 349 6.5 70 11 Dam and Lopes (2003) 225–276 68 5–19 Jónasdóttir et al. (2009) 20–60 Niehoff et al. (2015) 274 2.2 ± 0.2 25 van Someren Gréve (2013) 572 ± 55 86 000 ± 500 6.3 ± 0.2 32 ± 1 39 ± 9 This study Oxyrrhis marina 20–80 Niehoff et al. (2015) 286 4.7 ± 0.4 51 van Someren Gréve (2013) 906 ± 179 24 000 ± 800 13.1 ± 4.6 65 ± 23 61 ± 4 This study aUsing a carbon:chlorophyll α ratio of 50, according to Dam and Peterson (1991). Copepod waste production (or catabolic activities) Few studies are available on the metabolic activities of small copepods such as T. longicornis (e.g. Berner, 1962; Dam and Peterson, 1993; Nobili et al., 2013; Castellani and Altunbas, 2014), all reporting lower O2 consumption and C-equivalent respiration than the ones described herein (1–2 μL O2 ind−1 day−1 and 1% body C day−1). Our respiration and excretion incubations were performed without feeding the copepods, and probably underestimate the rates of fed copepods (Miller and Glibert, 1998; Thor, 2002a, 2002b; Nobili et al., 2013). High respiration rates have been suggested as a mechanism for removal of excess C in unbalanced nutrient conditions (Anderson et al., 2005; Hessen and Anderson, 2008), even though the mechanism itself is yet to be described (Malzahn et al., 2010). The results of the present study partially agree with this assumption. The second highest respiration rates were reported for copepods fed with the N-poor diets, but one of them was enriched in C in relation to N (Diat−) and the other had a C:N ratio close to that of the copepods (Dino−). Furthermore, the highest rates were measured for another one of the diets whose C:N more closely resembled that of the copepods (Dino+). The type of substrate catabolized by starved copepods in order to obtain energy influences respiration (Castellani and Altunbas, 2014) and excretion (Anderson, 1992) rates. Proteins are the main substrates catabolized in small calanoids (Thor, 2002). The RQ of proteins is higher than that of lipids (Gnaiger, 1983) and would, thus, generate higher C-equivalent respiration rates in starved individuals that were previously fed N-rich diets, as observed for O. marina by Meunier et al. (2012). Our results thus suggest that O2 consumption increases with decreasing food quality, regardless of an excess of C or not. If prey is of high nutritional quality, then O2 consumption seems to be modulated by the amount of food ingested. The preferential utilization of proteins by copepods previously fed with a N-rich diet should also result in higher ammonia excretion rates than those of copepods previously fed N-poor diets, but the opposite was recorded. It is possible that the high food C:N ratio of 19 in the Diat− treatment led to an increase in ingestion rates and a decrease in gut passage time. This, in turn, reduced the efficiency with which nutrients were absorbed and resulted in higher excretion rates (Plath and Boersma, 2001). FP production by copepods is an important source of POM and DOC in the epipelagic (Møller et al., 2003; Thor et al., 2003), contributing to microbial production with recycled nutrients (Smetacek, 1980; Strom et al., 1997). Although it has been claimed that undisturbed FP do not release DOC (Strom et al., 1997), it has been shown that 50% of the total C content of FP is released as DOC at the onset of defecation (Thor et al., 2003). DOC can be generated by copepods before absorption, as leakage from egested FP, and after absorption, as excretion in the form of nitrogenous organic compounds (Frangoulis et al., 2004). The methodology did not allow for the separation of both components. Even though some DOC may have been excreted, it was considered that the majority of DOC measured was leaked from FP, which is why DOC values were added to the C content of FP to calculate the egestion term (“F”) of the C budget. In our study, the percentage of total C egested (“F”) that corresponded to DOC varied widely between treatments, going from approximately 8% in Diat+ to 16% in Dino+, 31% in Dino− and 50% in Diat−. It would seem, thus, that the level of DOC leakage from FP is directly or indirectly influenced by food quality. Small calanoids such as T. longicornis are unable to store significant amounts of energy reserves, and must return to the environment any C consumed in excess of its needs and ability to absorb. Our results suggest that the primary pathway for T. longicornis to eliminate excess C was egestion, which removed twice as much C as that burnt via respiration (Fig. 3A–D). Furthermore, if all C had been assimilated and metabolized and no excess was left, assuming that all N was also assimilated, the molar C:N ratio of FP would not have differed from that of the diet treatments (Checkley, 1980). The fact that it was higher in the FP (except for Diat−) indicates that N was used constantly and efficiently for production and that the excess C was egested (Checkley, 1980). This pattern is further supported by the statistically similar nitrogen AE in the treatments, contrary to what stoichiometric theory would have predicted. The carbon AE, on the other hand, was significantly lower in the Dino− treatment. It could be that the resulting biochemical composition of the Dino− diet indirectly caused an inefficient absorption of C in copepods and led to an egestion similar to that of the other treatments despite a lower ingestion rate. Our estimates for the carbon AE were greater than the assimilation efficiency estimates for another small copepod (A. tonsa) feeding on the same prey species (Besiktepe and Dam, 2002). Copepod growth and reproduction (or anabolic activities) Most studies assume negligible increases in body weight in adult copepods which do not store energy reserves and only investigate reproductive investment as a measure of growth (Hirst and McKinnon, 2001). The C and N somatic growth rates observed in the present study, though lower than the EPRs, contest this assumption, as do other studies (Hirst and McKinnon, 2001; Dam and Lopes, 2003). A comparison between the net efficiencies for somatic growth (NSGE) and egg production (NEPE) emphasizes how the majority of assimilated energy is used in reproduction, but also that the portion destined to somatic growth is not negligible. The only absence of somatic growth was observed in terms of N for the Diat− treatment, as expected. The EPR recorded for T. longicornis in this study were within the range of those obtained when this copepod was fed with natural plankton (Peterson and Dam, 1996), similar prey species (Niehoff et al., 2015) and with water from the spring phytoplankton bloom (Castellani and Altunbas, 2014; Peterson and Kimmerer, 1994), but higher than when it was fed water from a dinoflagellate bloom (Jansen et al., 2006) (Table IV). The highest EPR in our study was obtained with the nutrient-rich dinoflagellate diet, as opposed to results from other studies (Turner et al., 2001; Dam and Lopes, 2003; Jónasdóttir et al., 2009). It must be noted that great controversy involves the topic of diatoms being good or bad quality diet items (for reviews, see Jónasdóttir et al., 1998; Paffenhöfer et al., 2005), with many suggesting its suitability for egg production but reduced success for hatching (Miralto et al., 1999; Ban et al., 1997). The quantity of body C invested into reproduction was similar across treatments, but body N investment was significantly different between the Diat− and the Dino+ treatments. This indicates N-limitation in egg production, as also suggested by Nobili et al. (2013) for T. longicornis. The nitrogen NEPE observed in our study further indicates a clear limitation of this element in copepods fed with the N-depleted diets. This contrasts the assumption that nutrients in limited amounts should be used with higher efficiency. Other studies have reported similar trends, with lower egg production efficiencies associated with higher food C:N ratios and with P-limitation (Anderson et al., 2005; Nobili et al., 2013). This pattern might be due to increased maintenance requirements for copepods fed with nutrient-limited diets (Anderson et al., 2005; Wendt and Thor, 2015). Even so, the NEPE values reported herein seem to be mostly higher than those obtained from the literature as either egg production efficiency or gross growth efficiency (e.g. Dam and Lopes, 2003; Thor et al., 2007; Nobili et al., 2013; Wendt and Thor, 2015). Few studies have approached the effect of N-limitation on EPR, which has been said to be both deleterious (Koski et al., 2006; Nobili et al., 2013) and advantageous (Augustin and Boersma, 2006). EPR does not provide information on egg viability (hatching success), so it would be necessary to conduct further investigations in order to understand the importance of diet quality for secondary production by T. longicornis. Climate change and marine C and N cycles Long-term monitoring data from Helgoland have shown a drastic decrease in calanoid copepod densities since 1985, a decline most likely caused by the decrease in nutrient (N and P) loading coupled to the increase in light penetration in the region (Boersma et al., 2015). These would have resulted in phytoplankton with higher C:nutrient ratios and, thus, of lower nutritional value for zooplankton (Boersma et al., 2015). Recent studies show controversial results regarding the relative dominance in recent years of diatoms and dinoflagellates in the southern North Sea (Wiltshire et al., 2010; Alvarez-Fernandes et al. 2012; Hinder et al., 2012; Boersma et al., 2015). Changes in the abundance of these prey species can affect secondary production, and a further increase in their C:N ratio could have major consequences for food web processes (Jones and Flynn, 2005; Malzahn and Boersma, 2011; Nobili et al., 2013; Boersma et al., 2015). On the other hand, increasing local temperatures (Wiltshire et al., 2010) could result in higher metabolic costs for copepods (Castellani and Altunbas, 2014), which can only be met by an increased C consumption (Boersma et al., 2016; Malzahn et al., 2016). In that scenario, increasing temperatures would dampen the effects of prey with higher C:nutrient ratios (Boersma et al., 2016; Malzahn et al., 2016). In N-limitation conditions (e.g. late spring bloom), dinoflagellates are still of better nutritional value than diatoms (Jones and Flynn, 2005; this study), but at higher temperatures, copepods might need to preferentially consume autotrophs to supply metabolic demand, as observed for T. longicornis (Boersma et al., 2016) and A. tonsa (Malzahn et al., 2016). All of these variables make it difficult to say at this point how T. longicornis (and possibly other calanoid copepods) will react to future changes in prey composition, even though its omnivorous strategy and related adaptations allow it to switch food sources depending on their availability (Daan et al., 1988; Gentsch et al., 2009). CONCLUSIONS The results obtained in the present study indicate that, under nutrient-replete conditions, dinoflagellates such as O. marina are a better food source for T. longicornis than diatoms. This major finding is, however, dependent upon environmental conditions. Furthermore, low-quality food also leads to higher respiration rates, regardless of its C-to-nutrient ratio, and to faster leakage of DOC from copepod FP. In addition, egestion seems to be the main pathway for eliminating excess C, contrary to the common belief of excess C being mostly respired. The vital rates measured herein for T. longicornis provide important information on this species’ food utilization efficiency and maintenance costs in relation to diet quality. The budget approach used herein is not free of flaws, and future research in the field should investigate more complex scenarios (mixed diets, different temperatures, varying food concentrations, include all life stages). SUPPLEMENTARY DATA Supplementary data are available at Journal of Plankton Research online. ACKNOWLEDGEMENTS We kindly thank the crew of the Aade research vessel (AWI-BAH) for providing us with zooplankton samples and the technicians at the AWI, BAH and Claudia Burau for the help with sample analyses. Funding R.M.F.-S. was supported by a doctoral research grant from the Doctoral Programme on Marine Ecosystem Health and Conservation (MARES, Framework Agreement Number 2011-0016). The work was further supported by Ghent University (BOF-GOA project 01G02617). C.L.M. was supported by the Bundesministerium für Bildung und Forschung (BMBF grant no. 01LN1702A). Data archiving The laboratory experimental raw data and metadata for this study are available through the PANGAEA repository under https://doi.pangaea.de/10.1594/PANGAEA.886050. Ethical standards The experiments described in the present study comply with the current laws of the country in which they were performed. Conflict of interest The authors declare that they have no conflict of interest. References Abou Debs , C. ( 1984 ) Carbon and nitrogen budget of the calanoid copepod Temora stylifera: effect of concentration and composition of food . Mar. Ecol. Prog. Ser. , 15 , 213 – 223 . 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Journal of Plankton ResearchOxford University Press

Published: May 21, 2018

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