Sex-specific starvation tolerance of copepods with different foraging strategies

Sex-specific starvation tolerance of copepods with different foraging strategies Abstract Planktonic copepods have sexual dimorphism that can lead to differences in starvation tolerance between genders. Additionally, mating may be energetically costly and thus reduce starvation tolerance. We investigated the influence of sexual dimorphism and mating on starvation tolerance of copepods with different feeding behaviours: Oithona nana (ambusher), Temora longicornis (feeding-current feeder) and Centropages typicus (cruiser). Males of C. typicus and O. nana had a starvation tolerance lower than females, whereas T. longicornis had a similar starvation tolerance between genders. Only O. nana males and females had reduced starvation tolerance when both genders were incubated together, which suggests that mating activities in ambushers have an energetic cost higher than in active feeding copepods. C:N ratios showed a non-significant difference between genders, which indicates that gender differences in starvation tolerance are not due to dissimilarities in lipid reserves. Gender differences in starvation tolerance can be partially explained by body size differences between sexes. This indicates a minor influence of mate-seeking behaviour on male starvation tolerance, likely due to reduced mate-searching behaviour under prolonged starvation. Our results demonstrate that sexual dimorphism can result in different starvation tolerance between copepod genders and that a negative effect of mating on starvation tolerance depends on the foraging strategy. INTRODUCTION Food in marine environments is generally scarce, patchy and fluctuating, often because of seasonal cycles (Cebrián and Valiela, 1999; Winder and Cloern, 2010). Therefore, planktonic organisms have to cope with the risk of starvation for short and long periods. In fact, starvation is considered one of the main non-predatory causes of mortality in zooplankton (Tang et al., 2014). Most studies on starvation tolerance of planktonic copepods focus only on females and little is known about the ability of male copepods to cope with starvation (Dagg, 1977; Finiguerra et al., 2013). The abundance of copepod males in field populations is low compared to females in many families (Kiørboe, 2008), which can limit population growth (Kiørboe, 2007). Therefore, a better knowledge of the potential causes of mortality (e.g. starvation) of males is crucial for understanding skewed sex ratios and the dynamics of copepod populations. Planktonic copepods have sexually dimorphic traits and males and females commonly differ in body size and motile behaviour (e.g. Gilbert and Williamson, 1983; Ohtsuka and Huys, 2001; van Someren Gréve et al., 2017b). A smaller body size in copepods often implies a higher weight-specific respiration rate (Almeda et al., 2011) since mass-specific metabolic rates commonly decline with size with a power of −1⁄4 (Kleiber, 1961; Kiørboe and Hirst, 2014). Copepod males are commonly smaller than females and males might have a higher mass-specific energy expenditure rate and, consequently, a lower starvation tolerance than females. Differences lipid storage between copepod genders can also cause differences in starvation tolerance between males and females (Gismervik, 1997). Copepod males typically display a mate-searching behaviour and swim more frequently and faster than females (Katona, 1973; Ohtsuka and Huys, 2001; Kiørboe et al., 2005; Bagøien and Kiørboe, 2005a). Differences in motile behaviour between genders may result in different energy expenditure (Paffenhöfer, 2006) and consequently in different levels of starvation tolerance. Behavioural asymmetry between copepod genders depends on their foraging strategy (Kiørboe, 2008; van Someren Gréve et al., 2017a). We can broadly classify the feeding behaviours of copepods in two main foraging strategies in terms of motility: “sit-and-wait” (ambushing) vs. “searching” (active feeding). Ambush feeding copepods remain motionless in the water waiting for prey to enter their perceptive sphere and when perceiving the prey they perform a fast jump to capture it (Kiørboe et al., 2010; Kiørboe, 2011, 2016). Active feeding copepods cruise through the water and capture encountered prey (“cruising feeders”) and/or can hover while generating a feeding current (“feeding-current feeders”) (Kiørboe, 2011, 2016). Thus, the conflict between feeding and mate-seeking behaviour varies depending on the copepod foraging strategy (Kiørboe et al., 2010; van Someren Gréve et al., 2017b). In strict ambushers (e.g. Oithonidae), there is a strong conflict between feeding and mate-seeking behaviour because feeding and searching for mates are mutually exclusive activities (Kiørboe, 2008). Hence, ambush feeding males need to alternate between stationary feeding and a potentially energy expensive female-seeking behaviour (Kiørboe, 2008). For example, ambush feeding Oithona males move ~20–30% of the time (Kiørboe, 2008; van Someren Gréve et al., 2017b) and 15-fold faster than females (Kiørboe, 2008) whereas females spent most of the time motionless or sinking. In copepods with active feeding behaviour, both genders move/swim actively and the conflict between feeding and mate searching is expected to be low (Bagøien and Kiørboe, 2005a; Kiørboe, 2007, 2008; Bjærke et al., 2016; van Someren Gréve et al., 2017b). However, gender-specific starvation tolerance of marine planktonic copepods with different foraging strategies has not been systematically examined. Mating behaviour of copepods commonly includes search, encounter, capture, escape behaviour and/or copulation (Buskey, 1998; Titelman et al., 2007; Dur et al., 2011) and involves mate recognition by mechanical or chemical sensorial perception (Bagøien and Kiørboe, 2005a). Upon mate encounter, the male grabs the female with his geniculated antennae and the female can thereafter swim around with the male attached for minutes to hours, depending on the maturation of the spermatophores (Blades, 1977). Females can also display different escape behaviours, including jumps, shaking off males and calmly swimming away, but these mating escape behaviours have rarely been quantified (Titelman et al., 2007). These behaviours associated to mating (copulation) can also affect the energetic expense and therefore influence starvation tolerance in both genders. Kiørboe et al. (2015) found that the lifespan of males and females in some copepod species was significantly reduced when the both genders were incubated together, which indicates that mating has a cost in terms of longevity. However, it is still unknown if mating (copulation)-related activities can also reduce starvation tolerance in planktonic copepods. In this study, we experimentally investigated the influence of gender and mating on starvation tolerance of planktonic copepods with different feeding behaviours: cruise feeding (Centropages typicus), feeding-current (Temora longicornis) and ambush feeding (Oithona nana). We hypothesize that (i) copepod males have lower starvation tolerance than females due to their smaller size and active mate-seeking behaviour, (ii) difference in starvation tolerance between genders is particularly high in ambush feeders due to the high asymmetry in motile behaviour between genders and (iii) mating (copulation) decreases starvation tolerance of both males and females, independently of their foraging strategy. To test our hypotheses, we determined the starvation tolerance of males and females of copepods with different feeding behaviours incubated separately or together in the absence of food. We evaluate the effect of elemental composition (C:N ratios), body size and motile behaviour on gender-specific starvation tolerance in copepods with different feeding behaviours, and discuss the ecological implications of our results in the context of food seasonality and copepod sex ratios in field populations. METHOD Experimental organisms Copepod males and females were obtained from continuous stock cultures at DTU Aqua. Stock cultures of C. typicus, T. longicornis and O. nana were established from specimens originally collected in Gullmar fjord (Sweden, 2014), Tjärnö (Sweden, 2013) and the Port of Gijón (Spain, 2012), respectively. Cultures of C. typicus (cruise feeder) and T. longicornis (feeding-current) were fed a mixture of phytoplankton: Akashiwo sanguinea, Heterocapsa triquetra, Prorocentrum minimum, Thalassiosira weissflogii and R. salina and, in the case of C. typicus also with Oxyrrhis marina. O. nana (ambush feeder) was fed the heterotrophic dinoflagellate O. marina, which was in turn fed R. salina. All copepods were fed three times weekly at saturating levels and kept at 15°C in the dark. The phytoplankton cultures were kept in exponential growth in B1 culture medium (Hansen, 1989) at 18°C on a 12:12-h light/dark cycle. Experimental procedures Before starting the experiment, copepodites (CIII–CV) were picked and isolated in 12-well plates [5 mL of 0.2 μm-filtered seawater (FSW) per well, salinity 32‰], and fed daily a mixture of O. marina and R. salina at saturating levels until reaching maturation (~2–4 days depending on the species). Upon maturity, the adult copepods were gently rinsed three times with FSW by sequential transferring of individual copepods to cuvettes with FSW under a dissecting microscope. The absence of phytoplankton after rinsing the copepods was verified under the microscope. The following experimental treatments were investigated: females alone (FF), males alone (MM) and females together with males (FM) and males together with females (MF) (Table I). Starvation experiments were conducted in six well multi-well plates where adult, either as a combination of both genders or two specimens of the same gender, were placed in wells filled with FSW (10 mL). We incubated a minimum of 37 wells (range: 37–68 replicates) of each experimental treatment (Table I). The few eggs and faecal pellets occasionally produced during the first day of starvation were removed. Dead copepods were removed daily and fixed with a 1% acid Lugol solution for later determination of body size (prosome length). Pictures of male and female copepods (28–67, depending on the species) were taken using a camera attached to an inverted microscope and the prosome length was measured by image analysis using the ImageJ software (Schneider et al., 2012). Table I: Summary of the characteristics of the copepods used in this study and the main results obtained from the starvation experiments Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b L is the average prosome length, W is the average body weight in carbon estimated using length to carbon conversion factors shown in Table II, SD is the standard deviation, n is the number of specimens per treatment. MST is the median survival time (~starvation tolerance, in days) and W-MST is the weight-normalized median survival time (in days μgC−1). Superscripted letters denote statistical differences between experimental treatments (Bonferroni test, α = 0.0167). Table I: Summary of the characteristics of the copepods used in this study and the main results obtained from the starvation experiments Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b L is the average prosome length, W is the average body weight in carbon estimated using length to carbon conversion factors shown in Table II, SD is the standard deviation, n is the number of specimens per treatment. MST is the median survival time (~starvation tolerance, in days) and W-MST is the weight-normalized median survival time (in days μgC−1). Superscripted letters denote statistical differences between experimental treatments (Bonferroni test, α = 0.0167). CHN elemental analysis We conducted CHN elemental analysis to estimate the carbon body weight, nitrogen body weight, C:N ratios and size to weight conversion factors of males and females. The culture of Oithona nana was lost (dead) and thus Oithona davisae was used for the carbon analyses. Oithona davisae has a body size, morphology and sexual dimorphism similar to O. nana. Oithona davisae stock cultures were established at DTU Aqua from cultures originally created at the Marine Sciences Institute of Barcelona from zooplankton samples collected in the harbour of Barcelona. Oithona davisae was reared in the laboratory as described for O. nana. Adults of C. typicus, T. longicornis and O. davisae were sieved from the continuous cultures using a 100, 200 or 250-μm mesh, respectively, and gently rinsed with 0.2 μm-filtered seawater (FSW, 32 PSU). Groups of 60–1100 individuals per gender (60 C. typicus, 170 T. longicornis, 1100 O. davisae) (triplicates) were sorted under a stereomicroscope and placed in autoclaved FSW for at least 2 hours to ensure gut evacuation. Only non-ovigerous females were selected for O. davisae. Copepods were rinsed several times and then filtered onto pre-incinerated (450°C, 6 h) glass-fibre filters (GF/A grade). Filters were dried (60°C, 24 h) and stored inside a vacuum desiccator for further CHN analysis. Additionally, 50 individuals per gender and species were fixed with acid Lugol´s solution (1% final) for the length (prosome) determinations using image analysis. The CHN content in the copepod samples was determined by a Thermo Fisher Scientific FLASH 2000 Organic Elemental Analyzer. Calculations and statistics Survival curves (% survivors as a function of starvation time) and median survival time were calculated using the Kaplan–Meier estimator (Kaplan and Meier, 1958). Starvation tolerance was defined as the time where 50% of the initial number of copepods had survived in absence of food (“median survival time”). To evaluate the effect of differences in size between genders, starvation tolerance was normalized to body weight by dividing the starvation time (days) with the average body weight (μg carbon) estimated from the prosome length to carbon conversion factors (μgC μm-1) calculated in the carbon analysis. The effect of body weight on starvation tolerance was examined by plotting the individual survival time vs. body weight for 28–67 copepods, depending on species and gender. We also examined the influence of exposure time to males on survival time of females to evaluate the effect of gender interactions/mating time on starvation tolerance of females. Differences between survival curves were tested using the Gehan–Breslow–Wilcoxon test. T-tests were used to determine significant differences between average values in the CHN analyses. Differences in size between genders were tested using a Kolmogorov–Smirnoff test, since the data for prosome length did not follow a normal distribution (D’Agostino–Pearson test for normality). Individual starvation tolerance (days) and body weight (μgC ind−1) data were normally distributed (D’Agostino–Pearson test) and a Pearson Correlation coefficient test was applied to estimate significant lineal correlation between both variables. To test differences between survival curves as a function of treatment and gender (FF vs. FM, MM vs. MF and FF vs. MM), pairwise comparisons were corrected for multiple pairwise comparisons using the Bonferroni test by setting the significance level (α) = 0.0167 (=alpha-level divided by the number of comparisons = 0.05/3). For all other statistical tests, α was set to 0.05. RESULTS The relationship between the fraction of survivors (%) and their starvation time (“survival curves”) varied depending on the species and experimental treatments (Fig. 1). Centropages typicus showed the lowest starvation tolerance (Fig. 1). Survival curves of C. typicus males and females were significantly different (P = 0.0157) and males had a median starvation tolerance of 1–2 days lower than females (Fig. 1A, Table I). However, although median starvation tolerance of males was lower when incubated together with females (Table I), survival curves of both genders did not significantly differ in the presence of the opposite sex (P = 0.0614 and P = 0.9545 for males and females, respectively) (Table I, Fig. 2A). In the case of T. longicornis, median starvation tolerance was 8 days with no significant difference between genders (P = 0.2029) or the presence of the opposite gender (P = 0.8746 and P = 0.9853 for males and females, respectively) (Fig. 1B and Table I). Survival curves of O. nana males and females were significantly different (P < 0.0001) (Fig. 1C) and median starvation tolerance of O. nana males was 2 days lower than starvation tolerance of females (Table I). Median starvation tolerance in both genders of O. nana was significantly reduced by 2 days when they were incubated together (P = 0.0025 and P = 0.0122 for males and females, respectively) (Fig. 1C and Table I). Fig. 1. View largeDownload slide Survival curves (% survivors as a function of starvation time in days) of copepod male and females with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the median starvation tolerance (MST, Table I). Fig. 1. View largeDownload slide Survival curves (% survivors as a function of starvation time in days) of copepod male and females with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the median starvation tolerance (MST, Table I). Fig. 2. View largeDownload slide Body carbon content (A–C), nitrogen content (D–F) and C:N ratios (G–I) of males and females of C. typicus (A, D, G), Temora longicornis (B, F, H), and O. davisae (C, F, I). The bars are the average of three replicates and error bars the standard deviation. Fig. 2. View largeDownload slide Body carbon content (A–C), nitrogen content (D–F) and C:N ratios (G–I) of males and females of C. typicus (A, D, G), Temora longicornis (B, F, H), and O. davisae (C, F, I). The bars are the average of three replicates and error bars the standard deviation. Individual C and N content varied depending on the species and gender (Fig. 2) and was positively related to prosome length (Fig. 2, Table II). C:N ratios were close to four in males and females of the three species, with no significant differences between genders. The estimated gender-specific size to carbon conversion factors (μgC μm−1) was higher for females than for males of C. typicus and T. longicornis and similar for O. davisae females and males (Table II). Table II: Summary of the characteristics of the copepods used the CHN analysis and the size to carbon conversion factors (C/L3) obtained for copepod males and females. Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 L, average prosome length (μm); C, carbon content per copepod; SD, standard deviation. Table II: Summary of the characteristics of the copepods used the CHN analysis and the size to carbon conversion factors (C/L3) obtained for copepod males and females. Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 L, average prosome length (μm); C, carbon content per copepod; SD, standard deviation. In the starvation experiments, the body size (prosome length, μm) differed between genders, with males being significantly smaller than females in the three species (Table I). When normalizing survival time with the estimated carbon body weights, the weight-specific starvation tolerance of the small ambush feeding O. nana was one order of magnitude higher than for the larger active species (Fig. 3). After normalizing survival time with C body weights, the difference in starvation tolerance between genders was reduced for C. typicus and O. nana (Fig. 3A and C, Table I) and increased for T. longicornis towards males with a weigh-specific starvation tolerance higher than females (Fig. 3B, Table I). Gender interactions significantly reduced the weight-specific starvation tolerance of both genders in O. nana (Fig. 3C, Table I). Fig. 3. View largeDownload slide Survival (%) as a function of weight-normalized starvation time (days μgC−1) of copepods with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the weight-specific median starvation tolerance (W-MST, Table I). Fig. 3. View largeDownload slide Survival (%) as a function of weight-normalized starvation time (days μgC−1) of copepods with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the weight-specific median starvation tolerance (W-MST, Table I). There was a significant positive relationship between individual survival time and body carbon weight for females of C. typicus (Fig. 4A). However, we did not find a statistically significant effect of individual carbon body weight on starvation tolerance for any other genders/species (Fig. 4), although a positive tendency was observed in other species and genders (Fig. 4B and D). The exposure time to males did not significantly affect the starvation tolerance of females in any of the studied species (Fig. 5). Fig. 4. View largeDownload slide Relationship between individual body weight (μgC ind−1) and starvation tolerance for females of Centropages typicus (A), Temora longicornis (B) and Oithona nana (C) and for males of C. typicus (D), T. longicornis (E) and O. nana (F). Continuous line is the linear regression model fitted to the data and the discontinuous lines are the 95% confidence intervals (A). Fig. 4. View largeDownload slide Relationship between individual body weight (μgC ind−1) and starvation tolerance for females of Centropages typicus (A), Temora longicornis (B) and Oithona nana (C) and for males of C. typicus (D), T. longicornis (E) and O. nana (F). Continuous line is the linear regression model fitted to the data and the discontinuous lines are the 95% confidence intervals (A). Fig. 5. View largeDownload slide Relationship between individual females survival time and their exposure time to males, based on the treatments where both genders were incubated together for Centropages typicus (A), Temora longicornis (B) and Oithona nana (C). Fig. 5. View largeDownload slide Relationship between individual females survival time and their exposure time to males, based on the treatments where both genders were incubated together for Centropages typicus (A), Temora longicornis (B) and Oithona nana (C). DISCUSSION Influence of elemental composition on gender-specific starvation tolerance in copepods Lipid accumulation is an important strategy for organisms to cope with periods of food limitation and thus differences in lipid content can cause differences in starvation tolerance between planktonic copepods. The C:N ratio is commonly used as an index of lipid content in copepods (Båmstedt, 1986; Gismervik, 1997; Postel et al., 2000). In the species studied, we found a C:N ratio of ~4 that is in the range of C:N ratios typically found in copepods from mid latitudes (C:N ratio: 3–4, Gismervik, 1997) and indicates a low lipid content compare to some copepods from high latitudes (C:N up to 13, Bamstedt, 1986; Gismervik, 1997). In our study, copepod males and females showed no significant differences in C:N ratio, which indicates that the observed differences in starvation tolerance are not related to differences in lipid reserves between genders. Influence of body size on gender-specific starvation tolerance in copepods A difference in body size between genders (sexual size dimorphism) is a widespread phenomenon in animals with sexual reproduction. In invertebrates, including copepods, males are commonly smaller than females (Gilbert and Williamson, 1983; Hirst and Kiørboe, 2014). A smaller body size commonly implies a higher weight-specific respiration rate (higher metabolic expenses) (Kleiber, 1961; Kiørboe and Hirst, 2014). Previous studies with Acartia spp. have also shown that males have a lower starvation tolerance than females (Parrish and Wilson, 1978; Colin and Dam, 2005; Finiguerra et al., 2013), and these differences have been suggested to be explained by size differences between genders (Finiguerra et al., 2013). Our results indicate that differences in starvation tolerance between genders in C. typicus and O. nana can be mainly explained by differences in body size. A positive tendency between individual survival time and body carbon was also observed in some genders/species. However, male T. longicornis showed a similar median survival time and a higher weight-specific starvation tolerance than females, despite their smaller size. This suggests that weight-specific metabolic expenses of T. longicornis females are higher than for males (they do not follow the “3/4 power-law” of metabolic rates vs. body mass, Kleiber, 1961) or that other factors besides size affect the gender difference in starvation tolerance in this species. Measurements of weight-specific respiration rates of males and females are required to better evaluate if size is one of the main drivers of gender differences in starvation tolerance of planktonic copepods. Effect of behaviour on gender-specific starvation tolerance in copepods Kiørboe et al. (2015) found that the male lifespan is up to 50% shorter than that of females in copepods with strong differences in motile behaviour between genders (e.g. Oithona). In contrast, in copepod species where both genders have relatively similar swimming activity (e.g. C. typicus and T. longicornis), male lifespan is only ~10% shorter than females (Kiørboe et al., 2015). This indicates that motile behaviour affects gender-specific longevity in planktonic copepods. However, our results indicate a minor effect of male motile behaviour (mate-seeking behaviour) on the gender-specific starvation tolerance of the copepods studied. Mate-seeking behaviour under prolonged starvation was not recorded, but our visual observations under the microscope indicate that O. nana males kept their female-searching behaviour at least during the first days of starvation but it was reduced after prolonged starvation. This suggests that mate-seeking behaviour of males is minimized after long starvation periods, which can explain the minimal effect of sexual behaviour on gender differences in starvation compared to the effect of gender behaviour on longevity. However, when comparing among species, we found that the small ambush feeding copepod O. nana has a weight-specific starvation tolerance one order of magnitude higher than the large active swimming copepods. Our previous video observations demonstrate that females of O. nana (low motility) and T. longicornis (active swimmer) change their motile behaviour/activity budgets slightly during the starvation. This suggests that motile behaviour (foraging strategy), and not only size, can also significantly influence starvation tolerance in planktonic copepods when comparing between species. Influence of mating on starvation tolerance in copepods Mating and reproduction can reduce the lifespan of planktonic copepods (12–20%) depending on their foraging strategy (Kiørboe et al., 2015). However, we did not observe a decrease in starvation tolerance when both genders of C. typicus and T. longicornis were incubated together, suggesting that the energetic cost of copulation and pre-copulation in cruise feeders and for copepods that utilize a feeding current is low, albeit C. typicus males showed a tendency toward a decreased starvation tolerance in the presence of females. The act of copulation is expected to be similar in the three species (e.g. Blades, 1977; Uchima and Murano, 1988) but only males and females of the ambush feeding O. nana had a lower starvation tolerance because of gender interactions. Female copepods produce either hydro-mechanical or chemical signals (pheromones) that males can perceive (Kiørboe et al., 2005; Bagøien and Kiørboe, 2005a, 2005b). When a male encounters a female signal, they increase they swimming activity and/or speed (Kiørboe and Bagøien, 2005); therefore, motile behaviour of males should increase due to the presence of female signal. In C. typicus and T. longicornis, which are active swimmers, differences in swimming activity and associated metabolic expenses between “normal” swimming and pheromone-tracking swimming behaviour are probably low because males increase speed for few minutes or change their swimming patterns in the presence of pheromones. However, swimming activity in Oithona spp. males, which alternate between motionless feeding and fast mate-seeking, can notably increase in the presence of pheromones from unmated females (Heuschele and Kiørboe, 2012), and, therefore, the presence of females must be energetically costly for the males. Similarly, females of Oithona spp., which spend most of the time motionless, would increase their motile activity (e.g. escape jumps) in the presence of males. Kiørboe et al. (2015) found that O. davisae females had a reduced lifespan of 15% when incubated together with males. Therefore, increased mate-seeking behaviour by males in the presence of pheromones and pre-copulation motile activities in females may explain the reduction in starvation tolerance observed in both genders of O. nana when both genders are incubated together. There was no correlation between how long females had been incubated together with males and their starvation tolerance, which suggests that the effect of gender interactions must be the result of initial interactions. Thus, this indicates that reproduction and associated mating behaviour is not prioritized when copepods are starving, but that the initial interactions between genders are costly enough to reduce the starvation tolerance of males and females in ambush feeding copepods. Ecological implications Planktonic copepods are exposed to food limitation at different levels and for short (from hours to days) and extended periods (from weeks to months) (Winder and Cloern, 2010). However, in the marine environment, copepods are never exposed to the complete absence of food. In addition, although the copepods studied displayed their characteristic behaviours even in the smaller volumes used in this study, the limited experimental volume could have decreased mate-searching behaviour and increased encounter rates/mating compared to field conditions, particularly for the large active species. Therefore, the estimated absolute median starvation tolerance should not be directly extrapolated to field conditions, but they indicate the relative differences in starvation between genders and among copepods with difference foraging strategies. Although differences in starvation tolerance between genders (2 days) and species (up to 5 days) initially may seem small, they reflect their different capabilities to cope with short-term food limitation, and are likely to affect survival in the field where food availability is highly fluctuating. The larger species, with active feeding behaviour, have a lower starvation tolerance than ambush feeders and thus need other strategies such as the production of resting eggs (Mauchline, 1998; Lindley and Reid, 2002) to survive through periods or seasons of low primary production. Small ambush feeding copepods as Oithona spp., which have an energetically low-cost behaviour (Paffenhöfer, 2006; Almeda et al., 2011), have relative high starvation tolerance and can better tolerate seasons of low primary production without storing large amounts of lipids or using resting eggs. However, mating in ambush feeders comes at the cost of reduced starvation tolerance, which can affect reproduction and survival of Oithona spp. during periods of low food availability. Therefore, the relative differences in starvation tolerance between genders and species observed can potentially influence the seasonal abundance and distribution of the studied species. In field populations, Oithona species typically have a low male to female sex ratio of abundance (<0.2), whereas Centropages and Temora species have a sex ratio close to 1 (Kuwenberg, 1993; Kiørboe, 2008; Hirst et al., 2010). Our results suggest that gender-specific starvation tolerance can also influence sex ratios in populations of Oithona (female-biased ratios, O. nana males have a lower starvation tolerance than females) and Temora (sex ratio close to 1, males and females have a similar starvation tolerance). However, we found that C. typicus males have a lower starvation tolerance than females but a sex ratio typically close to 1 in field populations. Therefore, although differences in starvation tolerance between genders can be a factor that affects sex ratios in some species, copepod sex ratios are likely the result of numerous factors and the relative contribution of each factor may vary between species. For instance, other factors such as an elevated predation risk (Hirst et al. 2010; Almeda et al., 2017), and a shorter lifespan (Rodríguez-Graña et al., 2010; Kiørboe et al., 2015) of males compared to females also explain the strong female-skewed sex ratios of Oithona spp. in field populations. CONCLUSIONS Our results demonstrate that starvation tolerance can significantly differ between copepod genders. The most pronounced difference in starvation tolerance between genders was observed in the ambush feeding copepod Oithona nana. Our results indicate that sexual size dimorphism can partially explain gender-specific starvation tolerance in planktonic copepods, and that mate-seeking behaviour have a minor influence on the starvation tolerance of males under prolonged starvation. The presence of the opposite gender reduced starvation tolerance in both males and females of O. nana, but not in active feeding copepods. This suggests that the energetic cost of mating behaviour in ambushers is higher than in active feeders due to the strong conflict between stationary feeding and mating activities in ambush feeding copepods. ACKNOWLEDGEMENTS We would like to thank Jack Melby for maintaining the plankton cultures, Anne Busk Faarborg for helping with the CHN analyses, Enric Saiz and Albert Calbet for providing the O. davisae cultures, Sara Ceballos for providing the O. nana cultures and Thomas Kiørboe for valuable comments on this manuscript. FUNDING This work was funded through The Centre for Ocean Life, which is a VKR Centre of Excellence supported by the Villum Kann Rasmussen Foundation. This work was also supported by a individual postdoctoral grant (17023) from the Danish Council for Independent Research (DFF) to R.A., a Marie Curie Intra-European fellowship from the People Programme of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement number 6240979 to R.A., a Hans Christian Ørsted Postdoctoral fellowship from Technical University of Denmark to R.A and an internship from the European Social Fund to RRT. REFERENCES Almeda , R. , Alcaraz , M. , Calbet , A. and Saiz , E. ( 2011 ) Metabolic rates and carbon budget of early developmental stages of the marine cyclopoid copepod Oithona davisae . Limnol. Oceanogr. , 56 , 403 – 414 . Google Scholar CrossRef Search ADS Almeda , R. , van Someren Gréve , H. and Kiørboe , T. ( 2017 ) Behaviour is a major determinant of predation risk in zooplankton . Ecosphere , 8 , e01668 . Google Scholar CrossRef Search ADS Bagøien , E. and Kiørboe , T. ( 2005 a) Blind dating—mate finding in planktonic copepods. I. Tracking the pheromone trail of Centropages typicus . Mar. Ecol. Prog. Ser. , 300 , 105 – 115 . Google Scholar CrossRef Search ADS Bagøien , E. and Kiørboe , T. ( 2005 b) Blind dating—mate finding in planktonic copepods. III. Hydromechanical communication in Acartia tonsa . Mar. Ecol. Prog. Ser. , 300 , 129 – 133 . Google Scholar CrossRef Search ADS Båmstedt , U. ( 1986 ) Chemical composition and energy content. In Corner , E. D. S. and O’Hara , S. C. M. (eds) , The Biological Chemistry of Marine Copepods . Clarendon , Oxford , pp. 1 – 58 . Bjærke , O. , Andersen , T. , Bækkedal , K. S. , Nordbotten , M. , Skau , L. F. and Titelman , J. ( 2016 ) Paternal energetic investments in copepods . Limnol. Oceanogr. , 61 , 508 – 517 . Google Scholar CrossRef Search ADS Blades , P. I. ( 1977 ) Mating behavior of Centropages typicus (Copepoda: Calanoida) . Mar. Biol. , 40 , 57 – 64 . Google Scholar CrossRef Search ADS Buskey , E. J. ( 1998 ) Components of mating behavior in planktonic copepods . J. Mar. Syst. , 15 , 13 – 21 . Google Scholar CrossRef Search ADS Cebrián , J. and Valiela , I. ( 1999 ) Seasonal patterns in phytoplankton biomass in coastal ecosystems . J. Plankton Res. , 21 , 429 – 444 . Google Scholar CrossRef Search ADS Colin , S. P. and Dam , H. G. ( 2005 ) Testing for resistance of pelagic marine copepods to a toxic dinoflagellate . Evol. Ecol. , 18 , 355 – 377 . Google Scholar CrossRef Search ADS Dagg , M. ( 1977 ) Some effects of patchy food environments on copepods . Limnol. Oceanogr. , 22 , 99 – 107 . Google Scholar CrossRef Search ADS Dur , G. , Souissi , S. , Schmitt , F. G. , Beyrend-Dur , D. and Hwang , J.-S. ( 2011 ) Mating and mate choice in Pseudodiaptomus annandalei (Copepoda: Calanoida) . J. Exp. Mar. Bio. Ecol. , 402 , 1 – 11 . Google Scholar CrossRef Search ADS Finiguerra , M. B. , Dam , H. G. , Avery , D. E. and Burris , Z. ( 2013 ) Sex-specific tolerance to starvation in the copepod Acartia tonsa . J. Exp. Mar. Bio. Ecol. , 446 , 17 – 21 . Google Scholar CrossRef Search ADS Gilbert , J. J. and Williamson , C. E. ( 1983 ) Sexual dimorphism in zooplankton (Copepoda, Cladocera, and Rotifera) . Annu. Rev. Ecol. Syst. , 14 , 1 – 33 . Google Scholar CrossRef Search ADS Gismervik , I. ( 1997 ) Stoichiometry of some planktonic crustaceans . J. Plankton Res. , 19 , 279 – 285 . Google Scholar CrossRef Search ADS Hansen , P. J. ( 1989 ) The red tide dinoflagellate Alexandrium tamarense: effects on behaviour and growth of a tintinnid ciliate . Mar. Ecol. Prog. Ser. , 53 , 105 – 116 . Google Scholar CrossRef Search ADS Heuschele , J. and Kiørboe , T. ( 2012 ) The smell of virgins: mating status of females affects male swimming behaviour in Oithona davisae . J. Plankton Res. , 34 , 929 – 935 . Google Scholar CrossRef Search ADS Hirst , A. G. , Bonnet , D. , Conway , D. V. P. and Kiørboe , T. ( 2010 ) Does predation controls adult sex ratios and longevities in marine pelagic copepods? Limnol. Oceanogr. , 55 , 2193 – 2206 . Google Scholar CrossRef Search ADS Hirst , A. G. and Kiørboe , T. ( 2014 ) Macroevolutionary patterns of sexual size dimorphism in copepods . Proc. R. Soc. B. , 281 , 20140739 . Google Scholar CrossRef Search ADS Kaplan , E. L. and Meier , P. ( 1958 ) Nonparametric estimation from incomplete observations . J. Am. Stat. Assoc. , 53 , 457 – 481 . Google Scholar CrossRef Search ADS Katona , S. K. ( 1973 ) Evidence for sex pheromones in planktonic copepods . Limnol. Oceanogr. , 18 , 574 – 583 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2007 ) Mate finding, mating, and population dynamics in a planktonic copepod Oithona davisae: there are too few males . Limnol. Oceanogr. , 52 , 1511 – 1522 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2008 ) Optimal swimming strategies in mate-searching pelagic copepods . Oecologia , 155 , 179 – 192 . Google Scholar CrossRef Search ADS PubMed Kiørboe , T. ( 2011 ) How zooplankton feed: mechanisms, traits and trade-offs . Biol. Rev. , 86 , 311 – 339 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2016 ) Foraging mode and prey size spectra in suspension feeding copepods and other zooplankton . Mar. Ecol. Prog. Ser. , 558 , 15 – 20 . Google Scholar CrossRef Search ADS Kiørboe , T. and Bagøien , E. ( 2005 ) Motility patterns and mate encounter rates in planktonic copepods . Limnol. Oceanogr. , 50 , 1999 – 2007 . Google Scholar CrossRef Search ADS Kiørboe , T. , Bagøien , E. and Thygesen , U. H. ( 2005 ) Blind dating—mate finding in planktonic copepods. II. The pheromone cloud of Pseudocalanus elongatus . Mar. Ecol. Prog. Ser. , 300 , 117 – 128 . Google Scholar CrossRef Search ADS Kiørboe , T. , Ceballos , S. and Thygesen , U. H. ( 2015 ) Interrelations between senescence, life history traits, and behaviour in planktonic copepods . Ecology , 96 , 2225 – 2235 . Google Scholar CrossRef Search ADS PubMed Kiørboe , T. and Hirst , A. G. ( 2014 ) Shifts in mass scaling of respiration, feeding, and growth rates across life-form transitions in marine pelagic organisms . Am. Nat. , 183 , 118 – 130 . Google Scholar CrossRef Search ADS Kiørboe , T. , Jiang , H. and Colin , S. P. ( 2010 ) Danger of zooplankton feeding: the fluid signal generated by ambush-feeding copepods . Proc. R. Soc. B. Biol. Sci. , 277 , 3229 – 3237 . Google Scholar CrossRef Search ADS Kleiber , M. ( 1961 ) The Fire of Life . Wiley , New York . Kuwenberg , J. H. M. ( 1993 ) Sex ratio of calanoid copepods in relation to population composition in the northwestern Mediterranean . Crustaceana , 64 , 281 – 299 . Google Scholar CrossRef Search ADS Lindley , J. A. and Reid , P. C. ( 2002 ) Variations in the abundance of Centropages typicus and Calanus helgolandicus in the North Sea: deviations from close relationships with temperature . Mar. Biol. , 141 , 153 – 165 . Google Scholar CrossRef Search ADS Mauchline , J. ( 1998 ) Biology of Calanoid Copepods . Academic Press , San Diego London Boston New York Sydney Tokyo Toronto , p. 710 . Ohtsuka , S. and Huys , R. ( 2001 ) Sexual dimorphism in calanoid copepods: morphology and function . Hydrobiologia , 453 , 441 – 466 . Google Scholar CrossRef Search ADS Paffenhöfer , G. A. ( 2006 ) Oxygen consumption in relation to motion of marine planktonic copepods . Mar. Ecol. Prog. Ser. , 317 , 187 – 192 . Google Scholar CrossRef Search ADS Parrish , K. K. and Wilson , D. F. ( 1978 ) Fecundity studies on Acartia tonsa (Copepoda: Calanoida) in standardized culture . Mar. Biol. , 46 , 65 – 81 . Google Scholar CrossRef Search ADS Postel , L. , Fock , H. and Hagen , W. ( 2000 ) Biomass and abundance. In Harris , R. P. , Wiebe , P. H. , Lenz , J. , Skjoldal , H. R. and Huntley , M. (eds) , ICES Zooplankton Methodology Manual . Academic Press , San Diego , pp. 83 – 192 . Google Scholar CrossRef Search ADS Rodríguez-Graña , L. , Calliari , D. , Tiselius , P. , Hansen , B. W. and Sköld , H. N. ( 2010 ) Gender-specific ageing and non-Mendelian inheritance of oxidative damage in marine copepods . Mar. Ecol. Prog. Ser. , 401 , 1 – 13 . Google Scholar CrossRef Search ADS Schneider , C. A. , Rasband , W. S. and Eliceiri , K. W. ( 2012 ) NIH Image to ImageJ: 25 years of image analysis . Nat. Methods , 9 , 671 – 675 . Google Scholar CrossRef Search ADS PubMed Tang , K. W. , Gladyshev , M. I. , Dubovskaya , O. P. , Kirillin , G. , Grossart , H.-P. and Beisner , B. E. ( 2014 ) Zooplankton carcasses and non-predatory mortality in freshwater and inland sea environments . J. Plankt. Res. , 36 , 597 – 612 . Google Scholar CrossRef Search ADS Titelman , J. , Varpe , O. , Eliassen , S. and Fiksen , O. ( 2007 ) Copepod mating: chance or choice? J. Plankton Res. , 29 , 1023 – 1030 . Google Scholar CrossRef Search ADS Uchima , M. and Murano , M. ( 1988 ) Mating behavior of the marine copepod Oithona davisae . Mar. Biol. , 99 , 39 – 45 . Google Scholar CrossRef Search ADS van Someren Gréve , H. , Almeda , R. and Kiørboe , T. ( 2017 a) Motile behavior and predation risk in planktonic copepods . Limnol. Oceanogr. , 62 , 1810 – 1824 . Google Scholar CrossRef Search ADS van Someren Gréve , H. , Almeda , R. , Lindegren , M. and Kiørboe , T. ( 2017 b) Gender-specific feeding rates in planktonic copepods with different feeding behavior . J. Plankton Res ., 39 , 631 – 644 . Google Scholar CrossRef Search ADS Winder , M. and Cloern , J. E. ( 2010 ) The annual cycles of phytoplankton biomass . Philos. Trans. R. Soc., B. , 365 , 3215 – 3226 . Google Scholar CrossRef Search ADS Author notes Corresponding editor: Roger Harris © 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

Sex-specific starvation tolerance of copepods with different foraging strategies

Loading next page...
 
/lp/ou_press/sex-specific-starvation-tolerance-of-copepods-with-different-foraging-Ll8tvSj3Z2
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
0142-7873
eISSN
1464-3774
D.O.I.
10.1093/plankt/fby006
Publisher site
See Article on Publisher Site

Abstract

Abstract Planktonic copepods have sexual dimorphism that can lead to differences in starvation tolerance between genders. Additionally, mating may be energetically costly and thus reduce starvation tolerance. We investigated the influence of sexual dimorphism and mating on starvation tolerance of copepods with different feeding behaviours: Oithona nana (ambusher), Temora longicornis (feeding-current feeder) and Centropages typicus (cruiser). Males of C. typicus and O. nana had a starvation tolerance lower than females, whereas T. longicornis had a similar starvation tolerance between genders. Only O. nana males and females had reduced starvation tolerance when both genders were incubated together, which suggests that mating activities in ambushers have an energetic cost higher than in active feeding copepods. C:N ratios showed a non-significant difference between genders, which indicates that gender differences in starvation tolerance are not due to dissimilarities in lipid reserves. Gender differences in starvation tolerance can be partially explained by body size differences between sexes. This indicates a minor influence of mate-seeking behaviour on male starvation tolerance, likely due to reduced mate-searching behaviour under prolonged starvation. Our results demonstrate that sexual dimorphism can result in different starvation tolerance between copepod genders and that a negative effect of mating on starvation tolerance depends on the foraging strategy. INTRODUCTION Food in marine environments is generally scarce, patchy and fluctuating, often because of seasonal cycles (Cebrián and Valiela, 1999; Winder and Cloern, 2010). Therefore, planktonic organisms have to cope with the risk of starvation for short and long periods. In fact, starvation is considered one of the main non-predatory causes of mortality in zooplankton (Tang et al., 2014). Most studies on starvation tolerance of planktonic copepods focus only on females and little is known about the ability of male copepods to cope with starvation (Dagg, 1977; Finiguerra et al., 2013). The abundance of copepod males in field populations is low compared to females in many families (Kiørboe, 2008), which can limit population growth (Kiørboe, 2007). Therefore, a better knowledge of the potential causes of mortality (e.g. starvation) of males is crucial for understanding skewed sex ratios and the dynamics of copepod populations. Planktonic copepods have sexually dimorphic traits and males and females commonly differ in body size and motile behaviour (e.g. Gilbert and Williamson, 1983; Ohtsuka and Huys, 2001; van Someren Gréve et al., 2017b). A smaller body size in copepods often implies a higher weight-specific respiration rate (Almeda et al., 2011) since mass-specific metabolic rates commonly decline with size with a power of −1⁄4 (Kleiber, 1961; Kiørboe and Hirst, 2014). Copepod males are commonly smaller than females and males might have a higher mass-specific energy expenditure rate and, consequently, a lower starvation tolerance than females. Differences lipid storage between copepod genders can also cause differences in starvation tolerance between males and females (Gismervik, 1997). Copepod males typically display a mate-searching behaviour and swim more frequently and faster than females (Katona, 1973; Ohtsuka and Huys, 2001; Kiørboe et al., 2005; Bagøien and Kiørboe, 2005a). Differences in motile behaviour between genders may result in different energy expenditure (Paffenhöfer, 2006) and consequently in different levels of starvation tolerance. Behavioural asymmetry between copepod genders depends on their foraging strategy (Kiørboe, 2008; van Someren Gréve et al., 2017a). We can broadly classify the feeding behaviours of copepods in two main foraging strategies in terms of motility: “sit-and-wait” (ambushing) vs. “searching” (active feeding). Ambush feeding copepods remain motionless in the water waiting for prey to enter their perceptive sphere and when perceiving the prey they perform a fast jump to capture it (Kiørboe et al., 2010; Kiørboe, 2011, 2016). Active feeding copepods cruise through the water and capture encountered prey (“cruising feeders”) and/or can hover while generating a feeding current (“feeding-current feeders”) (Kiørboe, 2011, 2016). Thus, the conflict between feeding and mate-seeking behaviour varies depending on the copepod foraging strategy (Kiørboe et al., 2010; van Someren Gréve et al., 2017b). In strict ambushers (e.g. Oithonidae), there is a strong conflict between feeding and mate-seeking behaviour because feeding and searching for mates are mutually exclusive activities (Kiørboe, 2008). Hence, ambush feeding males need to alternate between stationary feeding and a potentially energy expensive female-seeking behaviour (Kiørboe, 2008). For example, ambush feeding Oithona males move ~20–30% of the time (Kiørboe, 2008; van Someren Gréve et al., 2017b) and 15-fold faster than females (Kiørboe, 2008) whereas females spent most of the time motionless or sinking. In copepods with active feeding behaviour, both genders move/swim actively and the conflict between feeding and mate searching is expected to be low (Bagøien and Kiørboe, 2005a; Kiørboe, 2007, 2008; Bjærke et al., 2016; van Someren Gréve et al., 2017b). However, gender-specific starvation tolerance of marine planktonic copepods with different foraging strategies has not been systematically examined. Mating behaviour of copepods commonly includes search, encounter, capture, escape behaviour and/or copulation (Buskey, 1998; Titelman et al., 2007; Dur et al., 2011) and involves mate recognition by mechanical or chemical sensorial perception (Bagøien and Kiørboe, 2005a). Upon mate encounter, the male grabs the female with his geniculated antennae and the female can thereafter swim around with the male attached for minutes to hours, depending on the maturation of the spermatophores (Blades, 1977). Females can also display different escape behaviours, including jumps, shaking off males and calmly swimming away, but these mating escape behaviours have rarely been quantified (Titelman et al., 2007). These behaviours associated to mating (copulation) can also affect the energetic expense and therefore influence starvation tolerance in both genders. Kiørboe et al. (2015) found that the lifespan of males and females in some copepod species was significantly reduced when the both genders were incubated together, which indicates that mating has a cost in terms of longevity. However, it is still unknown if mating (copulation)-related activities can also reduce starvation tolerance in planktonic copepods. In this study, we experimentally investigated the influence of gender and mating on starvation tolerance of planktonic copepods with different feeding behaviours: cruise feeding (Centropages typicus), feeding-current (Temora longicornis) and ambush feeding (Oithona nana). We hypothesize that (i) copepod males have lower starvation tolerance than females due to their smaller size and active mate-seeking behaviour, (ii) difference in starvation tolerance between genders is particularly high in ambush feeders due to the high asymmetry in motile behaviour between genders and (iii) mating (copulation) decreases starvation tolerance of both males and females, independently of their foraging strategy. To test our hypotheses, we determined the starvation tolerance of males and females of copepods with different feeding behaviours incubated separately or together in the absence of food. We evaluate the effect of elemental composition (C:N ratios), body size and motile behaviour on gender-specific starvation tolerance in copepods with different feeding behaviours, and discuss the ecological implications of our results in the context of food seasonality and copepod sex ratios in field populations. METHOD Experimental organisms Copepod males and females were obtained from continuous stock cultures at DTU Aqua. Stock cultures of C. typicus, T. longicornis and O. nana were established from specimens originally collected in Gullmar fjord (Sweden, 2014), Tjärnö (Sweden, 2013) and the Port of Gijón (Spain, 2012), respectively. Cultures of C. typicus (cruise feeder) and T. longicornis (feeding-current) were fed a mixture of phytoplankton: Akashiwo sanguinea, Heterocapsa triquetra, Prorocentrum minimum, Thalassiosira weissflogii and R. salina and, in the case of C. typicus also with Oxyrrhis marina. O. nana (ambush feeder) was fed the heterotrophic dinoflagellate O. marina, which was in turn fed R. salina. All copepods were fed three times weekly at saturating levels and kept at 15°C in the dark. The phytoplankton cultures were kept in exponential growth in B1 culture medium (Hansen, 1989) at 18°C on a 12:12-h light/dark cycle. Experimental procedures Before starting the experiment, copepodites (CIII–CV) were picked and isolated in 12-well plates [5 mL of 0.2 μm-filtered seawater (FSW) per well, salinity 32‰], and fed daily a mixture of O. marina and R. salina at saturating levels until reaching maturation (~2–4 days depending on the species). Upon maturity, the adult copepods were gently rinsed three times with FSW by sequential transferring of individual copepods to cuvettes with FSW under a dissecting microscope. The absence of phytoplankton after rinsing the copepods was verified under the microscope. The following experimental treatments were investigated: females alone (FF), males alone (MM) and females together with males (FM) and males together with females (MF) (Table I). Starvation experiments were conducted in six well multi-well plates where adult, either as a combination of both genders or two specimens of the same gender, were placed in wells filled with FSW (10 mL). We incubated a minimum of 37 wells (range: 37–68 replicates) of each experimental treatment (Table I). The few eggs and faecal pellets occasionally produced during the first day of starvation were removed. Dead copepods were removed daily and fixed with a 1% acid Lugol solution for later determination of body size (prosome length). Pictures of male and female copepods (28–67, depending on the species) were taken using a camera attached to an inverted microscope and the prosome length was measured by image analysis using the ImageJ software (Schneider et al., 2012). Table I: Summary of the characteristics of the copepods used in this study and the main results obtained from the starvation experiments Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b L is the average prosome length, W is the average body weight in carbon estimated using length to carbon conversion factors shown in Table II, SD is the standard deviation, n is the number of specimens per treatment. MST is the median survival time (~starvation tolerance, in days) and W-MST is the weight-normalized median survival time (in days μgC−1). Superscripted letters denote statistical differences between experimental treatments (Bonferroni test, α = 0.0167). Table I: Summary of the characteristics of the copepods used in this study and the main results obtained from the starvation experiments Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b Species Foraging strategy Gender L ± SD (μm) W ± SD (μgC) n Treatment MST (days) W-MST (days μgC−1) Centropages typicus Cruise Females 1014 ± 68 7.87 ± 1.52 66 FF 7a 0.89a 52 FM 7a 0.89a Males 989 ± 51 6.92 ± 1.07 68 MM 6b 0.87a 52 MF 5b 0.72a Temora longicornis Feeding- current Females 659 ± 48 3.83 ± 0.84 42 FF 8a 2.09a 50 FM 8a 2.09a Males 634 ± 43 3.01 ± 0.61 50 MM 8a 2.66b 50 MF 8a 2.66b Oithona nana Ambush Females 330 ± 18 0.40 ± 0.07 68 FF 11a 27.57a 37 FM 10b 25.06b Males 309 ± 20 0.35 ± 0.07 42 MM 9c 25.75a 38 MF 8d 22.89b L is the average prosome length, W is the average body weight in carbon estimated using length to carbon conversion factors shown in Table II, SD is the standard deviation, n is the number of specimens per treatment. MST is the median survival time (~starvation tolerance, in days) and W-MST is the weight-normalized median survival time (in days μgC−1). Superscripted letters denote statistical differences between experimental treatments (Bonferroni test, α = 0.0167). CHN elemental analysis We conducted CHN elemental analysis to estimate the carbon body weight, nitrogen body weight, C:N ratios and size to weight conversion factors of males and females. The culture of Oithona nana was lost (dead) and thus Oithona davisae was used for the carbon analyses. Oithona davisae has a body size, morphology and sexual dimorphism similar to O. nana. Oithona davisae stock cultures were established at DTU Aqua from cultures originally created at the Marine Sciences Institute of Barcelona from zooplankton samples collected in the harbour of Barcelona. Oithona davisae was reared in the laboratory as described for O. nana. Adults of C. typicus, T. longicornis and O. davisae were sieved from the continuous cultures using a 100, 200 or 250-μm mesh, respectively, and gently rinsed with 0.2 μm-filtered seawater (FSW, 32 PSU). Groups of 60–1100 individuals per gender (60 C. typicus, 170 T. longicornis, 1100 O. davisae) (triplicates) were sorted under a stereomicroscope and placed in autoclaved FSW for at least 2 hours to ensure gut evacuation. Only non-ovigerous females were selected for O. davisae. Copepods were rinsed several times and then filtered onto pre-incinerated (450°C, 6 h) glass-fibre filters (GF/A grade). Filters were dried (60°C, 24 h) and stored inside a vacuum desiccator for further CHN analysis. Additionally, 50 individuals per gender and species were fixed with acid Lugol´s solution (1% final) for the length (prosome) determinations using image analysis. The CHN content in the copepod samples was determined by a Thermo Fisher Scientific FLASH 2000 Organic Elemental Analyzer. Calculations and statistics Survival curves (% survivors as a function of starvation time) and median survival time were calculated using the Kaplan–Meier estimator (Kaplan and Meier, 1958). Starvation tolerance was defined as the time where 50% of the initial number of copepods had survived in absence of food (“median survival time”). To evaluate the effect of differences in size between genders, starvation tolerance was normalized to body weight by dividing the starvation time (days) with the average body weight (μg carbon) estimated from the prosome length to carbon conversion factors (μgC μm-1) calculated in the carbon analysis. The effect of body weight on starvation tolerance was examined by plotting the individual survival time vs. body weight for 28–67 copepods, depending on species and gender. We also examined the influence of exposure time to males on survival time of females to evaluate the effect of gender interactions/mating time on starvation tolerance of females. Differences between survival curves were tested using the Gehan–Breslow–Wilcoxon test. T-tests were used to determine significant differences between average values in the CHN analyses. Differences in size between genders were tested using a Kolmogorov–Smirnoff test, since the data for prosome length did not follow a normal distribution (D’Agostino–Pearson test for normality). Individual starvation tolerance (days) and body weight (μgC ind−1) data were normally distributed (D’Agostino–Pearson test) and a Pearson Correlation coefficient test was applied to estimate significant lineal correlation between both variables. To test differences between survival curves as a function of treatment and gender (FF vs. FM, MM vs. MF and FF vs. MM), pairwise comparisons were corrected for multiple pairwise comparisons using the Bonferroni test by setting the significance level (α) = 0.0167 (=alpha-level divided by the number of comparisons = 0.05/3). For all other statistical tests, α was set to 0.05. RESULTS The relationship between the fraction of survivors (%) and their starvation time (“survival curves”) varied depending on the species and experimental treatments (Fig. 1). Centropages typicus showed the lowest starvation tolerance (Fig. 1). Survival curves of C. typicus males and females were significantly different (P = 0.0157) and males had a median starvation tolerance of 1–2 days lower than females (Fig. 1A, Table I). However, although median starvation tolerance of males was lower when incubated together with females (Table I), survival curves of both genders did not significantly differ in the presence of the opposite sex (P = 0.0614 and P = 0.9545 for males and females, respectively) (Table I, Fig. 2A). In the case of T. longicornis, median starvation tolerance was 8 days with no significant difference between genders (P = 0.2029) or the presence of the opposite gender (P = 0.8746 and P = 0.9853 for males and females, respectively) (Fig. 1B and Table I). Survival curves of O. nana males and females were significantly different (P < 0.0001) (Fig. 1C) and median starvation tolerance of O. nana males was 2 days lower than starvation tolerance of females (Table I). Median starvation tolerance in both genders of O. nana was significantly reduced by 2 days when they were incubated together (P = 0.0025 and P = 0.0122 for males and females, respectively) (Fig. 1C and Table I). Fig. 1. View largeDownload slide Survival curves (% survivors as a function of starvation time in days) of copepod male and females with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the median starvation tolerance (MST, Table I). Fig. 1. View largeDownload slide Survival curves (% survivors as a function of starvation time in days) of copepod male and females with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the median starvation tolerance (MST, Table I). Fig. 2. View largeDownload slide Body carbon content (A–C), nitrogen content (D–F) and C:N ratios (G–I) of males and females of C. typicus (A, D, G), Temora longicornis (B, F, H), and O. davisae (C, F, I). The bars are the average of three replicates and error bars the standard deviation. Fig. 2. View largeDownload slide Body carbon content (A–C), nitrogen content (D–F) and C:N ratios (G–I) of males and females of C. typicus (A, D, G), Temora longicornis (B, F, H), and O. davisae (C, F, I). The bars are the average of three replicates and error bars the standard deviation. Individual C and N content varied depending on the species and gender (Fig. 2) and was positively related to prosome length (Fig. 2, Table II). C:N ratios were close to four in males and females of the three species, with no significant differences between genders. The estimated gender-specific size to carbon conversion factors (μgC μm−1) was higher for females than for males of C. typicus and T. longicornis and similar for O. davisae females and males (Table II). Table II: Summary of the characteristics of the copepods used the CHN analysis and the size to carbon conversion factors (C/L3) obtained for copepod males and females. Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 L, average prosome length (μm); C, carbon content per copepod; SD, standard deviation. Table II: Summary of the characteristics of the copepods used the CHN analysis and the size to carbon conversion factors (C/L3) obtained for copepod males and females. Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 Species Foraging strategy Gender L ± SD (μm) C ± SD (μgC cop−1) Conversion factor (μgC mm−3) Centropages typicus Cruise Females 1075 ± 51 9.39 ± 0.41 7.55 ± 0.33 Males 1034 ± 33 7.91 ± 0.65 7.16 ± 0.59 Temora longicornis Feeding-current Females 650 ± 53 3.67 ± 1.48 13.38 ± 5.39 Males 616 ± 53 2.77 ± 1.11 11.82 ± 4.76 Oithona davisae Ambush Females 299 ± 21 0.30 ± 0.01 11.10 ± 0.24 Males 311 ± 9 0.36 ± 0.04 11.85 ± 1.32 L, average prosome length (μm); C, carbon content per copepod; SD, standard deviation. In the starvation experiments, the body size (prosome length, μm) differed between genders, with males being significantly smaller than females in the three species (Table I). When normalizing survival time with the estimated carbon body weights, the weight-specific starvation tolerance of the small ambush feeding O. nana was one order of magnitude higher than for the larger active species (Fig. 3). After normalizing survival time with C body weights, the difference in starvation tolerance between genders was reduced for C. typicus and O. nana (Fig. 3A and C, Table I) and increased for T. longicornis towards males with a weigh-specific starvation tolerance higher than females (Fig. 3B, Table I). Gender interactions significantly reduced the weight-specific starvation tolerance of both genders in O. nana (Fig. 3C, Table I). Fig. 3. View largeDownload slide Survival (%) as a function of weight-normalized starvation time (days μgC−1) of copepods with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the weight-specific median starvation tolerance (W-MST, Table I). Fig. 3. View largeDownload slide Survival (%) as a function of weight-normalized starvation time (days μgC−1) of copepods with different feeding behaviours: Centropages typicus (cruising feeder) (A), Temora longicornis (feeding-current feeder) (B) and Oithona nana (ambush feeder) (C). The grey horizontal line indicates survival = 50%, which was used to estimate the weight-specific median starvation tolerance (W-MST, Table I). There was a significant positive relationship between individual survival time and body carbon weight for females of C. typicus (Fig. 4A). However, we did not find a statistically significant effect of individual carbon body weight on starvation tolerance for any other genders/species (Fig. 4), although a positive tendency was observed in other species and genders (Fig. 4B and D). The exposure time to males did not significantly affect the starvation tolerance of females in any of the studied species (Fig. 5). Fig. 4. View largeDownload slide Relationship between individual body weight (μgC ind−1) and starvation tolerance for females of Centropages typicus (A), Temora longicornis (B) and Oithona nana (C) and for males of C. typicus (D), T. longicornis (E) and O. nana (F). Continuous line is the linear regression model fitted to the data and the discontinuous lines are the 95% confidence intervals (A). Fig. 4. View largeDownload slide Relationship between individual body weight (μgC ind−1) and starvation tolerance for females of Centropages typicus (A), Temora longicornis (B) and Oithona nana (C) and for males of C. typicus (D), T. longicornis (E) and O. nana (F). Continuous line is the linear regression model fitted to the data and the discontinuous lines are the 95% confidence intervals (A). Fig. 5. View largeDownload slide Relationship between individual females survival time and their exposure time to males, based on the treatments where both genders were incubated together for Centropages typicus (A), Temora longicornis (B) and Oithona nana (C). Fig. 5. View largeDownload slide Relationship between individual females survival time and their exposure time to males, based on the treatments where both genders were incubated together for Centropages typicus (A), Temora longicornis (B) and Oithona nana (C). DISCUSSION Influence of elemental composition on gender-specific starvation tolerance in copepods Lipid accumulation is an important strategy for organisms to cope with periods of food limitation and thus differences in lipid content can cause differences in starvation tolerance between planktonic copepods. The C:N ratio is commonly used as an index of lipid content in copepods (Båmstedt, 1986; Gismervik, 1997; Postel et al., 2000). In the species studied, we found a C:N ratio of ~4 that is in the range of C:N ratios typically found in copepods from mid latitudes (C:N ratio: 3–4, Gismervik, 1997) and indicates a low lipid content compare to some copepods from high latitudes (C:N up to 13, Bamstedt, 1986; Gismervik, 1997). In our study, copepod males and females showed no significant differences in C:N ratio, which indicates that the observed differences in starvation tolerance are not related to differences in lipid reserves between genders. Influence of body size on gender-specific starvation tolerance in copepods A difference in body size between genders (sexual size dimorphism) is a widespread phenomenon in animals with sexual reproduction. In invertebrates, including copepods, males are commonly smaller than females (Gilbert and Williamson, 1983; Hirst and Kiørboe, 2014). A smaller body size commonly implies a higher weight-specific respiration rate (higher metabolic expenses) (Kleiber, 1961; Kiørboe and Hirst, 2014). Previous studies with Acartia spp. have also shown that males have a lower starvation tolerance than females (Parrish and Wilson, 1978; Colin and Dam, 2005; Finiguerra et al., 2013), and these differences have been suggested to be explained by size differences between genders (Finiguerra et al., 2013). Our results indicate that differences in starvation tolerance between genders in C. typicus and O. nana can be mainly explained by differences in body size. A positive tendency between individual survival time and body carbon was also observed in some genders/species. However, male T. longicornis showed a similar median survival time and a higher weight-specific starvation tolerance than females, despite their smaller size. This suggests that weight-specific metabolic expenses of T. longicornis females are higher than for males (they do not follow the “3/4 power-law” of metabolic rates vs. body mass, Kleiber, 1961) or that other factors besides size affect the gender difference in starvation tolerance in this species. Measurements of weight-specific respiration rates of males and females are required to better evaluate if size is one of the main drivers of gender differences in starvation tolerance of planktonic copepods. Effect of behaviour on gender-specific starvation tolerance in copepods Kiørboe et al. (2015) found that the male lifespan is up to 50% shorter than that of females in copepods with strong differences in motile behaviour between genders (e.g. Oithona). In contrast, in copepod species where both genders have relatively similar swimming activity (e.g. C. typicus and T. longicornis), male lifespan is only ~10% shorter than females (Kiørboe et al., 2015). This indicates that motile behaviour affects gender-specific longevity in planktonic copepods. However, our results indicate a minor effect of male motile behaviour (mate-seeking behaviour) on the gender-specific starvation tolerance of the copepods studied. Mate-seeking behaviour under prolonged starvation was not recorded, but our visual observations under the microscope indicate that O. nana males kept their female-searching behaviour at least during the first days of starvation but it was reduced after prolonged starvation. This suggests that mate-seeking behaviour of males is minimized after long starvation periods, which can explain the minimal effect of sexual behaviour on gender differences in starvation compared to the effect of gender behaviour on longevity. However, when comparing among species, we found that the small ambush feeding copepod O. nana has a weight-specific starvation tolerance one order of magnitude higher than the large active swimming copepods. Our previous video observations demonstrate that females of O. nana (low motility) and T. longicornis (active swimmer) change their motile behaviour/activity budgets slightly during the starvation. This suggests that motile behaviour (foraging strategy), and not only size, can also significantly influence starvation tolerance in planktonic copepods when comparing between species. Influence of mating on starvation tolerance in copepods Mating and reproduction can reduce the lifespan of planktonic copepods (12–20%) depending on their foraging strategy (Kiørboe et al., 2015). However, we did not observe a decrease in starvation tolerance when both genders of C. typicus and T. longicornis were incubated together, suggesting that the energetic cost of copulation and pre-copulation in cruise feeders and for copepods that utilize a feeding current is low, albeit C. typicus males showed a tendency toward a decreased starvation tolerance in the presence of females. The act of copulation is expected to be similar in the three species (e.g. Blades, 1977; Uchima and Murano, 1988) but only males and females of the ambush feeding O. nana had a lower starvation tolerance because of gender interactions. Female copepods produce either hydro-mechanical or chemical signals (pheromones) that males can perceive (Kiørboe et al., 2005; Bagøien and Kiørboe, 2005a, 2005b). When a male encounters a female signal, they increase they swimming activity and/or speed (Kiørboe and Bagøien, 2005); therefore, motile behaviour of males should increase due to the presence of female signal. In C. typicus and T. longicornis, which are active swimmers, differences in swimming activity and associated metabolic expenses between “normal” swimming and pheromone-tracking swimming behaviour are probably low because males increase speed for few minutes or change their swimming patterns in the presence of pheromones. However, swimming activity in Oithona spp. males, which alternate between motionless feeding and fast mate-seeking, can notably increase in the presence of pheromones from unmated females (Heuschele and Kiørboe, 2012), and, therefore, the presence of females must be energetically costly for the males. Similarly, females of Oithona spp., which spend most of the time motionless, would increase their motile activity (e.g. escape jumps) in the presence of males. Kiørboe et al. (2015) found that O. davisae females had a reduced lifespan of 15% when incubated together with males. Therefore, increased mate-seeking behaviour by males in the presence of pheromones and pre-copulation motile activities in females may explain the reduction in starvation tolerance observed in both genders of O. nana when both genders are incubated together. There was no correlation between how long females had been incubated together with males and their starvation tolerance, which suggests that the effect of gender interactions must be the result of initial interactions. Thus, this indicates that reproduction and associated mating behaviour is not prioritized when copepods are starving, but that the initial interactions between genders are costly enough to reduce the starvation tolerance of males and females in ambush feeding copepods. Ecological implications Planktonic copepods are exposed to food limitation at different levels and for short (from hours to days) and extended periods (from weeks to months) (Winder and Cloern, 2010). However, in the marine environment, copepods are never exposed to the complete absence of food. In addition, although the copepods studied displayed their characteristic behaviours even in the smaller volumes used in this study, the limited experimental volume could have decreased mate-searching behaviour and increased encounter rates/mating compared to field conditions, particularly for the large active species. Therefore, the estimated absolute median starvation tolerance should not be directly extrapolated to field conditions, but they indicate the relative differences in starvation between genders and among copepods with difference foraging strategies. Although differences in starvation tolerance between genders (2 days) and species (up to 5 days) initially may seem small, they reflect their different capabilities to cope with short-term food limitation, and are likely to affect survival in the field where food availability is highly fluctuating. The larger species, with active feeding behaviour, have a lower starvation tolerance than ambush feeders and thus need other strategies such as the production of resting eggs (Mauchline, 1998; Lindley and Reid, 2002) to survive through periods or seasons of low primary production. Small ambush feeding copepods as Oithona spp., which have an energetically low-cost behaviour (Paffenhöfer, 2006; Almeda et al., 2011), have relative high starvation tolerance and can better tolerate seasons of low primary production without storing large amounts of lipids or using resting eggs. However, mating in ambush feeders comes at the cost of reduced starvation tolerance, which can affect reproduction and survival of Oithona spp. during periods of low food availability. Therefore, the relative differences in starvation tolerance between genders and species observed can potentially influence the seasonal abundance and distribution of the studied species. In field populations, Oithona species typically have a low male to female sex ratio of abundance (<0.2), whereas Centropages and Temora species have a sex ratio close to 1 (Kuwenberg, 1993; Kiørboe, 2008; Hirst et al., 2010). Our results suggest that gender-specific starvation tolerance can also influence sex ratios in populations of Oithona (female-biased ratios, O. nana males have a lower starvation tolerance than females) and Temora (sex ratio close to 1, males and females have a similar starvation tolerance). However, we found that C. typicus males have a lower starvation tolerance than females but a sex ratio typically close to 1 in field populations. Therefore, although differences in starvation tolerance between genders can be a factor that affects sex ratios in some species, copepod sex ratios are likely the result of numerous factors and the relative contribution of each factor may vary between species. For instance, other factors such as an elevated predation risk (Hirst et al. 2010; Almeda et al., 2017), and a shorter lifespan (Rodríguez-Graña et al., 2010; Kiørboe et al., 2015) of males compared to females also explain the strong female-skewed sex ratios of Oithona spp. in field populations. CONCLUSIONS Our results demonstrate that starvation tolerance can significantly differ between copepod genders. The most pronounced difference in starvation tolerance between genders was observed in the ambush feeding copepod Oithona nana. Our results indicate that sexual size dimorphism can partially explain gender-specific starvation tolerance in planktonic copepods, and that mate-seeking behaviour have a minor influence on the starvation tolerance of males under prolonged starvation. The presence of the opposite gender reduced starvation tolerance in both males and females of O. nana, but not in active feeding copepods. This suggests that the energetic cost of mating behaviour in ambushers is higher than in active feeders due to the strong conflict between stationary feeding and mating activities in ambush feeding copepods. ACKNOWLEDGEMENTS We would like to thank Jack Melby for maintaining the plankton cultures, Anne Busk Faarborg for helping with the CHN analyses, Enric Saiz and Albert Calbet for providing the O. davisae cultures, Sara Ceballos for providing the O. nana cultures and Thomas Kiørboe for valuable comments on this manuscript. FUNDING This work was funded through The Centre for Ocean Life, which is a VKR Centre of Excellence supported by the Villum Kann Rasmussen Foundation. This work was also supported by a individual postdoctoral grant (17023) from the Danish Council for Independent Research (DFF) to R.A., a Marie Curie Intra-European fellowship from the People Programme of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement number 6240979 to R.A., a Hans Christian Ørsted Postdoctoral fellowship from Technical University of Denmark to R.A and an internship from the European Social Fund to RRT. REFERENCES Almeda , R. , Alcaraz , M. , Calbet , A. and Saiz , E. ( 2011 ) Metabolic rates and carbon budget of early developmental stages of the marine cyclopoid copepod Oithona davisae . Limnol. Oceanogr. , 56 , 403 – 414 . Google Scholar CrossRef Search ADS Almeda , R. , van Someren Gréve , H. and Kiørboe , T. ( 2017 ) Behaviour is a major determinant of predation risk in zooplankton . Ecosphere , 8 , e01668 . Google Scholar CrossRef Search ADS Bagøien , E. and Kiørboe , T. ( 2005 a) Blind dating—mate finding in planktonic copepods. I. Tracking the pheromone trail of Centropages typicus . Mar. Ecol. Prog. Ser. , 300 , 105 – 115 . Google Scholar CrossRef Search ADS Bagøien , E. and Kiørboe , T. ( 2005 b) Blind dating—mate finding in planktonic copepods. III. Hydromechanical communication in Acartia tonsa . Mar. Ecol. Prog. Ser. , 300 , 129 – 133 . Google Scholar CrossRef Search ADS Båmstedt , U. ( 1986 ) Chemical composition and energy content. In Corner , E. D. S. and O’Hara , S. C. M. (eds) , The Biological Chemistry of Marine Copepods . Clarendon , Oxford , pp. 1 – 58 . Bjærke , O. , Andersen , T. , Bækkedal , K. S. , Nordbotten , M. , Skau , L. F. and Titelman , J. ( 2016 ) Paternal energetic investments in copepods . Limnol. Oceanogr. , 61 , 508 – 517 . Google Scholar CrossRef Search ADS Blades , P. I. ( 1977 ) Mating behavior of Centropages typicus (Copepoda: Calanoida) . Mar. Biol. , 40 , 57 – 64 . Google Scholar CrossRef Search ADS Buskey , E. J. ( 1998 ) Components of mating behavior in planktonic copepods . J. Mar. Syst. , 15 , 13 – 21 . Google Scholar CrossRef Search ADS Cebrián , J. and Valiela , I. ( 1999 ) Seasonal patterns in phytoplankton biomass in coastal ecosystems . J. Plankton Res. , 21 , 429 – 444 . Google Scholar CrossRef Search ADS Colin , S. P. and Dam , H. G. ( 2005 ) Testing for resistance of pelagic marine copepods to a toxic dinoflagellate . Evol. Ecol. , 18 , 355 – 377 . Google Scholar CrossRef Search ADS Dagg , M. ( 1977 ) Some effects of patchy food environments on copepods . Limnol. Oceanogr. , 22 , 99 – 107 . Google Scholar CrossRef Search ADS Dur , G. , Souissi , S. , Schmitt , F. G. , Beyrend-Dur , D. and Hwang , J.-S. ( 2011 ) Mating and mate choice in Pseudodiaptomus annandalei (Copepoda: Calanoida) . J. Exp. Mar. Bio. Ecol. , 402 , 1 – 11 . Google Scholar CrossRef Search ADS Finiguerra , M. B. , Dam , H. G. , Avery , D. E. and Burris , Z. ( 2013 ) Sex-specific tolerance to starvation in the copepod Acartia tonsa . J. Exp. Mar. Bio. Ecol. , 446 , 17 – 21 . Google Scholar CrossRef Search ADS Gilbert , J. J. and Williamson , C. E. ( 1983 ) Sexual dimorphism in zooplankton (Copepoda, Cladocera, and Rotifera) . Annu. Rev. Ecol. Syst. , 14 , 1 – 33 . Google Scholar CrossRef Search ADS Gismervik , I. ( 1997 ) Stoichiometry of some planktonic crustaceans . J. Plankton Res. , 19 , 279 – 285 . Google Scholar CrossRef Search ADS Hansen , P. J. ( 1989 ) The red tide dinoflagellate Alexandrium tamarense: effects on behaviour and growth of a tintinnid ciliate . Mar. Ecol. Prog. Ser. , 53 , 105 – 116 . Google Scholar CrossRef Search ADS Heuschele , J. and Kiørboe , T. ( 2012 ) The smell of virgins: mating status of females affects male swimming behaviour in Oithona davisae . J. Plankton Res. , 34 , 929 – 935 . Google Scholar CrossRef Search ADS Hirst , A. G. , Bonnet , D. , Conway , D. V. P. and Kiørboe , T. ( 2010 ) Does predation controls adult sex ratios and longevities in marine pelagic copepods? Limnol. Oceanogr. , 55 , 2193 – 2206 . Google Scholar CrossRef Search ADS Hirst , A. G. and Kiørboe , T. ( 2014 ) Macroevolutionary patterns of sexual size dimorphism in copepods . Proc. R. Soc. B. , 281 , 20140739 . Google Scholar CrossRef Search ADS Kaplan , E. L. and Meier , P. ( 1958 ) Nonparametric estimation from incomplete observations . J. Am. Stat. Assoc. , 53 , 457 – 481 . Google Scholar CrossRef Search ADS Katona , S. K. ( 1973 ) Evidence for sex pheromones in planktonic copepods . Limnol. Oceanogr. , 18 , 574 – 583 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2007 ) Mate finding, mating, and population dynamics in a planktonic copepod Oithona davisae: there are too few males . Limnol. Oceanogr. , 52 , 1511 – 1522 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2008 ) Optimal swimming strategies in mate-searching pelagic copepods . Oecologia , 155 , 179 – 192 . Google Scholar CrossRef Search ADS PubMed Kiørboe , T. ( 2011 ) How zooplankton feed: mechanisms, traits and trade-offs . Biol. Rev. , 86 , 311 – 339 . Google Scholar CrossRef Search ADS Kiørboe , T. ( 2016 ) Foraging mode and prey size spectra in suspension feeding copepods and other zooplankton . Mar. Ecol. Prog. Ser. , 558 , 15 – 20 . Google Scholar CrossRef Search ADS Kiørboe , T. and Bagøien , E. ( 2005 ) Motility patterns and mate encounter rates in planktonic copepods . Limnol. Oceanogr. , 50 , 1999 – 2007 . Google Scholar CrossRef Search ADS Kiørboe , T. , Bagøien , E. and Thygesen , U. H. ( 2005 ) Blind dating—mate finding in planktonic copepods. II. The pheromone cloud of Pseudocalanus elongatus . Mar. Ecol. Prog. Ser. , 300 , 117 – 128 . Google Scholar CrossRef Search ADS Kiørboe , T. , Ceballos , S. and Thygesen , U. H. ( 2015 ) Interrelations between senescence, life history traits, and behaviour in planktonic copepods . Ecology , 96 , 2225 – 2235 . Google Scholar CrossRef Search ADS PubMed Kiørboe , T. and Hirst , A. G. ( 2014 ) Shifts in mass scaling of respiration, feeding, and growth rates across life-form transitions in marine pelagic organisms . Am. Nat. , 183 , 118 – 130 . Google Scholar CrossRef Search ADS Kiørboe , T. , Jiang , H. and Colin , S. P. ( 2010 ) Danger of zooplankton feeding: the fluid signal generated by ambush-feeding copepods . Proc. R. Soc. B. Biol. Sci. , 277 , 3229 – 3237 . Google Scholar CrossRef Search ADS Kleiber , M. ( 1961 ) The Fire of Life . Wiley , New York . Kuwenberg , J. H. M. ( 1993 ) Sex ratio of calanoid copepods in relation to population composition in the northwestern Mediterranean . Crustaceana , 64 , 281 – 299 . Google Scholar CrossRef Search ADS Lindley , J. A. and Reid , P. C. ( 2002 ) Variations in the abundance of Centropages typicus and Calanus helgolandicus in the North Sea: deviations from close relationships with temperature . Mar. Biol. , 141 , 153 – 165 . Google Scholar CrossRef Search ADS Mauchline , J. ( 1998 ) Biology of Calanoid Copepods . Academic Press , San Diego London Boston New York Sydney Tokyo Toronto , p. 710 . Ohtsuka , S. and Huys , R. ( 2001 ) Sexual dimorphism in calanoid copepods: morphology and function . Hydrobiologia , 453 , 441 – 466 . Google Scholar CrossRef Search ADS Paffenhöfer , G. A. ( 2006 ) Oxygen consumption in relation to motion of marine planktonic copepods . Mar. Ecol. Prog. Ser. , 317 , 187 – 192 . Google Scholar CrossRef Search ADS Parrish , K. K. and Wilson , D. F. ( 1978 ) Fecundity studies on Acartia tonsa (Copepoda: Calanoida) in standardized culture . Mar. Biol. , 46 , 65 – 81 . Google Scholar CrossRef Search ADS Postel , L. , Fock , H. and Hagen , W. ( 2000 ) Biomass and abundance. In Harris , R. P. , Wiebe , P. H. , Lenz , J. , Skjoldal , H. R. and Huntley , M. (eds) , ICES Zooplankton Methodology Manual . Academic Press , San Diego , pp. 83 – 192 . Google Scholar CrossRef Search ADS Rodríguez-Graña , L. , Calliari , D. , Tiselius , P. , Hansen , B. W. and Sköld , H. N. ( 2010 ) Gender-specific ageing and non-Mendelian inheritance of oxidative damage in marine copepods . Mar. Ecol. Prog. Ser. , 401 , 1 – 13 . Google Scholar CrossRef Search ADS Schneider , C. A. , Rasband , W. S. and Eliceiri , K. W. ( 2012 ) NIH Image to ImageJ: 25 years of image analysis . Nat. Methods , 9 , 671 – 675 . Google Scholar CrossRef Search ADS PubMed Tang , K. W. , Gladyshev , M. I. , Dubovskaya , O. P. , Kirillin , G. , Grossart , H.-P. and Beisner , B. E. ( 2014 ) Zooplankton carcasses and non-predatory mortality in freshwater and inland sea environments . J. Plankt. Res. , 36 , 597 – 612 . Google Scholar CrossRef Search ADS Titelman , J. , Varpe , O. , Eliassen , S. and Fiksen , O. ( 2007 ) Copepod mating: chance or choice? J. Plankton Res. , 29 , 1023 – 1030 . Google Scholar CrossRef Search ADS Uchima , M. and Murano , M. ( 1988 ) Mating behavior of the marine copepod Oithona davisae . Mar. Biol. , 99 , 39 – 45 . Google Scholar CrossRef Search ADS van Someren Gréve , H. , Almeda , R. and Kiørboe , T. ( 2017 a) Motile behavior and predation risk in planktonic copepods . Limnol. Oceanogr. , 62 , 1810 – 1824 . Google Scholar CrossRef Search ADS van Someren Gréve , H. , Almeda , R. , Lindegren , M. and Kiørboe , T. ( 2017 b) Gender-specific feeding rates in planktonic copepods with different feeding behavior . J. Plankton Res ., 39 , 631 – 644 . Google Scholar CrossRef Search ADS Winder , M. and Cloern , J. E. ( 2010 ) The annual cycles of phytoplankton biomass . Philos. Trans. R. Soc., B. , 365 , 3215 – 3226 . Google Scholar CrossRef Search ADS Author notes Corresponding editor: Roger Harris © 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)

Journal

Journal of Plankton ResearchOxford University Press

Published: Mar 28, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off