Effects of prey trophic mode on the gross-growth efficiency of marine copepods: the case of mixoplankton.

Copepod reproductive success largely depends on food quality, which also reflects the prey trophic mode. As such, modelling simulations postulate a trophic enhancement to higher trophic levels when mixotrophy is accounted in planktonic trophodynamics. Here, we tested whether photo-phagotrophic protists (mixoplankton) could enhance copepod gross-growth efficiency by nutrient upgrading mechanisms compared to obligate autotrophs and heterotrophs. To validate the hypothesis, we compared physiological rates of the copepod Paracartia grani under the three functional nutrition types. Ingestion and egg production rates varied depending on prey size and species, regardless of the diet. The gross-growth efficiency was variable and not significantly different across nutritional treatments, ranging from 3 to 25% in the mixoplanktonic diet compared to autotrophic (11-36%) and heterotrophic (8-38%) nutrition. Egg hatching and egestion rates were generally unaffected by diet. Overall, P. grani physiological rates did not differ under the tested nutrition types due to the large species-specific variation within trophic mode. However, when we focused on a single species, Karlodinium veneficum, tested as prey under contrasting trophic modes, the actively feeding dinoflagellate boosted the egestion rate and decreased the copepod gross-growth efficiency compared to the autotrophic ones, suggesting possible involvement of toxins in modulating trophodynamics other than stoichiometric constraints.

Introduction

Copepods are the dominant mesozooplanktonic grazers in the marine realm, showing a wide variety of feeding modes and dietary habits[1], and bridging unicellular producers and consumers with fish populations. Copepod feeding and reproductive performance depends on specific food traits, such as prey size[2-4], prey concentration[5], prey motility[6], and food quality[7,8]. For copepods, food quality (e.g., biochemical composition and relative contribution of micro- and macronutrients of their prey[9]) is typically related to prey-type specific characteristics, ultimately associated to the prey trophic mode and nutrient availability. According to ecological stoichiometry, heterotrophic prey would benefit copepods in terms of stoichiometric balance[10]. However, from previous studies, no apparent enhancement in the egg production efficiency was observed in copepods fed heterotrophic protists compared to strict autotrophic prey[7].

Overall, both autotrophic and heterotrophic protists make up copepod diet in different relative contributions according to the envclass="Chemical">ironmental nutrient regime and the class="Chemical">particular food web tyclass="Chemical">pe (classic herbivore vs. microbial) established[11,12]. In class="Chemical">productive systems, such as uclass="Chemical">pwellings, diatoms reclass="Chemical">present the dominant class="Chemical">pan class="Chemical">nitrogen and lipid source in copepod diets[11,13]. In oligotrophic environments, heterotrophic nano- and microplankton, such as many dinoflagellates and ciliates are the prevailing food[11]. Nevertheless, the dichotomic paradigm placing strict phototrophs and heterotrophs as major players in nutrient cycling within planktonic communities is challenged by the presence of mixotrophy. In fact, nowadays a large proportion of marine protists is acknowledged to be capable of performing both photosynthesis and phagotrophy[14], and therefore referred herein to as mixoplankton[15]. Assigning mixoplankton to a fixed functional role in planktonic communities is a hard task due to the great variety of mixotypes existing[16] and their flexible reliance on either nutritional strategy[15]. As a consequence, by overlooking mixoplankton as key drivers in ocean production, the predicted contribution of species to nutrient fluxes would only reflect an incomplete dichotomic food web structure[15].

Hitherto, experimental evidences[17,18] and model simulations[16,19] reinforce the idea that mixoplankton do influence trophic interactions and nutrient cycling via trophic upgrade and channelling of nutrients to higher predators from unfavourably-sized prey. Nonetheless, how mixoplankton affect copepod and other zooplankton vital rates is not yet fully understood, and the outcome of the experimental evidence available is diverse. Some studies have reported either enhancement[17] or the lack of any remarpan class="Species">kable effect on coclass="Chemical">peclass="Chemical">pod class="Chemical">physiology when fed different class="Chemical">potential mixotroclass="Chemical">phic dinoflagellates[20,21], whereas when the mixoclass="Chemical">planktonic sclass="Chemical">pecies tested may contain toxins, as tyclass="Chemical">pically occur during the onset of harmful class="Chemical">pan class="Disease">algal blooms[22-24], other assays have showed impairment of copepod vital rates. It is worth noting, however, that mixoplankton tested in previous studies were usually not grown under phagotrophic status, rather they were reared as functional autotrophs (i.e., in inorganic nutrient-rich media and high irradiance levels without additional prey supply)[15]. Therefore, up to date, the nutritional value of functional (actively feeding) mixoplankton on copepod physiology is still under-investigated[17] and the integration of mixoplankton as independent trophic level is only partially addressed on ecosystem scale by a few numerical models and network analyses[25,26].

In the present study, we determined differences in the gross-growth efficiency of the calanoid copepod class="Species">Paracartia grani (Sars) when offered functional mixotroclass="Chemical">phic class="Chemical">prey in comclass="Chemical">parison to tyclass="Chemical">pically-used autotroclass="Chemical">phic and heterotroclass="Chemical">phic diets. We restricted our study to mixoclass="Chemical">plankton that do not class="Chemical">produce (acute) toxic resclass="Chemical">ponses in coclass="Chemical">peclass="Chemical">pods, and ensured the mixotroclass="Chemical">phic class="Chemical">prey used in the exclass="Chemical">periments were actively feeding. To achieve this aim, we conducted grazing exclass="Chemical">periments to quantify ingestion, egg class="Chemical">production and egg hatching rates under the aforementioned dietary conditions. We also determined the coclass="Chemical">peclass="Chemical">pod fecal class="Chemical">pellet class="Chemical">production and fecal class="Chemical">pellet size under the different diets tested to assess variations in egestion rate due the likely differences in the degree of class="Chemical">prey digestion and class="Chemical">pacclass="Chemical">pan class="Species">kaging. Finally, we also explored the effects of intraspecific variability of the prey trophic mode (in both oligotrophic and eutrophic conditions) on the copepod physiological performance.

Results

Elemental content and stoichiometric ratios

Size, elemental contents and stoichiometric molar ratios of the prey species are listed in Table 1. The C content normalized to cell volume was lower in heterotrophs (0.11 ± 0.004 pg C μm−3) compared to the other trophic modes (0.15 ± 0.01 and 0.17 ± 0.02 pg C μm−3 in autotrophs and mixotrophs, respectively; Kruspan class="Species">kal–Wallis, Dunn’s test, P = 0.041). N and P contents and stoichiometric ratios (C:N, C:P and N:P), however, did not differ among troclass="Chemical">phic modes (one-way ANOVA, P > 0.05). We further exclass="Chemical">plored class="Chemical">potential differences between the class="Chemical">prey troclass="Chemical">phic modes by Princiclass="Chemical">pal Comclass="Chemical">ponent Analysis (PCA). Figure 1 shows the contribution of class="Chemical">prey stoichio-morclass="Chemical">phometric traits on the grouclass="Chemical">ping of sclass="Chemical">pecies within an orthogonal 2D-sclass="Chemical">pace along the two most relevant class="Chemical">princiclass="Chemical">pal comclass="Chemical">ponents (PCs). C:P ratio and N content exclass="Chemical">plained > 90% of variation along the PC1 and PC2 resclass="Chemical">pectively (Fig. 1a). However, no class="Chemical">particular distinction among troclass="Chemical">phic modes emerged as auto-, mixo- and heterotroclass="Chemical">phic class="Chemical">prey were clustered together (95% CI) without significant differences (P > 0.05) (Fig. 1b).

Table 1

Size (equivalent spherical diameter, ESD), size class, elemental content and stoichiometric molar ratios of the prey species. The prey carbon and cell concentrations used in the copepod feeding incubations are also provided. Average ± 1 s.e.m.

Diet	ESD (µm)	Size class	pg C µm−3	pg N µm−3	pg P µm−3	C:N	C:P	N:P	µg C L−1	cells mL−1
Rhodomonas salina	7.2	Small	0.17 ± 0.01	0.04 ± 0.003	0.004 ± 0.00001	5.3 ± 0.05	98 ± 8.02	18.6 ± 1.343	945 ± 4.4	29,964 ± 141
Karlodinium veneficum f/2	10.5	Small	0.13 ± 0.008	0.03 ± 0.002	0.006 ± 0.00002	5.7 ± 0.20	58 ± 3.86	10.2 ± 0.984	546 ± 3.7	6741 ± 46
Karlodinium veneficum FSW	11.5	Small	0.12 ± 0.007	0.02 ± 0.001	0.003 ± 0.00002	7.3 ± 0.03	97 ± 5.80	13.2 ± 0.776	498 ± 1.9	4821 ± 19
Thalassiosira weissflogii	13.5	Medium	0.17 ± 0.004	0.03 ± 0.001	0.004 ± 0.00002	6.0 ± 0.02	105 ± 2.602	17.4 ± 0.396	711 ± 4.9	3053 ± 21
Heterocapsa sp.	13.7	Medium	0.15 ± 0.005	0.02 ± 0.001	0.004 ± 0.00002	9.0 ± 0.05	110 ± 3.775	12.2 ± 0.383	539 ± 2.1	2490 ± 10
Karlodinium veneficum mixo	12.0	Small	0.18 ± 0.01	0.03 ± 0.001	0.004 ± 0.00003	7.1 ± 0.05	109 ± 5.751	15.4 ± 0.727	690 ± 8.3	4270 ± 12
Karlodinium armiger	16.1	Medium	0.18 ± 0.005	0.04 ± 0.002	0.006 ± 0.00005	5.1 ± 0.07	84 ± 2.48	16.5 ± 0.667	410 ± 1.6	845 ± 3
Mesodinium rubrum	18.9	Large	0.13 ± 0.006	0.03 ± 0.001	0.003 ± 0.00009	6.1 ± 0.10	113 ± 5.874	18.4 ± 0.865	616 ± 5.7	1297 ± 29
Oxyrrhis marina	16.4	Medium	0.10 ± 0.001	0.02 ± 0.000	0.004 ± 0.00006	5.1 ± 0.03	71 ± 1.47	13.9 ± 0.242	506 ± 3.7	2202 ± 19
Gyrodinium dominans	18.9	Large	0.12 ± 0.003	0.02 ± 0.002	0.004 ± 0.0003	5.7 ± 0.19	80 ± 6.55	14.1 ± 1.388	686 ± 6.0	1514 ± 18
Strombidium arenicola	30.7	Large	0.11 ± 0.006	0.03 ± 0.003	0.006 ± 0.0003	4.8 ± 0.24	49 ± 3.47	10.3 ± 1.104	300 ± 25.0	179 ± 15

Figure 1

Principal component analysis. (a) Component loadings of the original variables and (b) component scores of combinations of the original variables along the two principal component axes. The circle in (b) depicts the Hotelling's T2 with 95% confidence interval. Abbreviations in (b) are described in Table 3.

Size (equivalent spherical diameter, ESD), size class, elemental content and stoichiometric molar ratios of the prey species. The prey pan class="Chemical">carbon and cell concentrations used in the coclass="Chemical">peclass="Chemical">pod feeding incubations are also class="Chemical">provided. Average ± 1 s.e.m.

Table 3

Growth conditions of the different prey species.

FTM	Species	Strain	Culture medium	Irradiance (µE)	Diet	Prey:Predator ratio	Culture volume (L)
Auto	Rhodomonas salina (R)	K-0294	f/2	110	–	–	5
Auto	Karlodinium veneficum f/2 (Kv f/2)	K21-ICMB-274	f/2	90	–	–	4
Auto	Karlodinium veneficum fsw (Kv fsw)	K21-ICMB-274	FSW	90	–	–	4
Auto	Thalassiosira weissflogii (T)		f/2 + Si	90	–	–	1
Auto	Heterocapsa sp. (H)		f/2	90	–	–	1
Mixo	Karlodinium veneficum mixo (Kv mixo)	K21-ICMB-274	FSW	90	R. salina	0.3	4
Mixo	Karlodinium armiger (Ka)	ICM-ZOO-KA001	FSW	40	R. salina	6	10
Mixo	Mesodinium rubrum 1 (M1, M2)	DK-2009	FSW	80	T. amphioxeia	2	20
Hetero	Oxyrrhis marina (O)	ICM-ZOO-OM001	FSW	20	R. salina	15	1
Hetero	Gyrodinium dominans 1 (G1, G2)	ICM-ZOO-GD001	FSW	20	R. salina	20	3
Hetero	Strombidium arenicola (S)	ICM-ZOO-SA001	FSW	20	R. salina	100	8

FTM functional trophic mode, Auto autotrophic, Mixo mixotrophic, Hetero heterotrophic. Letters in parenthesis after the species name refer to the abbreviated initials used in graphical representations. FSW plain filtered seawater containing 8.8 µM N (NO2− + NO3−) and 0.5 µM PO43−; µE µmol photons m−1 s-1. Notice that two experiments were carried out with M. rubrum (M1 and M2) and also with G. dominans (G1 and G2).

Regarding the variation within class="Species">Karlodinium veneficum grown in different conditions, the C content of class="Chemical">pan class="Species">K. veneficum increased significantly when acting as mixotroph (0.18 ± 0.01 pg C μm−3), whereas the cell quota was similar under high (0.13 ± 0.08 pg C μm−3) and low (0.12 ± 0.007 pg C μm−3) nutrient load in both autotrophic cultures (one-way ANOVA, Tukey HSD, P = 0.008; Table 1). The P content was statistically different among the three functionally growing K. veneficum cultures (one-way ANOVA, Tukey HSD, P < 0.001), being two-fold lower in the poor-nutrient autotrophic dinoflagellate with respect to the nutrient-replete one (respectively, 0.003 ± 0.00002 pg P μm−3 and 0.006 ± 0.0002 pg  P μm−3; Table 1). In the K. veneficum reared as functional autotroph in high-nutrients, C:N and C:P molar ratios were significantly lower than the nutrient-depleted K. veneficum, regardless of their nutritional strategies (one-way ANOVA, Tukey HSD, P < 0.001). However, the N:P ratio increased significantly in the mixotrophic K. veneficum compared to the nutrient-rich autotrophic dinoflagellate (Kruskal–Wallis, Tukey HSD, P = 0.03; Table 1).

Ingestion rates

Figure 2a shows the ingestion rates of P. grani adult females, in terms of class="Chemical">carbon intake, as a function of class="Chemical">prey size. In general, there were significant differences in the feeding rates between size classes (7–13 µm; 13–18 µm; 18–31 µm) (one-way ANOVA, Bonferroni, P = 0.026). Mean values ranged between 3.7 ± 0.1 µg C ind−1 d−1 when fed the small-sized autotroclass="Chemical">phic class="Chemical">pan class="Species">K. veneficum (grown in FSW) and 18 ± 2 µg C ind−1 d−1 when fed the large-sized mixotrophic Mesodinium rubrum. Nevertheless, despite the large size, the aloricate ciliate Strombidium arenicola was eaten at moderately lower rates compared to the other large prey species (Fig. 2a). Even within the intermediate prey size range, substantial variation (ca. 3.5-fold change) was found among species. The ingestion rates did not vary significantly among functional trophic modes (one-way ANOVA, P = 0.547), most likely because of the high variability observed, especially among the different autotrophic and mixotrophic prey. Regarding stoichiometric ratios, we only found a moderate but significant linear relationship between C ingestion and prey N:P ratio (one-way ANOVA, P = 0.046, Fig. 2b; Table S1). Regarding K. veneficum, which was tested under the 3 different trophic conditions (Fig. 2a, Table 2), significant differences emerged between autotrophic and mixotrophic nutrition (Kruskal–Wallis, Tukey HSD, P = 0.038). In fact, the C ingestion of the adult female P. grani on the mixotrophic K. veneficum (5.9 ± 0.1 µg C ind−1 d−1) was ca. 1.6-fold higher than the one measured on the dinoflagellates grown autotrophically in f/2 (3.8 ± 0.3 µg C ind−1 d−1) and nutrient-poor (i.e. FSW) medium (3.7 ± 0.1 µg C ind−1 d−1).

Figure 2

Ingestion rate of adult female P. grani as a function of (a) prey size and (b) prey N:P ratio. Non-linear fit with peak Sigma-Plot function in (a) and simple linear regression fit and corresponding equation in (b) are provided. Abbreviations are described in Table 3. Error bars are ± 1 s.e.m.

Table 2

Copepod physiological responses under the three K. veneficum diets.

Physiological response	K. veneficum treatments
	Auto (f/2)	Auto (FSW)	Mixo (FSW + R. salina)
Ingestion rate (µg C ind−1 d−1)	3.8 ± 0.32	3.7 ± 0.14	5.9 ± 0.14*
Fecal pellet production rate (fecal pellets ind−1 d−1)	12 ± 0.5	5 ± 0.2**	8 ± 0.3
Fecal pellet volume (106 µm3 fecal pellet−1)	0.14 ± 0.013	0.16 ± 0.032	0.34 ± 0.042**
Egestion rate (106 µm3 ind−1 d−1)	1.7 ± 0.07	0.9 ± 0.05*	2.8 ± 0.12**
E/I ratio (%)	5.8 ± 0.23	2.8 ± 0.15	8.4 ± 0.44*
Egg production rate (eggs ind−1 d−1)	27 ± 1.8	30 ± 2.2	25 ± 1.1
Gross-growth efficiency (%)	23 ± 2.5	29 ± 2.9	15 ± 0.5*
Hatching success (%)	71 ± 2.5	74 ± 2.0	72 ± 2.7

Auto autotrophic, Mixo mixotrophic, FSW plain filtered seawater.

Significant comparisons are indicated by *. *P < 0.05, **P < 0.001. Average ± 1 s.e.m.

Copepod physiological responses under the three pan class="Species">K. veneficum diets.

Auto autotrophic, Mixo mixotrophic, FSW plain filtered seapan class="Chemical">water.

Fecal pellet production rates

Fecal pellet production rate ranged between 5 ± 0.3 pellets ind−1 d−1 on a diet of class="Species">K. veneficum (FSW) and 84 ± 4 class="Chemical">pellets ind−1 d−1 when fed on class="Chemical">pan class="Species">Thalassiosira weissflogii (Fig. 3a). Fecal pellet size was variable and ranged between (geometric) mean values of 0.09 × 106 µm3 pellet−1 for M. rubrum and 0.44 × 106 µm3 pellet−1 for the diatom T. weissflogii (Fig. 3b). When egestion rate is expressed as total biovolume of fecal pellet produced, most of the prey tested rendered rather similar and low egestion rates (with values ranging between 0.9 ± 0.1 and 3.9 ± 0.3 × 106 µm3 ind−1 d−1 for K. veneficum (FSW) and Oxyrrhis marina, respectively, Fig. 3c), with the exception of the cryptophyte Rhodomonas salina and the diatom T. weissflogii. On these two diets, in fact, the egestion rate of adult female P. grani accounted for 12.6 ± 0.7 × 106 µm3 ind−1 d−1 (R. salina) and 37 ± 2 × 106 µm3 ind−1 d−1 (T. weissflogii). These egestion rates were ca. 5 and 17 times higher than the average rates shown on the other prey. When comparing K. veneficum diets, as a consequence of increased pellet size, the mixotrophic K. veneficum yielded significantly higher egestion outputs compared to the autotrophic ones (one-way ANOVA, Tukey HSD, P < 0.001, Fig. 3b,c, Table 2). No correlation was found between defecation traits (pellet volume, pellet production and egestion rate) and either prey size or ingestion rate (data not shown; one-way ANOVA, P > 0.05 in all cases), nor with prey stoichiometry (one-way ANOVA, P > 0.05, Table S1).

Figure 3

Egestion of P. grani. (a) Fecal pellet production rate, (b) fecal pellet volume as function of fecal pellet production rate, (c) volumetric egestion rate in the different dietary treatments, (d) egestion/ingestion ratio as function of prey size. Abbreviations are described in Table 3. Error bars are ± 1 s.e.m.

The ratio of egested biovolume to total ingested biovolume (E/I ratio; Fig. 3d), expressed as a percentage, is as a proxy for discerning potential differences in either the absorption efficiency, the degree of pellet pacclass="Species">kaging or the class="Chemical">presence of coclass="Chemical">peclass="Chemical">pod sloclass="Chemical">pclass="Chemical">py feeding under the different diets. With the exceclass="Chemical">ption of class="Chemical">pan class="Species">T. weissflogii and R. salina, with E/I ratios of respectively 38 ± 2% and 34 ± 2%, for the rest of species the mean value was 4.2 ± 0.8% and the range of variation was moderate (1–10%; Fig. 3d), without significant differences across trophic modes or prey size classes (one-way ANOVA, P > 0.05) nor prey stoichiometry (one-way ANOVA, P > 0.05, Table S1). At a finer scale, intraspecific variations in E/I ratio emerged when the different K. veneficum diets were compared. In fact, a higher E/I ratio was observed under mixotrophic K. veneficum diet (8.4 ± 0.3%) compared to the autotrophic K. veneficum diets, either in high or low nutrient (FSW) media (respectively, 5.8 ± 0.2% and 2.8 ± 0.1%; Kruskal–Wallis, Tukey HSD, P = 0.020, Fig. 3d, Table 2).

Egg production rates, egg hatching success and gross-growth efficiency

P. grani egg production rates were variable and ranged between 15 ± 2 eggs ind−1 d−1 when the diet consisted of class="Species">M. rubrum and 78 ± 3 eggs ind−1 d−1 when fed the heterotroclass="Chemical">phic ciliate class="Chemical">pan class="Species">S. arenicola (Fig. 4a). The three K. veneficum cultures showed similar reproductive outputs (mean: 27 ± 1 eggs ind−1 d−1, Table 2). In either case, no significant difference in egg production rates among diets was found when comparing among trophic modes or between the three K. veneficum diets (one-way ANOVA, P > 0.05). Neither food quality (prey stoichiometric ratios) significantly influence egg production rate (one-way ANOVA, P > 0.05, Table S1).

Figure 4

Reproduction and recruitment in the different dietary treatments. (a) P. grani egg production rate and (b) egg hatching success after 48 h from collection. Error bars are ± 1 s.e.m.

Egg hatching success was overall high under most of the dietary treatments (nearly 70%; Fig. 4b), but declined when copepods were fed class="Species">M. rubrum (27% ± 4) and to less extent when fed the autotroclass="Chemical">phic class="Chemical">pan class="Species">Heterocapsa sp. (54% ± 1). The three differentially growing K. veneficum did not exhibit any significant difference in hatching success among them (one-way ANOVA, P > 0.05; Fig. 4b, Table 2). Slightly significant relationship was found between hatching success and prey C:P ratio (one-way ANOVA, P = 0.042, Table S1). However, the amount of variance explained was rather low and the relationship was no longer significant when one outlier species (M. rubrum) was removed.

P. grani females showed high values (25–40%) of gross-growth efficiency when feeding on class="Species">R. salina, class="Chemical">pan class="Species">K. veneficum (FSW), Heterocapsa sp., Karlodinium armiger and S. arenicola (Fig. 5a). Very low efficiencies (< 10%) were found when fed M. rubrum and Gyrodinium dominans. Surprisingly, the latter two prey were ingested at reasonably high rates, but the reproductive output was comparatively very low (Fig. 5b). No significant differences were found in gross-growth efficiencies of P. grani on the different diets according to the nutritional mode of the offered prey (one-way ANOVA, P > 0.05; Fig. 5a) and no significant relationship existed with prey food quality (one-way ANOVA, P > 0.05, Table S1). Contrarily to this general picture, however, when we focused only on K. veneficum, the consumption of the autotrophic and mixotrophic cultures yielded different gross-growth efficiency outputs. In fact, the mixotrophic diet resulted in lower P. grani gross-growth efficiency (15% ± 1) compared to autotrophic nutrition in either high or low nutrient conditions (respectively, 23% ± 2 and 29% ± 3), without differences between nutrient treatments (Kruskal–Wallis, Tukey HSD, P = 0.03; Fig. 5a, Table 2).

Figure 5

Gross-growth efficiency of P. grani. (a) Carbon gross-growth efficiency in the different dietary conditions and (b) egg production rate as a function of ingestion rate. The dashed line in (b) represents a 30% gross-growth efficiency illustrated for comparative purposes. Abbreviations are described in Table 3. Error bars are ± 1 s.e.m.

Gross-growth efficiency of P. grani. (a) pan class="Chemical">Carbon gross-growth efficiency in the different dietary conditions and (b) egg class="Chemical">production rate as a function of ingestion rate. The dashed line in (b) reclass="Chemical">presents a 30% gross-growth efficiency illustrated for comclass="Chemical">parative class="Chemical">purclass="Chemical">poses. Abbreviations are described in Table 3. Error bars are ± 1 s.e.m.

Discussion

Phagotrophic organisms are expected to accumulate more N and P compared to autotrophs that store C-rich structural class="Chemical">polysaccharides and class="Chemical">photosynthates[27]. However, we found no significant differences in the elemental contents and stoichiometric ratios among the different nutrition modes of class="Chemical">protist class="Chemical">prey (Fig. 1, Table 1). Only the C content of the heterotroclass="Chemical">phs was significantly lower than that for the autotroclass="Chemical">phic and mixotroclass="Chemical">phic class="Chemical">prey, but this difference did not translate into significant stoichiometric changes (Table 1). The lack of clear differences in the stoichiometric ratios observed when comclass="Chemical">paring our autotroclass="Chemical">phic and mixotroclass="Chemical">phic sclass="Chemical">pecies (and strains) may be due to (a) stronger but sclass="Chemical">pecies-sclass="Chemical">pecific declass="Chemical">pendency of autotroclass="Chemical">phs on external inorganics and (b) flexible reliance of mixoclass="Chemical">plankton on either nutritional mode under the aclass="Chemical">pclass="Chemical">plied growing conditions. In fact, mixoclass="Chemical">plankton can lie in the class="Chemical">phototroclass="Chemical">phic or heterotroclass="Chemical">phic sclass="Chemical">pectrum of the troclass="Chemical">phic continuum according to the mixotyclass="Chemical">pe[16]. Furthermore, beside the well-known role of nutrients and light, also the interclass="Chemical">play between these two and the size class is acknowledged to influence stoichiometry in class="Chemical">protists within a given troclass="Chemical">phic category[27]. Among the sclass="Chemical">pecies tested, cell volume-normalised C content (including N and P also when reclass="Chemical">ported) was coherent with class="Chemical">previously-reclass="Chemical">ported values in the literature for the autotroclass="Chemical">phs class="Chemical">pan class="Species">R. salina and T. weissflogii[28], Heterocapsa sp.[29] and the heterotrophs G. dominans and O. marina[30], and the closely-related Strombidium sulcatum[31] under similar growth conditions than those in this study. In the kleptoplastidic M. rubrum, elemental quotas normalised to cell volume were slightly different from those of a previous study[32], perhaps due to the differences between works in the nutrient load of the growth medium. We could not find any elemental content profile in the literature for K. armiger, but our C estimate agreed well with the ones calculated on the basis of general equations developed for athecate dinoflagellates[33].

Regarding the elemental composition of our autotrophic class="Species">K. veneficum strain, our values matched those reclass="Chemical">ported by Li et al. under Redfield nutrient class="Chemical">proclass="Chemical">portions[34]. Some authors have reclass="Chemical">ported a correlation between the ingestion rate of class="Chemical">pan class="Species">K. veneficum and the predator C quota[34], which might explain the increase in C content observed in our mixotrophic K. veneficum compared to the phototrophic cultures (Table 1). Generally, small-intermediate sized mixoplankton tend to feed on C-rich phototrophs when light or nitrogen-depleted[27]. Furthermore, it is worth noting that karlotoxins are C-rich structures, which are produced in higher proportion under nutrient limitation and during active feeding[35,36]. Then, it is possible that the C quota of the mixotrophic K. veneficum was higher than that of autotrophic cultures due to the use of karlotoxins for phagotrophy[34]. We unfortunately did not measure toxin content in our experiments.

The ingestion rates of P. grani female fell within the range of values previously reported for this copepod species under high concentrations of class="Species">R. salina[37], class="Chemical">pan class="Species">Heterocapsa sp.[38], T. weissflogii[39] and O. marina[40], and for the closely-related Acartia tonsa on similarly-sized prey[39,41,42]. Regarding stoichiometric ratios as proxy of food quality, we found a relationship between copepod ingestion rate and prey N:P ratio (Fig. 2b), indicating that P-poor prey were ingested at higher rate, probably as a means to compensate for the lack of the specific nutrient[43], although copepod response to nutrient deficient-prey may also be unaffected as previously demonstrated[38]. However, the amount of variance in ingestion rates explained by prey N:P ratio was rather modest (30%), and was basically driven by the data relative to M. rubrum and T. weissflogii. Conversely, as could be anticipated, prey size appeared to be the most relevant factor to consider when interpreting our results. Thus, ingestion rates of P. grani increased with prey size up to an optimum, and then declined when fed the largest prey tested, S. arenicola, resulting in values similar to those exhibited for much smaller prey species (i.e., Heterocapsa sp.) (Fig. 2a). Such dome-shaped relationship between feeding rates and prey size are already known for copepods[3,28,44]. Very small prey are hardly perceived or retained efficiently, whereas larger prey may either exhibit strong escape responses or require longer handling by the copepod[45] or being too big to be fully ingested[3]. The optimal prey size range (13–18 µm) for P. grani observed in our study fits well with the values reported for adult A. tonsa[44].

Although egestion rates and pellet size of P. grani varied largely among prey, they were not correlated with either ingestion rates or prey size and were independent of prey stoichiometry (Table S1) or of prey nutritional strategy (Fig. 3a–c), as also reported for class="Species">A. tonsa fed different autotroclass="Chemical">phic and heterotroclass="Chemical">phic class="Chemical">protists[42,46]. Coclass="Chemical">peclass="Chemical">pod fecal class="Chemical">pellet size and density have been shown to be either related to[47] or indeclass="Chemical">pendent of food concentration. However, in our exclass="Chemical">periments, coclass="Chemical">peclass="Chemical">pods were always above satiation level, thus the differences reclass="Chemical">ported herein must result from sclass="Chemical">pecies-sclass="Chemical">pecific class="Chemical">particularities in the degree of class="Chemical">prey class="Chemical">pacclass="Chemical">pan class="Species">kaging. Our pellet volume estimates agree with previous reports on A. tonsa fed saturating concentration of various prey over a size range between 7 and 20 µm[42]. The large pellet volume on a diatom-diet was expected, since diatom siliceous materials are not digested within copepod intestinal tract and fecal pellets appear larger and more resistant compared to the ones egested under soft diet[42,48,49]. The increase in pellet size recorded in the mixotrophic K. veneficum diet, compared to the two autotrophic cultures of this species, may be the consequence of the phagotrophic activity of the dinoflagellate on R. salina. It is possible that the dinoflagellate might change composition when grown mixotrophically[50], yielding different pellet volume in copepods. In general terms, enhanced egestion rates due to increase in pellet dimension under functionally mixotrophic K. veneficum diet (Table 2) would have an influence in the recycling of organic matter within the microbial food web, as fecal pellet size (and density) normally determines their sinking rates, modifying the export production out of the photic layer[42].

The analysis of the egestion/ingestion (E/I) ratio, based on volumetric data (Fig. 3d), may be more informative than the sole comparison of egestion rates, as it considers the amount (volume) of prey ingested. The calculated E/I ratios overall had no clear relationship with prey size, prey stoichiometry or prey trophic mode, showing distinctively high values when copepods were fed the cryptophyte class="Species">R. salina and the diatom class="Chemical">pan class="Species">T. weissflogii. Comparing our E/I ratio for T. weissflogii (44%) with those obtained by Besiktepe and Dam (58%)[42], it is rather clear that the diatom is largely undigested by Acartia spp. due to the presence of a siliceous frustule. In contrast, our E/I ratio for R. salina (33%) is higher than those obtained in their study for similarly-sized soft prey such as Dunaliella tertiolecta (4%) and Uronema sp. (7%)[42]. The reason for the high E/I ratio when fed R. salina is less clear. Assuming the copepod absorption efficiency to be the reciprocal of the E/I ratio, our estimated copepod absorption efficiency (on a volumetric basis) would be 63% for R. salina and 62% for T. weissflogii, whereas in all the other prey tested the values fell within the very high range (> 90%). In this regard, Thor and Wendt[51] compared the carbon absorption efficiency of A. tonsa fed different prey[51]. These authors found that at high R. salina concentrations the copepod carbon absorption efficiency was particularly lower (53%) than when fed other species (T. weiisflogii: 63%, D. terctiolecta: 77%), hence resulting in much higher expected egestion rates, in agreement with our results[51].

Ingested food is eventually respired, egested and excreted as discarded materials and solutes, and when exceeding the basal metabolic requirements, it is finally incorporated into the body as reserves, growth or reproductive output. In our experiments, P. grani egg production rate increased with ingestion rate, maximum values reached on a diet of the heterotrophic ciliate class="Species">S. arenicola, closely followed by class="Chemical">pan class="Species">R. salina, O. marina and Heterocapsa sp. (Fig. 4a). The egg production rates obtained in our experiments match previous values reported on P. grani feeding on some of the tested prey (O. marina, R. salina, Heterocapsa sp.)[38,40,52]. However, the increase in egg production was not always coherent with ingestion rate. In fact, surprisingly, T. weissflogii, M. rubrum and G. dominans, which fall in the preferred prey size range (13–18 µm), yielded rather low reproductive output compared to the corresponding ingestion rate (Fig. 5b). In the case of the diatom, the presence of silica frustule[48] and the abovementioned drop in absorption efficiency may help explain the lower fecundity. Since no relationship was found between egg production and prey stoichiometric traits (Table S1), it is possible that the prey which resulted in comparatively low egg production rates were presumably deficient in some essential fatty acids or other compounds essential to copepod reproduction and recruitment[8]. Unexpectedly, the egg production rate of P. grani when offered K. veneficum was similar between mixotrophic and autotrophic diets, regardless of the nutrient conditions (Table 2). We cannot discard, however, that the lack of apparent advantage in the reproductive output for copepods fed mixoplankton could be related to the degree of mixotrophic behaviour exhibited by our K. veneficum, with rather moderate ingestion rates (maximum daily intakes of 1 R. salina grazer−1[53]). K. armiger, on the other hand, promoted higher egg production rate. K. armiger is known to graze on a greater extent (ca. 9 R. salina grazer−1 d−1) compared to the congeneric K. veneficum[54], thus, perhaps contributing to enhanced nutrient transfer to the predator in our study.

Our main aim was to assess the gross-growth efficiency of copepods when fed diets of different nutritional mode. Although egg production rates per se are a very useful proxy to understand the overall impact of a prey on secondary production, the normalization of the reproductive rate per amount of food ingested allows a better picture of the prey nutritional quality. The gross-growth efficiency for C in marine calanoid copepods under optimal conditions usually falls around 30–40%[44,55]. Without distinction on the basis of prey trophic mode, several of the diets we tested reached these high efficiency values in agreement with other studies[56,57]. Other diets, however, proved to be less efficient, e.g., class="Species">M. rubrum and class="Chemical">pan class="Species">G. dominans (Fig. 5a). Considering the dependency of ingestion on prey size, one may argue that prey size constraints (i.e. sloppy feeding) could be the source of the particularly low gross-growth efficiency (3–8%) measured for these large species. Large prey might not be ingested in their entirety, therefore resulting in overestimation of actual ingestion rates when the food removal methods is applied[3,4]. Using the equations provided by Møller[4] to calculate the expected prey size-dependent loss of organic matter by copepod sloppy feeding, the overestimation of C intake due to sloppy feeding in our study would account for up to 13%. Such augmented values would negligibly affect our gross-growth efficiency estimates (only 0.6 and 1.4% increase for the M. rubrum and G. dominans diets, respectively). Therefore, these corrections assuming sloppy feeding would not be sufficient to explain the huge discrepancy found between ingestion and reproduction under these diets. Moreover, we did not observe lower gross-growth efficiency in P. grani when fed even larger prey, S. arenicola, confirming that sloppy feeding alone cannot be the source of low reproductive output under M. rubrum and G. dominans diets. Alternatively, we may suggest toxicity (as observed in some diatom species[58]) or deficient food quality in these prey[7,30,56]. Concerning low nutritional quality arguments (stoichiometric constraints), one would expect some compensatory feeding as demonstrated to occur in A. tonsa fed nutrient-deficient algae[43]. In our study, high N:P prey such as M. rubrum were ingested at higher rates (Fig. 2b). Yet, no significant relationship emerged between gross-growth efficiency and prey N:P ratio (Table S1), hence rendering stoichiometric arguments rather weak in explaining the variations observed. Additionally, as mentioned before, fatty acids are essential dietary constituents in copepod reproduction. We did not determine the fatty acid contents and composition of our tested prey, but for some of them fatty acid data can be found in the literature and may help to interpret our results. Thus, in accordance with our results for M. rubrum and G. dominans, Broglio et al.[7] found that diets of the heterotrophic M. pulex and G. dominans were less nutritional to A. tonsa compared to the cryptophyte R. salina. A positive correlation was found between reproduction and ingested fatty acids (especially the ratio between 22:6ω3 and 20:5ω3), but no specific dependency was determined on the basis of prey nutritional strategy, reinforcing our observations[7]. In their study the low performance of M. pulex and G. dominans was attributed to the lower 22:20 ratio, which is known to influence the reproductive performance in crustaceans[7].

Concerning the effects of trophic mode on the nutritional value of class="Species">K. veneficum, we observed a droclass="Chemical">p in gross-growth efficiency when coclass="Chemical">peclass="Chemical">pods were fed on the dinoflagellate acting as mixoclass="Chemical">plankton, contrarily to literature and model exclass="Chemical">pectations (Fig. 5a, Table 2). As mentioned before, class="Chemical">pan class="Species">K. veneficum exhibits variation in the phagotrophic tendency[22,53] and produces karlotoxins (although with a large variability between strains[35]) to immobilize its prey prior to capture. Therefore, it could be hypothesized a possible involvement of toxic weapons against R. salina on which the dinoflagellate was feeding before being offered to the copepods[36]. Nevertheless, in our experiments any possible allelopathic effects were not enough to trigger deadly outcomes on the copepods throughout the 5 days of exposure and, although the gross-growth efficiency of the copepod was clearly lower, this was still comparable to those yielded under other non-toxic diets (O. marina, Heterocapsa sp., T. weissflogii). Although K. veneficum has been reported toxic in occasions for copepods, in our experiments we did not observe any enhanced mortality or sublethal effects (abnormal motility) compared to the other prey. Furthermore, in other independent experiments (unpublished data), we observed that the mortality of P. grani along ontogeny on a diet of both functionally autotrophic (20%) and mixotrophic K. veneficum (16%) was overall comparable to that registered on a monodiet of R. salina (20%) (Traboni et al., unpubl). This renders K. veneficum suitable prey for P. grani and valid for comparison with the other species tested, despite its acknowledged toxicity. The gross-growth efficiency on a diet of K. armiger was considerably high, despite the species has been demonstrated to have detrimental acute effects on other Acartia species when offered at saturating concentrations[22]. Perhaps toxicity is boosted upon ammonium addition as nitrogen source[59], which was not the case in our culture medium. This hypothesis stresses the importance of nutrient types other than ratios for mixotrophic growth and toxicity to be tested in future experiments.

Finally, egg hatching success was, in most cases, relatively high and differences recorded were not related to the prey trophic mode neither across diets nor among class="Species">K. veneficum troclass="Chemical">phic tyclass="Chemical">pes (Fig. 4b). For class="Chemical">pan class="Species">Acartia sp., similar or slightly higher hatching rates were reported on natural plankton assemblage as well as on monodiets of R. salina, G. dominans and O. marina in the range between 67 and 91%[40,45,52,60]. Our hatching rates dropped in only a few cases, specifically on a diet of M. rubrum and Heterocapsa sp. In Broglio et al.[7], lower hatching (20–60%) was recorded in A. tonsa fed the ciliate S. sulcatum and more generally hatching was found to be related to the ingestion of certain polyunsaturated fatty acids (PUFAs), stressing the importance of fatty acid composition for egg viability[7]. The same hypothesis, however, cannot be made for Heterocapsa sp., whose reported content of cellular PUFA[57] is similar to that of other species which yielded high hatching rates in the above mentioned studies. Phosphorus depletion has been proposed as additional limiting conditions for copepod development in both field[61] and laboratory experiments[38]. Even if moderate, we observed increased hatching rate in copepods fed low C:P prey (Table S1); yet, this relationship seems to be no longer significant if high-C:P M. rubrum is removed. Although copepod C-ingestion and prey N:P ratio were positively related, the calculated P ingestion rates on M. rubrum and Heterocapsa sp. are not different from other species tested (between 1 and 4 × 10–7 µg P ind−1 d−1) and cannot explain the variation in hatching rates we observed.

To our knowledge this is the first attempt at evaluating the actual nutritional contribution that mixoplankton may have on marine copepods. Most studies that have focused on mixotrophic species as prey for copepod feeding experiments have not used functionally phagotrophic mixoplankton (i.e. feeding on other protists). Although until relatively recently mixoplankton was considered a rare fact, the current gap in the empirical observation of mixoplankton role in copepod diet is also a consequence of the challenge in reaching considerable standing pan class="Species">stocks for exclass="Chemical">perimentation under controlled class="Chemical">phagotroclass="Chemical">phic status[15].

Overall, we found that only moderate relationship existed between copepod ingestion rate with the prey N:P ratio and size. None of the reproductive parameters was substantially affected by nutritional quality proxies due to the intraspecific variability among prey traits and the lack of a clear pattern placing mixotrophs on a different nutritional quality level with respect to other protists, according to their stoichiometry. We conclude that in the conditions tested mixoplankton proved to be as nutritional as the strict phago- and autotrophs to P. grani. Mixotrophic class="Species">K. veneficum diet under our conditions did not enhance coclass="Chemical">peclass="Chemical">pod gross-growth efficiency in remarclass="Chemical">pan class="Species">kable fashion compared to the obligate autotrophic dinoflagellates; instead, it represented a less valuable prey, perhaps due to a presumably certain degree of toxicity associated to the phagotrophic behaviour, and it promoted larger fecal pellets with potential consequences on C export rate. We postulate that our experimental conditions could not trigger significant variation in copepod physiology because of the resemblance of our mixotrophic strains to strict photo- or heterotrophic food. In other words, the limited reliance of K. veneficum on phagotrophy could have flattened important differences on copepod performance relative to strict autotrophic K. veneficum diets. Additional studies with other mixoplankters and other copepod species may help to provide clearer and more robust conclusions on the role of mixotrophy on the carbon transfer mediated by copepods to upper trophic levels. Moreover, since nutrient ratios are known to be environmental forcing triggering a switch in protist trophic mode, we acknowledge the need to assess in future research whether mixoplankton may change their quality and influence higher predators as a result of stronger nutrient regime shifts and higher prey availability.

Materials and methods

Copepod cultures

The calanoid copepod class="Species">Paracartia grani used in the exclass="Chemical">periments originates from a culture maintained at the ICM[52]. The coclass="Chemical">peclass="Chemical">pods were grown in 20–40 L tanks in a cold room at 19 °C and 10:14 light:dark cycle, and routinely fed the cryclass="Chemical">ptoclass="Chemical">phyte class="Chemical">pan class="Species">Rhodomonas salina. New copepod cohorts were set-up by siphoning the bottom of the culture tanks, and transferring the newly-collected eggs into a new tank. After hatching, the copepods were supplied with increasing concentrations of R. salina along development up to approx. 30,000 cells mL−1 (1000 µg C L−1) when adults. Only less than 2-week old healthy adult females were used in the experiments.

Protist cultures

All prey species were grown non-axenically in batch cultures at 38 psu and 19 °C. The functionally autotrophic species tested were the cryptophyte class="Species">R. salina, the diatom class="Chemical">pan class="Species">Thalassiosira weissflogii and the thecate dinoflagellate Heterocapsa sp., all growing exponentially in constantly aerated f/2 medium (+Si for T. weissflogii) under high light (90–100 μmol photons m−2 s−1). Heterocapsa and Rhodomonas species have been reported to be able to feed on bacteria when environmental conditions challenge the strict autotrophic metabolism (5–13 bacteria protist−1 h−1 in Heterocapsa rotundata by food removal method[62] and < 1 bacteria protist−1 in Rhodomonas sp. analysing the food vacuole content[63]). However, in our conditions, we assume that plenty of inorganic nutrients and high irradiance levels would limit bacterial uptake to negligible rates, conferring both species in our study the role of autotrophs. As strict heterotrophic protists, cultures of the athecate dinoflagellates Gyrodinium dominans and Oxyrrhis marina and of the ciliate Strombidium arenicola were grown in non-aerated filtered seawater (FSW) with a daily supply of R. salina. Finally, we selected the non-constitutive mixoplanktonic ciliate Mesodinium rubrum (reared in non-aerated FSW on a diet of the cryptophyte Teleaulax amphioxeia) and two athecate dinoflagellates, Karlodinium veneficum and Karlodinium armiger as constitutive mixoplankton; both Karlodinium species were reared in FSW and supplied R. salina during the culturing period. In the case of K. veneficum, we further explored the effects of nutrient status and trophic strategy “within species”. Thus, K. veneficum was grown in constant aeration both as strict autotrophic under two contrasted nutrient environments (f/2 medium vs. FSW), and as mixotroph in low-nutrient conditions (FSW + R. salina). This species is more flexible than our K. armiger strain and M. rubrum as the latter depend on food supply for their growth[64,65], limiting our possibilities of testing them as pure autotrophs. Generally, to prevent overgrowth of prey in the mixoplankton and heterotrophic cultures, grazers were kept in semi-saturating food conditions. Further details in growth conditions can be found in Table 3. Standing stocks of the cultures were determined with a Multisizer III particle counter (Beckman Coulter) or by Sedgewick-Rafter counts.

FTM functional trophic mode, Auto autotrophic, Mixo mixotrophic, Hetero heterotrophic. Letters in parenthesis after the species name refer to the abbreviated initials used in graphical representations. FSW plain filtered seaclass="Chemical">water containing 8.8 µM N (NO2− + NO3−) and 0.5 µM class="Chemical">pan class="Chemical">PO43−; µE µmol photons m−1 s-1. Notice that two experiments were carried out with M. rubrum (M1 and M2) and also with G. dominans (G1 and G2).

Experimental setting

Copepod experiments consisted of a preconditioning period (4–5 days) followed by 24 h incubations for the determination of ingestion and egg production rates; both the preconditioning and experimental periods were carried out at saturating food concentrations (Table 1). We chose the long preconditioning to minimize food history effects on egg production[66]. Protist cultures were up-scaled to different final volumes based on experimental needs (Table 3). Batch cultures of the autotrophic prey were diluted on a daily basis to keep them in exponential growth. Regarding the mixo- and heterotrophs, when sufficient amounts were reached, we split the standing class="Species">stocks into 5 1-L Pyrex bottles (or round-bottom flasks). These 5 individual new class="Chemical">pan class="Species">stocks were served to feed the copepods during each of the 4 preconditioning days and the experimental day. All grazer cultures were routinely food-replenished except the ones to be immediately used, which were left 24 h in starvation before being offered to the copepods, allowing the depletion of their own prey. Only S. arenicola and O. marina, which exhibited very fast prey removal, were fed until the last 24 h to avoid potential nutritional impoverishment. By following this protocol, copepods ingested prey of the same food quality throughout the preconditioning and experimental periods. R. salina and T. amphioxeia concentrations in mixo- and heterotrophic protist cultures were determined with the particle counter and/or by microscopic counts, ensuring no substantial cryptophyte biomass would be available to copepods in food suspensions (< 0.1 µg C L−1). Prey suspensions for copepod experiments were prepared by diluting the culture stocks to the desired concentrations, and adjusted with the Multisizer particle counter, which served for both enumeration of prey suspensions and estimation of cell volume. In the case of the ciliates M. rubrum and S. arenicola, which may escape from the particle counter flow, 10 mL samples were preserved in acetic Lugol’s solution (2% final concentration), and their concentrations determined under the microscope. For these latter species, however, cell volume was measured from live counts with the particle counter.

For preconditioning, groups of adult females (60–200 according to experimental needs) were sorted from the cohorts and transferred into 4 L class="Chemical">Nalgene polycarbonate bottles filled with the resclass="Chemical">pective food susclass="Chemical">pensions class="Chemical">preclass="Chemical">pared in FSW (Table 1). Each bottle was amended with 5 mL of f/2 medium L−1 (f/2 + Si for class="Chemical">pan class="Species">T. weissflogii) to minimize the effects of copepod excretion[31]. Bottles were maintained at low irradiance (10 μmol photons m−2 s−1) to avoid prey overgrowth. Every day, 75% of the content of each preconditioning bottle was siphoned out with a silicone tube fitted with 20–40 µm mesh net (according to prey size) to avoid losing the copepods, and subsequently the bottles were refilled with fresh prey suspensions at the corresponding concentrations (Table 1). By this way, we ensured minimal stress and no loss of animals during refilling procedures.

The day of the experiment, 613 mL wide-mouth Pyrex bottles were carefully filled with new food suspensions (Table 1) in four sequential steps, gently mixing before refilling. The 24 h incubations comprised one start (to account for the initial prey concentration), four control (copepod-free) and four grazing (with added copepods) bottles. Copepods were retrieved from the preconditioning bottles by gentle filtration on a 200 µm mesh sieve, transferred to a Petri dish and then pipetted out into the experimental Pyrex bottles. The number of copepods used (8–26), reflected the prey size and the a priori expected clearance rates[37,40]. Thereafter, the start bottle was sacrificed to assess the initial prey concentration, and the remaining bottles were capped, checked for the presence of air bubbles and placed on a rotating Ferris wheel (0.2 rpm). Some extra copepods were fixed in pan class="Chemical">formaldehyde (4% final concentration) and class="Chemical">photograclass="Chemical">phed under the inverted microscoclass="Chemical">pe (40 × magnification) for size measurements. The class="Chemical">prosome length was determined with the software ImageJ.

After 20–24 h, the bottles were removed from the wheel and their contents filtered through 200 µm sieves to collect the copepods; control bottles were treated similarly for consistency. Copepods were checked for their condition, anesthetized with class="Chemical">MS222[52] and counted. Then, the 200-µm sieved class="Chemical">pan class="Chemical">water was thoroughly homogenized and 50–100 mL samples were taken for either the particle counter or microscopic counts (Lugol’s solution samples). Finally, the whole remaining content (400–500 mL) was filtered onto a 20 μm sieve (rinsed three times) to collect eggs and fecal pellets. These were transferred onto Petri dishes where fecal pellets were counted fresh after collection to calculate egestion rate. After counting, fecal pellets and eggs were photographed (100–200 × magnification; LEICA-MC170 HD) and then promptly incubated at 19 °C. After 48 h, the Petri dishes containing eggs and fecal pellets were preserved with Lugol’s solution; unhatched eggs and nauplii were enumerated to calculate egg production and hatching success. Egg shells contained in fecal pellets were taken into account for egg production rates[39]. Eggs and fecal pellet size was measured using the software ImageJ. Contours of eggs/pellets were traced and fit to a geometrical ellipsoid for the posterior calculation of biovolume.

Copepod feeding rates were calculated from prey removal according to Frost’s equations[67]. Egestion rates were expressed as number of fecal pellets and also as biovolume of fecal pellet produced. The ratio of egestion rate to ingestion rate in biovolume terms, E/I ratio, was calculated as the quotient between egestion rates and ingestion rates (both in µm3 ind−1 d−1 units), expressed as percentage.

Hatching success was estimated as percentage of nauplii hatched after 48 h, based on the initial egg number. Ingestion and egg production rates were converted into µg Cprey ind−1 d−1 using respectively the prey pan class="Chemical">carbon contents analysed in this study (see below) and the P. grani egg content class="Chemical">provided by Saiz et al.[68]. Gross-growth efficiency (as %) was calculated as the quotient between egg class="Chemical">production rates and ingestion rates, both exclass="Chemical">pressed in class="Chemical">pan class="Chemical">carbon terms.

Elemental analysis

Prior to each experimental incubation, we filtered aliquots of the prey class="Species">stocks onto class="Chemical">pre-combusted (450 °C, 5 h) GF/F filters (Whatman, 25 mm) for determination of the class="Chemical">pan class="Chemical">carbon (C), nitrogen (N) and phosphorus (P) elemental composition. The filters for CN analysis were oven-dried at 60 °C, 48–72 h and then stored in a desiccator until processing with a Flash EA1112 (Thermo Finnigan) CHNS analyser. The filters for P analysis were immediately frozen at −80 °C until processing. P samples were processed as in Isari et al.[38] applying the acid persulfate digestion method and posterior conversion to dissolved inorganic P with a Seal Analytical AA3 (Bran + Luebbe) analyser. In a few occasions, CNP profiles of the prey were not concurrent with the experiments, but done posteriorly. In those cases, we ensured similar culture growth conditions; a posteriori comparison with previously measured CNP contents from our culture collection revealed the suitability of elemental and stoichiometric data obtained. For the mixotrophic and heterotrophic protists, when small amounts of their own cryptophyte prey had remained at the time of preparing the experimental suspensions, their contribution to the elemental content of the copepod prey was subtracted after analysis. Stoichiometric ratios were calculated as molar ratios and propagation of error was taken into account due to independency between CN and P samples[68].

Statistical analysis

Graphics and statistical analyses were conducted with the software SigmaPlot 14.0. Data were checked for normality (Shapiro–Wilk) and homoscedasticity (Brown-Forsythe). One-way ANOVA and post-hoc comparisons (Tukey class="Disease">HSD, Bonferroni tests) were class="Chemical">performed to assess differences in the coclass="Chemical">peclass="Chemical">pod class="Chemical">physiological rates among functional troclass="Chemical">phic modes. Krusclass="Chemical">pan class="Species">kal–Wallis and Dunn’s post hoc test were performed instead of ANOVA when homoscedasticity assumptions were not met. More specifically, we compared copepod physiological responses across all the diets under the three functional trophic modes (auto-, mixo-, heterotrophic); we did the same kind of analysis also independently for the specific K. veneficum comparisons (autotrophic in f/2, autotrophic in FSW and mixotrophic in FSW + R. salina). In addition, we tested for differences of copepod ingestion rate in relation to prey size classes applying the same procedure as with functional trophic modes. The size classes, small (7–13 µm), intermediate (13–18 µm) and large (18–31 µm), were established on the basis of optimal prey:predator ratio from previous works and on the likely occurrence of sloppy feeding[4]. Results were considered significant at the 0.05 level. Generally, in case of non-significant comparisons, only P values from ANOVA and Kruskal–Wallis tests are reported; on the contrary, if those tests yielded significant differences (by default P < 0.05), only P values relative to the post-hoc analysis are reported. Simple linear regression analyses were conducted between copepod physiological responses and prey stoichiometric traits as proxies of food quality (one-way ANOVA output reported). Graphical exploration of morpho-stoichiometric characteristics of prey species and clustering of prey trophic modes was performed with a multivariate Principal Component Analysis (PCA).

Supplementary Table 1.