Shifts in survival and reproduction after chronic warming enhance the potential of a marine copepod to persist under extreme heat events.

The study of a species' thermal tolerance and vital rates responses provides useful metrics to characterize its vulnerability to ocean warming. Under prolonged thermal stress, plastic and adaptive processes can adjust the physiology of organisms. Yet it is uncertain whether the species can expand their upper thermal limits to cope with rapid and extreme changes in environmental temperature. In this study, we reared the marine copepod Paracartia grani at control (19°C) and warmer conditions (25°C) for >18 generations and assessed their survival and fecundity under short-term exposure to a range of temperatures (11-34°C). After multigenerational warming, the upper tolerance to acute exposure (24 h) increased by 1-1.3°C, although this enhancement decreased to 0.3-0.8°C after longer thermal stress (7 days). Warm-reared copepods were smaller and produced significantly fewer offspring at the optimum temperature. No shift in the thermal breadth of the reproductive response was observed. Yet the fecundity rates of the warm-reared copepods in the upper thermal range were up to 21-fold higher than the control. Our results show that chronic warming improved tolerance to stress temperatures and fecundity of P. grani, therefore, enhancing its chances to persist under extreme heat events.

Ecophysiological response of marine copepods to dietary elemental imbalances.

We assessed the effects of nutrient imbalanced diets on the feeding, reproduction and gross-growth efficiency of egg production of the copepod Paracartia grani. The cryptophyte Rhodomonas salina, cultivated under balanced (f/2) and imbalanced growth conditions (N and P limitation), served as prey. Copepod C:N and C:P ratios increased in the imbalanced treatments, particularly under P limitation. Feeding and egg production rates did not differ between the balanced and N-limited treatments but decreased under P limitation. We found no evidence of compensatory feeding in P. grani. C gross-growth efficiency averaged 0.34 in the balanced treatment and declined to values of 0.23 and 0.14 for the N- and P-limited treatments, respectively. Under N limitation, N gross-growth efficiency increased significantly to a mean value of 0.69, likely as a result of increasing the nutrient absorption efficiency. P gross-growth efficiency reached values > 1 under P limitation, involving the depletion of body P. Hatching success was >80%, with no differences among diets. Hatched nauplii, however, had lower size and slower development when the progenitor was fed a P-limited diet. This study highlights the effects of P limitation in copepods, which are more constraining than N, and the presence of maternal effects driven by prey nutritional composition that ultimately may affect population fitness.

The effect of short-term temperature exposure on vital physiological processes of mixoplankton and protozooplankton.

Sudden environmental changes like marine heatwaves will become more intense and frequent in the future. Understanding the physiological responses of mixoplankton and protozooplankton, key members of marine food webs, to temperature is crucial. Here, we studied two dinoflagellates (one protozoo- and one mixoplanktonic), two ciliates (one protozoo- and one mixoplanktonic), and two cryptophytes. We report the acute (24 h) responses on growth and grazing to a range of temperatures (5-34 °C). We also determined respiration and photosynthetic rates for the four grazers within 6 °C of warming. The thermal performance curves showed that, in general, ciliates have higher optimal temperatures than dinoflagellates and that protozooplankton is better adapted to warming than mixoplankton. Our results confirmed that warmer temperatures decrease the cellular volumes of all species. Q10 coefficients suggest that grazing is the rate that increases the most in response to temperature in protozooplankton. Yet, in mixoplankton, grazing decreased in warmer temperatures, whereas photosynthesis increased. Therefore, we suggest that the Metabolic Theory of Ecology should reassess mixoplankton's position for the correct parameterisation of future climate change models. Future studies should also address the multigenerational response to temperature changes, to confirm whether mixoplankton become more phototrophic than phagotrophic in a warming scenario after adaptation.

Reduction in thermal stress of marine copepods after physiological acclimation.

We studied the phenotypic response to temperature of the marine copepod Paracartia grani at the organismal and cellular levels. First, the acute (2 days) survival, feeding and reproductive performances at 6-35°C were determined. Survival was very high up to ca. 30°C and then dropped, whereas feeding and fecundity peaked at 23-27°C. An acclimation response developed after longer exposures (7 days), resulting in a decline of the biological rate processes. As a consequence, Q10 coefficients dropped from 2.6 to 1.6, and from 2.7 to 1.7 for ingestion and egg production, respectively. Due to the similarity in feeding and egg production thermal responses, gross-growth efficiencies did not vary with temperature. Respiration rates were less sensitive (lower Q10) and showed an opposite pattern, probably influenced by starvation during the incubations. The acclimation response observed in the organismal rate processes was accompanied by changes in body stoichiometry and in the antioxidant defense and cell-repair mechanisms. Predictions of direct effects of temperature on copepod performance should consider the reduction of Q10 coefficients due to the acclimation response. Copepod population dynamic models often use high Q10 values and may overestimate thermal effects.

Thermal Acclimation and Adaptation in Marine Protozooplankton and Mixoplankton.

Proper thermal adaptation is key to understanding how species respond to long-term changes in temperature. However, this is seldom considered in protozooplankton and mixoplankton experiments. In this work, we studied how two heterotrophic dinoflagellates (Gyrodinium dominans and Oxyrrhis marina), one heterotrophic ciliate (Strombidium arenicola), and one mixotrophic dinoflagellate (Karlodinium armiger) responded to warming. To do so, we compared strains adapted at 16, 19, and 22°C and those adapted at 16°C and exposed for 3 days to temperature increases of 3 and 6°C (acclimated treatments). Neither their carbon, nitrogen or phosphorus (CNP) contents nor their corresponding elemental ratios showed straightforward changes with temperature, except for a modest increase in P contents with temperature in some grazers. In general, the performance of both acclimated and adapted grazers increased from 16 to 19°C and then dropped at 22°C, with a few exceptions. Therefore, our organisms followed the "hotter is better" hypothesis for a temperature rise of 3°C; an increase of >6°C, however, resulted in variable outcomes. Despite the disparity in responses among species and physiological rates, 19°C-adapted organisms, in general, performed better than acclimated-only (16°C-adapted organisms incubated at +3°C). However, at 22°C, most species were at the limit of their metabolic equilibrium and were unable to fully adapt. Nevertheless, adaptation to higher temperatures allowed strains to maintain physiological activities when exposed to sudden increases in temperature (up to 25°C). In summary, adaptation to temperature seems to confer a selective advantage to protistan grazers within a narrow range (i.e., ca. 3°C). Adaptation to much higher increases of temperatures (i.e., +6°C) does not confer any clear physiological advantage (with few exceptions; e.g., the mixotroph K. armiger), at least within the time frame of our experiments.

The strengths and weaknesses of Live Fluorescently Labelled Algae (LFLA) to estimate herbivory in protozooplankton and mixoplankton.

The Live Fluorescently Labelled Algae (LFLA) technique has been used numerous times to estimate microzooplankton herbivory. Yet, it is unknown how mixoplankton (i.e., single-cell organisms that can combine phototrophy and phagotrophy) affect the outcome of this technique. Hence, we conducted a broad-spectrum assessment of the strengths and weaknesses of the LFLA technique, using several mixoplanktonic and protozooplanktonic grazers. Species from different taxonomic groups and different feeding mechanisms were tested in short-term experiments (ca. 5 h) in the laboratory, at different prey concentrations and during light and dark periods of the day. Overall, our findings suggest that the LFLA technique, due to its short-term nature, is an effective tracker of diel ingestion and digestion rates, and can detect new mixoplanktonic predators. We recommend that, irrespective of the prey concentration, incubations to measure grazing rates with this technique should generally be concluded within 1 h (adaptable to the environmental temperature). Nevertheless, our results also call for caution whenever using LFLA in the field: feeding mechanisms other than direct engulfment (like peduncle feeding) may provide severely biased ingestion rates. Furthermore, size and species selectivity are very hard to circumvent. To reduce the effects of selectivity, we propose the combined use of two distinctly coloured fluorochromes (i.e., distinct emission spectra). With this modification, one could either label different size ranges of prey or account for species-specific interactions in the food web.

Mixoplankton interferences in dilution grazing experiments.

It remains unclear as to how mixoplankton (coupled phototrophy and phagotrophy in one cell) affects the estimation of grazing rates obtained from the widely used dilution grazing technique. To address this issue, we prepared laboratory-controlled dilution experiments with known mixtures of phyto-, protozoo-, and mixoplankton, operated under different light regimes and species combinations. Our results evidenced that chlorophyll is an inadequate proxy for phytoplankton when mixoplankton are present. Conversely, species-specific cellular counts could assist (although not fully solve) in the integration of mixoplanktonic activity in a dilution experiment. Moreover, cell counts can expose prey selectivity patterns and intraguild interactions among grazers. Our results also demonstrated that whole community approaches mimic reality better than single-species laboratory experiments. We also confirmed that light is required for protozoo- and mixoplankton to correctly express their feeding activity, and that overall diurnal grazing is higher than nocturnal. Thus, we recommend that a detailed examination of initial and final plankton communities should become routine in dilution experiments, and that incubations should preferably be started at the beginning of both day and night periods. Finally, we hypothesize that in silico approaches may help disentangle the contribution of mixoplankton to the community grazing of a given system.

Predator Chemical Cue Effects on the Diel Feeding Behaviour of Marine Protists.

We have assessed the effect of copepod chemical cues on the diel feeding rhythms of heterotrophic and mixotrophic marine protists. All phagotrophic protists studied exhibited relatively high diurnal feeding rates. The magnitude of the diel feeding rhythm, expressed as the quotient of day and night ingestion rates, was inversely related to the time that phagotrophic protists were maintained in the laboratory in an environment without predators. In the case of the recently isolated ciliate Strombidium arenicola, the rhythm was lost after a few months. When challenged with chemical alarm signals (copepodamides) from the copepod Calanus finmarchicus at realistic concentrations (0.6-6 pM), S. arenicola partially re-established diurnal feeding. Conversely, the amplitude of the diel feeding rhythm for the ciliate Mesodinium rubrum was not affected by copepodamides, although the 24-h integrated food intake increased by approximately 23%. For the dinoflagellates Gyrodinium dominans and Karlodinium armiger, copepodamides significantly reduced the amplitude of their diel feeding rhythms; significant positive effects on total daily ingestion were only observed in G. dominans. Finally, the dinoflagellate Oxyrrhis marina, isolated >20 years ago, showed inconsistent responses to copepodamides, except for an average 6% increase in its total ingestion over 24 h. Our results demonstrate that the predation risk by copepods affects the diel feeding rhythm of marine protists and suggests a species-specific response to predation threats.

Non-lethal effects of the predator Meganyctiphanes norvegica and influence of seasonal photoperiod and food availability on the diel feeding behaviour of the copepod Centropages typicus.

Predators can induce changes in the diel activity patterns of marine copepods. Besides vertical migration, diel feeding rhythms have been suggested as an antipredator phenotypic response. We conducted experiments to assess the non-lethal direct effects of the predator Meganyctiphanes norvegica (northern krill) on the diel feeding patterns of the calanoid copepod Centropages typicus. We also analysed the influence of seasonal photoperiod and prey availability on the intensity of copepod feeding rhythms. We did not detect any large effect of krill presence on the diel feeding behaviour of copepods, either in day-night differences or total daily ingestions. Seasonal photoperiod and prey availability, however, significantly affected the magnitude of copepod feeding cycles, with larger diel differences in shorter days and at lower prey concentrations. Therefore, the role of non-lethal direct effects of predators on the diel feeding activity of marine copepods remain debatable and might not be as relevant as in freshwater zooplankton.

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.

Caveats on the use of rotenone to estimate mixotrophic grazing in the oceans.

Phagotrophic mixotrophs (mixoplankton) are now widely recognised as important members of food webs, but their role in the functioning of food webs is not yet fully understood. This is due to the lack of a well-established technique to estimate mixotrophic grazing. An immediate step in this direction would be the development of a method that separates mixotrophic from heterotrophic grazing that can be routinely incorporated into the common techniques used to measure microplankton herbivory (e.g., the dilution technique). This idea was explored by the addition of rotenone, an inhibitor of the respiratory electron chain that has been widely used to selectively eliminate metazoans, both in the field and in the laboratory. Accordingly, rotenone was added to auto-, mixo-, and heterotrophic protist cultures in increasing concentrations (ca. 24 h). The results showed that mixotrophs survived better than heterotrophs at low concentrations of rotenone. Nevertheless, their predation was more affected, rendering rotenone unusable as a heterotrophic grazing deterrent. Additionally, it was found that rotenone had a differential effect depending on the growth phase of an autotrophic culture. Altogether, these results suggest that previous uses of rotenone in the field may have disrupted the planktonic food web.

Towards an Understanding of Diel Feeding Rhythms in Marine Protists: Consequences of Light Manipulation.

Temporal programs synchronised with the daily cycle are of adaptive importance for organisms exposed to periodic fluctuations. This study deepens into several aspects of the exogenous and endogenous nature of microbial grazers. We investigated the diel rhythms of cell division and feeding activity of four marine protists under different light regimes. In particular, we tested if the feeding cycle of protistan grazers could be mediated by a light-aided enhancement of prey digestion, and also explored the consequences of cell division on diel feeding rhythms. Cell division occurred at night for the heterotrophic dinoflagellates Gyrodinium dominans and Oxyrrhis marina. In contrast, the mixotrophic dinoflagellate Karlodinium armiger and the ciliate Strombidium sp. mostly divided during the day. Additionally, a significant diurnal feeding rhythm was observed in all species. When exposed to continuous darkness, nearly all species maintained the cell division rhythm, but lost the feeding cycle within several hours/days (with the exception of O. marina that kept the rhythm for 9.5 days). Additional feeding experiments under continuous light also showed the same pattern. We conclude that the feeding rhythms of protistan grazers are generally regulated not by cell division nor by the enhancement of digestion by light. Our study, moreover, indicates that the cell division cycle is under endogenous control, whereas an external trigger is required to maintain the feeding rhythm, at least for most of the species studied here.

Planktonic food web structure and trophic transfer efficiency along a productivity gradient in the tropical and subtropical Atlantic Ocean.

Oligotrophic and productive areas of the ocean differ in plankton community composition and biomass transfer efficiency. Here, we describe the plankton community along a latitudinal transect in the tropical and subtropical Atlantic Ocean. Prochlorococcus dominated the autotrophic community at the surface and mixed layer of oligotrophic stations, replaced by phototrophic picoeukaryotes and Synechococcus in productive waters. Depth-integrated biomass of microzooplankton was higher than mesozooplankton at oligotrophic stations, showing similar biomasses in productive waters. Dinoflagellates dominated in oligotrophic waters but ciliates dominated upwelling regions. In oligotrophic areas, microzooplankton consumed ca. 80% of the production, but ca. 66% in upwelling zones. Differences in microzooplankton and phytoplankton communities explain microzooplankton diel feeding rhythms: higher grazing rates during daylight in oligotrophic areas and diffuse grazing patterns in productive waters. Oligotrophic areas were more efficient at recycling and using nutrients through phytoplankton, while the energy transfer efficiency from nutrients to mesozooplankton appeared more efficient in productive waters. Our results support the classic paradigm of a shorter food web, and more efficient energy transfer towards upper food web levels in productive regions, but a microbially dominated, and very efficient, food web in oligotrophic regions. Remarkably, both models of food web exist under very high microzooplankton herbivory.

Effects of concentration and size of suspended particles on the ingestion, reproduction and mortality rates of the copepod, Acartia tonsa.

Suspended sediments are a common occurrence in the marine environment. They can be generated by natural causes, including waves and currents, or brought about by anthropogenic activities such as reclamation and dredging. High sediment concentrations are known to have negative consequences on copepods; however, the impact of sediment size has largely been overlooked. Here we examine the effects of sediment size and concentration in combination with varying algae concentrations on the ingestion rate, egg production, hatching success and survivorship of the copepod species, Acartia tonsa. High concentration of 'small' sediments at 'low' food availability had the greatest negative impact on all parameters except hatching success. Greater food concentration was able to mitigate some of these effects. High concentrations of 'large' sediments also reduced egg production rates, possibly due to A. tonsa avoiding falling particles. We conclude that it is important to examine the particle size distribution when evaluating the impacts of suspended sediments on copepods.

Publisher Correction: Sex-Dependent Effects of Caloric Restriction on the Ageing of an Ambush Feeding Copepod.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Sex-Dependent Effects of Caloric Restriction on the Ageing of an Ambush Feeding Copepod.

Planktonic copepods are a very successful group in marine pelagic environments, with a key role in biogeochemical cycles. Among them, the genus Oithona is one of the more abundant and ubiquitous. We report here on the effects of caloric (food) restriction on the ageing patterns of the copepod Oithona davisae. The response of O. davisae to caloric restriction was sex dependent: under food limitation, females have lower age-specific mortality rates and longer lifespans and reproductive periods; male mortality rates and life expectancy were not affected. Males are more active swimmers than females, and given their higher energetic demands presumably generate reactive oxygen species at higher rates. That was confirmed by starvation experiments, which showed that O. davisae males burn through body reserves much faster, resulting in shorter life expectancy. Compared with common, coastal calanoid copepods, the effects of caloric restriction on O. davisae appeared less prominent. We think this difference in the magnitude of the responses is a consequence of the distinct life-history traits associated with the genus Oithona (ambush feeder, egg-carrier), with much lower overall levels of metabolism and reproductive effort.

A light-induced shortcut in the planktonic microbial loop.

Mixotrophs combine photosynthesis with phagotrophy to cover their demands in energy and essential nutrients. This gives them a competitive advantage under oligotropihc conditions, where nutrients and bacteria concentrations are low. As the advantage for the mixotroph depends on light, the competition between mixo- and heterotrophic bacterivores should be regulated by light. To test this hypothesis, we incubated natural plankton from the ultra-oligotrophic Eastern Mediterranean in a set of mesocosms maintained at 4 light levels spanning a 10-fold light gradient. Picoplankton (heterotrophic bacteria (HB), pico-sized cyanobacteria, and small-sized flagellates) showed the fastest and most marked response to light, with pronounced predator-prey cycles, in the high-light treatments. Albeit cell specific activity of heterotrophic bacteria was constant across the light gradient, bacterial abundances exhibited an inverse relationship with light. This pattern was explained by light-induced top-down control of HB by bacterivorous phototrophic eukaryotes (PE), which was evidenced by a significant inverse relationship between HB net growth rate and PE abundances. Our results show that light mediates the impact of mixotrophic bacterivores. As mixo- and heterotrophs differ in the way they remineralize nutrients, these results have far-reaching implications for how nutrient cycling is affected by light.

Effects of eutrophication on the planktonic food web dynamics of marine coastal ecosystems: The case study of two tropical inlets.

We studied the plankton dynamics of two semi-enclosed marine coastal inlets of the north of Jurong Island separated by a causeway (SW Singapore; May 2012-April 2013). The west side of the causeway (west station) has residence times of ca. one year and is markedly eutrophic. The east side (east station) has residence times of one month and presents lower nutrient concentrations throughout the year. The higher nutrient concentrations at the west station did not translate into significantly higher concentrations of chlorophyll a, with the exception of some peaks at the end of the South West Monsoon. Microzooplankton were more abundant at the west station. The west station exhibited more variable abundances of copepods during the year than did the east station, which showed a more stable pattern and higher diversity. Despite the higher nutrient concentrations at the west station (never limiting phytoplankton growth), the instantaneous phytoplankton growth rates there were generally lower than at the east station. The phytoplankton communities at the west station were top-down controlled, largely by microzooplankton grazing, whereas those of the east station alternated between top-down and bottom-up control, with mesozooplankton being the major grazers. Overall, the trophic transfer efficiency from nutrients to mesozooplankton in the eutrophic west station was less efficient than in the east station, but this was mostly because a poor use of inorganic nutrients by phytoplankton rather than an inefficient trophic transfer of carbon. Some hypotheses explaining this result are discussed.

Functional ecology of aquatic phagotrophic protists - Concepts, limitations, and perspectives.

Functional ecology is a subdiscipline that aims to enable a mechanistic understanding of patterns and processes from the organismic to the ecosystem level. This paper addresses some main aspects of the process-oriented current knowledge on phagotrophic, i.e. heterotrophic and mixotrophic, protists in aquatic food webs. This is not an exhaustive review; rather, we focus on conceptual issues, in particular on the numerical and functional response of these organisms. We discuss the evolution of concepts and define parameters to evaluate predator-prey dynamics ranging from Lotka-Volterra to the Independent Response Model. Since protists have extremely versatile feeding modes, we explore if there are systematic differences related to their taxonomic affiliation and life strategies. We differentiate between intrinsic factors (nutritional history, acclimatisation) and extrinsic factors (temperature, food, turbulence) affecting feeding, growth, and survival of protist populations. We briefly consider intraspecific variability of some key parameters and constraints inherent in laboratory microcosm experiments. We then upscale the significance of phagotrophic protists in food webs to the ocean level. Finally, we discuss limitations of the mechanistic understanding of protist functional ecology resulting from principal unpredictability of nonlinear dynamics. We conclude by defining open questions and identifying perspectives for future research on functional ecology of aquatic phagotrophic protists.

Non-proportional bioaccumulation of trace metals and metalloids in the planktonic food web of two Singapore coastal marine inlets with contrasting water residence times.

We analyzed the concentrations of trace metals/metalloids (TMs) in the water, sediment and plankton of two semi-enclosed marine coastal inlets located north of Jurong Island and separated by a causeway (SW Singapore; May 2012-April 2013). The west side of the causeway (west station) has residence times of approximately one year, and the east side of the causeway (east station) has residence times of one month. The concentrations of most of the TMs in water and sediment were higher in the west than in the east station. In the water column, most of the TMs were homogeneously distributed or had higher concentrations at the surface. Preliminary evidence suggests that the TMs are primarily derived from aerosol depositions from oil combustion and industry. Analyses of TMs in seston (>0.7µm; mostly phytoplankton) and zooplankton (>100µm) revealed that the seston from the west station had higher concentrations of most TMs; however, the concentrations of TMs in zooplankton were similar at the two stations. Despite the high levels of TMs in water, sediment and seston, the bioaccumulation detected in zooplankton was moderate, suggesting either the presence of effective detoxification mechanisms or/and the inefficient transfer of TMs from primary producers to higher trophic levels as a result of the complexity of marine planktonic food webs. In summary, the TM concentrations in water and seston are not reliable indicators of the bioaccumulation at higher trophic levels of the food web.