1. Predation rates

2. From a trophic point of view, microzooplankton plays a fundamental role as principal carrier of energy from the primary producers to the upper trophic levels as it is the foremost predator of nanoplankton (2 – 20 m) and picoplankton (0.2 – 2 m) in the “Microbial loop”, as well as consumer of microphytoplankton and prey of mesozooplankton in the “Classic food web”. Microzooplankton is important in mesozooplankton diet, providing an essential food supply, but the reported contribution of microzooplankton to mesozooplankton carbon ration is very variable. Trophic role of microzooplankton

3. Grazing assessment The main problem to estimate grazing impact of micro- zooplankton (and heterotrophic nanoplankton - HNAN) on their prey is that prey is in the same dimensional range of predators and thus it is impossible to separate them. The dilution method, as introduced by Landry & Hasset in 1982, has become very popular over the last ten years and is now considered a standard protocol for the estimation of micro- zooplankton and HNAN herbivory and bacterivory. Besides the chlorophyll concentration classic method, HPL measurements were used to estimate taxon- or pigment– specific mortality rates, as well as flow cytometry, microscopical counts of micro- and nano-prey, epifluorescent microscopical enumeration of bacteria. Relatively few studies investigated the composition of the predator communities despite several authors pointed out at the relevance of this kind of investigations.

4. The dilution method The main advantage of the dilution method is that it does not require any manipulation of the thin organisms involved in the experiment. The dilution approach relies on the reduction of encounter rates between prey and their grazers. Natural water samples are amended with varying proportions of filtered seawater creating a dilution series, and grazing rate is estimated as the increase in apparent prey growth rate with dilution factor. Predators grazing rate is estimated as the slope of a regression of apparent prey growth in the various dilutions against dilution factor. Growth rate of the prey is estimated as apparent growth rate extrapolated to 100 % dilution (growth in the absence of grazers).

5. The preceding theoretical development involves three restrictive assumptions. First, the exponential model is assumed to apply in the broad sense. This allows that growth and mortality rates may vary on short time scales but provides a framework for computing average rates over incubation periods on the order of a day. Second, the mean specific growth rate of the prey is assumed to be constant, density independent. To satisfy this assumption, dissolved nutrients must remain non-limiting, or equally limiting, to growth at all dilutions during the experimental incubation. Third, the average clearance rate of individual consumer is assumed to be constant at all dilutions; ingestion rates of the grazers are directly related to the prey density. Growth rates of microphytoplankton and microzooplankton grazing rates were calculated from regression of apparent growth against dilution factor.

6. The growth of the prey is described by: C t = C 0 e (k–g)t or (1/t) ln (Ct/C 0 )= k-g Where C 0 is the carbon concentration of the prey at the beginning, C t the C concentration at the end of the experiment (time t), k the apparent growth coefficient, and g the grazing coefficient. Production (P) and ingestion (I) can be calculated as P = k*C m, and I = g*C m where C m is the average carbon concentration during the incubation. The apparent grow rate coefficient (1/t) ln(Ct/C 0 ) are on ordinate axis, while the dilution rates are on abscissa. The intercept of the regression line with ordinate axis, where g=0 represents the prey instantaneous growth rate; the regression line slope is the negative value of grazing instantaneous rate “g”.

7. Water is collected by means of Niskin bottles and immediately and kindly filtered through a 200 m mesh size net, and pored in large clean polyethylene bottles (20 l) kept at the temperature of the collection depth, usually near or at the surface. Bottles must be carefully cleaned and sterilized before using

8. Then part of the water is filtered through 0.22 m to obtained water free from any organisms and added in different percentage to “whole” water.

9. Dilution experiments: 5 concentrations (3 replicates) Start of incubation (Time zero) End of incubation (Time t = 24 h) Parameters observed in each replicates: autotrophic community (pico-, nano- and micro- phytoplankton) heterotrophic community (bacteria, heterotrophic nanoplankton, microzooplankton)

10. Bottles are incubated on the deck in flowing sea water to mime natural environment for 24 h

12. - -

13. By measuring each component we were able to recognize specific grazing rate on each taxonomic group or even species. In this case also on total phytoplankton we observed a significant grazing impact, but not on dinoflagellates. If dinoflagellates are the dominant taxa, if we use chl a only we probably don’t see any grazing effect.

14. Bacterial mortality induced by microzoo- and HNAN grazing Bacterial mortality induced by HNAN grazing We can assess the grazing impact on bacteria by the only heterotrophic nanoplankton fraction by filtering water through a 10 m mesh to eliminate nanoplankton consumers

16. Secondary production of microzooplankton and HNAN can be assessed by counting their abundance at the beginning and at the end of the dilution experiment. If their biomasses changed over the incubation period we must calculate the average abundance. In this case HNAN always increased, while microzooplankton only in August.

17. Ingestion efficiency

20. FWE = ratio between the production of primary producers and top predator production Surface - Microzooplankton top consumers Oligotrophic conditions = 0.03 Meso-eutrophic conditions = 0.10 Eutrophied conditions = 0.01 Meso-bathypelagic realm – Nanoplankton top consumers FEW = 0.13

21. Mesozooplankton grazing There are several methods to estimate mesozooplankton grazing rates (e.g. gut fluorescence, 14 C labeled algae, pigment analysis by High Performance Liquid Cromatography (HPLC), or dual labeling technique), but the most reliable method to quantify feeding rates also on non-pigmented taxa is the analysis of particle removal in bottle incubations. However, this method may still present some problems, besides the obvious bottle effect, because of interferences with microzooplankton feeding activity on the same prey. To overcome this problem it is necessary to simultaneously estimate the microzooplankton grazing rates in separate dilution experiments.

22. Grazing experiment 3 bottles filled with 2 l of filtered (200 µm) sea water The same + 20 copepods (cladocerans) in each bottle 24 h of incubation at environmental light and temperature Acartia clausi Penilia avirostris

23. A. clausi was the dominant organism in 3 of the 4 seasons, P. avirostris prevailed in summer. Knowing their C content we extrapolated grazing rates to the whole mesozooplankton community by multiplying for the total mesozooplankton C content.

24. 1226

25. Mesozooplankton diet is mostly constituted by diatoms

26. Percentage on total mesozooplankton diet constituted by microzooplankton. Microzooplankton is a source for mesozooplankton when diatom availability decreases

27. 1246 Total removal on autotrophic fraction

29. Holling curve model 3 Raw r 2 = 0.946 Mean correct r 2 = 0.889 It appears that there is a threshold around 30 gCl -1 and a food saturation around 300 gCl -.