Copepod ecophysiology experiments

The SZN sampling and analysis strategy was as follows (samples were taken at N1,N2,N3,N4, S1,S2,S3,S4, E1,E2,E3,E4 and SE01,SE02,SE03 SE04 stations):

• For egg production and hatching success and RNA extraction for stress gene analyses, zooplankton was collected from the bottom to the surface with a Nansen net (200 $\mu$m mesh size); at each station, 10 Calanus helgolandicus females were sorted from samples and incubated in 50 ml bottles with natural sea water phytoplankton; after 24 h females were removed and eggs counted, after another 24 h the number of hatched eggs was determined; at each station, 30 females were incubated in filtered sea water for 24 h and then fixed with Trizol (Invitrogen) and stored in liquid nitrogen;
• Phytoplankton was sampled at each station with a 30 $\mu$m net ( 40 cm diameter)by towing at the surface for ten minutes; samples were centrifuged at 3500 rpm for 3 minutes and pellets were stored in liquid nitrogen; each pellet will be analyzed for chemical costituents such as polyunsaturated aldehydes, epoxyalcohols, hydroxyacids and other oxylipins.

The 200 $\mu$m Nansen net sample was used to quantify the number of Calanus helgolandicus females, males and juveniles in order to estimate secondary production rates at the time for this important copepod species in the Adriatic Sea. Egg production and egg hatching success data will further allow the estimation of recruitment rates at each station in this period which generally coincides with the period of maximum population numbers for this species. To verify the possibility of using fluorescent markers to quantify live/dead eggs thereby avoiding cumbersome egg hatching success experiments that require 48h onboard ship incubations, 100 Calanus eggs were also collected at several stations and fixed in 4% formaldehyde for further Comet-assay analysis in the laboratory. This is a sensitive technique for the detection of DNA damage at the level of the individual cell and is a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. The test has rarely been used for copepods before and SZN is currently developing this method in the laboratory, together with new methods for stress gene analyses in copepods, particularly in C. helgolandicus. Frozen samples (adult females and males) collected at different stations during the cruise will be evaluated for up- or down-regulation of the following genes: Cytochrome P450, Glutathione S-transferase, Glutathione Synthase, Aldehyde Dehydrogenases, Catalase, Superoxide Dismutase, Alpha and Beta Tubulins, Inhibitor of Apoptosis Protein (IAP), Cell Cycle and Apoptosis Regulatory Protein (CARP), Cellular Apoptosis Susceptibility Protein (CAS). These results will allow to determine whether copepods were more "stressed" at some stations rather than others, and to identify the possible causes leading to reduced fitness in C. helgolandicus. Previous studies in SZN laboratory have shown that diatoms produce a number of toxic oxylipins deriving from the oxidation of polyunsaturated fatty acids which reduce copepod egg hatching success and later larval recruitment (reviewed by IANORA2010). Since the period of the cruise coincided with the end of a Skeletonema marinoi diatom bloom the objective of the studies was to evaluate oxylipin production at different stations and possible impacts on the C. helgolandicus population (e.g. reduced egg hatching success and population recruitment rates, down-regulation of specific stress genes).

Figure 13: ISMAR SW104 Water/sediment corers, long and short pipes.