Experimental protocol

2.3.1. Animals. Copepods were collected using a 250 µm, 1 m diameter plankton net with a non-filtering cod-end, hauled vertically from 100 m. The contents of the cod-end were gently poured into a 20 l bucket of fresh surface sea

Figure 2.1 Map of the study area (Irminger Basin) with cruise tracks from D262

shown in red and D264 shown in yellow. Scale on right hand side indicates

Figure 2.2. Locations of water sampling stations along the Reykjanes Ridge

in April (black circles) and July/August (red triangles). Number denotes

water (from non-toxic supply). Female C. finmarchicus for the feeding incubations were sorted into groups of ten under the dissection microscope using a wide-bore pipette. All animals were individually inspected to ensure that the antennules and sensilla were intact and that they were free from parasites.

The biochemistry of the animals at the start of the experiments (initial animals) was determined by sorting 5 replicate groups of 5 females from the same sample for C/N and fatty acid analysis. Upon termination of the 5 day incubation period, the animals from each experimental bottle were split into two groups: 5 for elemental and 5 for fatty acid analysis (final animals). Animals for C/N analysis were stored in tin cups, whilst those for fatty acid analysis were placed into 1.1 ml screw- capped, Teflon septum vials and completely filled with solvent (Chloroform:Methanol 2:1 v/v) before storage. All animals for biochemical investigation were stored at –80º until analysis.

2.3.2. Collection of water. Water containing the natural microplankton

assemblage from the chlorophyll a maximum (located by examining the downwards

fluorescence profile of the CTD cast) was collected in 10 l Niskin bottles with Teflon fittings. Seawater from the non-toxic supply (pumped by means of an impellor from ~5 m below the surface) was used only when the weather was too bad to deploy the CTD rosette. The water was gently screened with a submerged 90 µm mesh to remove other copepods, then carefully transferred via silicone tubing into the 2200 ml clear glass incubation bottles. Each bottle was filled a little at a time to ensure maximum homogeneity between bottles. All incubation bottles (experimental and control) were topped up with the screened seawater and sealed with clingfilm to remove air bubbles.

2.3.3. Incubation protocol. Bottles containing the screened microplankton

assemblage and 10 female C. finmarchicus copepods are referred to as experimental

bottles. Those containing only the microplankton assemblage without copepods are referred to as control bottles. Feeding rates were quantified by incubating 6 experimental bottles alongside 4 controls for 24 hrs on a water-cooled plankton

wheel illuminated by natural light at the in situ photoperiod. The water was sampled

at the start and end of each incubation for ‘initial’ and ‘final’ particulates (Section 2.3.5.). After each 24 h incubation period, the copepods were carefully transferred

via a dip-tube to bottles of fresh, screened seawater from the chlorophyll a maximum

consecutive days, and appeared intact and healthy upon termination of the experiments.

2.3.4. Eggs. Eggs and faecal pellets were removed from the experimental

bottles at the end of each incubation by gentle filtration (63 µm). Control bottles

were treated correspondingly. The water was then sampled for ‘final’ particulates (Section 2.3.5.). The eggs from each experimental bottle were counted under a dissection microscope. Half of the total eggs produced each day were stored on a pre- combusted GF/F filter (12 h in muffle furnace at 500 ºC to remove any organic

contamination: Feely and members of the working group, 1991) for elemental

analysis. The remainder were stored in a 1.1 ml screw-capped, Teflon septum vial filled with solvent (Chloroform:Methanol 2:1 v/v) for fatty acid analysis. All egg samples were stored at –80º.

2.3.5. Particulate sampling. To determine how the composition, abundance and biochemistry of the microplankton community changed during the 24 hr incubations and thus the quantity and quality of food consumed by the copepods, a suite of samples were taken from the water at the beginning and end of the incubations. ‘Initial’ samples refer to those taken from the screened in the incubation bottles at the start of the incubation period. ‘Final’ samples refer to those taken from the experimental and control bottles following the removal of eggs and faecal pellets at the end of the daily incubation period. The initial microplankton sample was a single 200 ml aliquot. Final microplankton samples were 100 ml aliquots taken from each of the experimental and control bottles, following the removal of eggs. All microplankton samples were preserved with 10 % acid Lugol’s solution (see Appendix 1) and stored in amber medicine bottles in the dark until analysis.

A particulate sample for biochemical analysis (C/N or lipid) consisted of the particulate matter from 1000 ml of water, collected on a pre-combusted GF/F filter under gentle vacuum. Triplicate initial samples for C/N and fatty acid analyses were taken at the start of each day. Following the removal of the final microplankton sample at the end of each incubation period, each bottle yielded a single, final particulate sample for C/N and fatty acid analyses.

Because of the sensitive nature of the elemental and fatty acid analyses, filters were only handled using clean stainless steel forceps (Ehrhardt and Koeve 1999). Those for fatty acid analysis were folded in half, then in half again, slotted into 2 ml screw-capped, Teflon septum vials and completely filled with solvent

(Chloroform:Methanol 2:1 v/v). Filters for C/N analysis were folded in half (sample side inwards). All particulate samples were stored in individual labelled polythene zip-lock bags and maintained at –80 ºC.