This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.

The diffusive boundary layers surrounding sessile marine organisms have been implicated in controlling an organism's metabolism and growth. We studied boundary layers surrounding hermatypic corals by monitoring oxygen concentrations on a submillimetric scale. Oxygen concentration within the boundary layers varied from supersaturation during the day to anoxia at night, although the ambient water composition remained constant. Detailed mapping and oxygen measurements revealed diel oxygen fluctuations from supersaturation (373% air saturation) in the light to complete oxygen depletion at darkness in the massive coral Favia favus. Exposure to a 5-cm/s current reduced the boundary layer thickness from 2.44 mm to 1.90 mm, allowing more rapid oxygen exchange across the diffusive boundary layer. Similar patterns were found in the branching coral Stylophora pistillata. In massive corals, the thickness of the diffusive boundary layer was negatively correlated with the size of the polyp. We suggest that the distribution of corals in areas of differential turbulence is related to the thickness of the diffusive boundary layers surrounding them.

The hermatypic coral Stylophora pistillata has a wide bathymetric distribution (0 to 70 m). Within this range, light intensity decreases exponentially. Deep-water colonies are generally planar in morphology, with the upper part being dark and the bottom-facing part pale. Shallow-water colonies are generally subspherical and ivory in coloration. We studied the effects of photoacclimation on photosynthesis, respiration, and calcification in S. pistillata colonies along its bathymetric range over a reef profile (5 to 65 m) in Eilat, Gulf of Aqaba, Red Sea, during winter and summer, using a submersible respirometer. Respiration rate, light-saturated rate of photosynthesis (P max ), compensation light intensity (E c ), and light intensity of incipient saturation (E k ), all decreased with depth. In contrast, the efficiency of photosynthesis (α) increased with depth. All colonies displayed 'lightenhanced calcification' during daytime and decreasing calcification rates with depth. These results indicate an adjustment in harvesting and utilization of light by the algal symbionts to the light environment. At all light intensities except the lowest ones, there was a consistent ratio of calcification to photosynthesis, in agreement with the concept of light-enhanced calcification. In the deepest, lowlight corals, there was no evidence for support of calcification by photosynthesis, and we assume that these colonies subsist mainly by preying on zooplankton.

Nitrogen fixation, as measured by acetylene reduct~on, has been detected to be associated with various hermatypic corals. Experiments were carried out on the massive coral Favia f a v u s both in situ and in the laboratory. Nitrogen fixation activity was found to be light dependent and fully inhibited by 5 X 10-6 M DCMU [3-(3,4-dichloropheny1)-1,l-dimethylurea Addition of glucose restored nitrogen fixation activity both in the dark and in the presence of DCMU. Removal of the coral tissue prevented acetylene reduction, while addition of glucose to the coral skeleton restored this activity. Bacteria isolated from the coral skeleton were found by dot blotting to contain the nif H gene. These results suggest that nitrogen-fixing bacteria found in the skeleton of corals benefit from organic carbon excreted by the coral tissue. The interaction between the nitrogen-fixing organisms and the coral may be of major lrnportance for the nitrogen budget of the corals.

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