Guidelines for the Care and Welfare of Cephalopods in Research -A consensus based on an initiative by CephRes, FELASA and the Boyd Group.

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.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

First report of a successful treatment of a mucodegenerative disease in the chambered nautilus (Nautilus pompilius).

A single Nautilus pompilius manifested a bacterial infection and nematode infestation soon after it was received from the wild, resulting in a significant buildup of mucus above the left eye and tentacles. This condition is known to lead to rapid mucodegeneration of the tentacles and epithelium, resulting in death. The specimen was quarantined 24 days after arrival. Initial topical treatments of 10% povidone solution were effective at slowing the progression of the mucus but did not eliminate it. After 26 days in quarantine, a new treatment regimen was developed that coupled a whole-animal dip in 25 mg/L oxytetracycline solution with the 10% povidone treatment on alternate days, for 5 days. After this treatment, mucus production ceased and nematodes were not present in tissue samples. The specimen was moved back to the original holding system after a quarantine period of 53 days.

The evolution of flexible behavioral repertoires in cephalopod molluscs.

Cephalopods are a large and ancient group of marine animals with complex brains. Forms extant today are equipped with brains, sensors, and effectors that allow them not to just exist beside modern vertebrates as predators and prey; they compete fiercely with marine vertebrates at every scale from small crustaceans to sperm whales. We review the evolution of this group's brains, learning ability and complex behavior. We outline evidence that although competition with vertebrates has left a deep impression on the brains and behavior of cephalopods, the original reorganization of their complex brains from their molluscan ancestors might have been forged in ancient seas millions of years before the advent of bony fishes.

Memory of visual and topographical features suggests spatial learning in nautilus (Nautilus pompilius L.).

Previous studies demonstrate that soft-bodied (coleoid) cephalopods are adept at learning and remembering features of their environment, but little is known about their primitive relative, nautilus. Nautilus makes nightly migrations from deep to shallow water along coral reef slopes, covering large areas of varied substrate. Memory of its surroundings may be advantageous, but the nautilus brain is the simplest among extant cephalopods, lacking dedicated neural regions that support learning and memory in other cephalopods. The authors hypothesize that the absence of these regions in nautilus may affect memory storage. Here the authors report the first evidence for spatial memory in 2- and 3-dimensional arenas. In a small open-field maze, nautiluses learned the location of a goal within 3 trials, and memory was stable for at least 2 weeks. In 3-dimensional environments, animals habituated within and across trials when their surroundings were unchanged, but activity increased when the environment changed topographically, although not when the change was visual only. These results are comparable to performances of coleoids in similar tasks and are surprising given the far simpler neuroanatomy of nautilus.

A biphasic memory curve in the chambered nautilus, Nautilus pompilius L. (Cephalopoda: Nautiloidea).

Cephalopods are an exceptional taxon for examining the competing influences of ecology and evolutionary history on brain and behaviour. Coleoid cephalopods (octopuses, cuttlefishes and squids) have evolved specialised brains containing dedicated learning and memory centres, and rely on plastic behaviours to hunt prey effectively and communicate intricate visual displays. Their closest living relative, the primitive nautilus, is the sole remnant of an ancient lineage that has persisted since the Cambrian. Nautilus brains are the simplest among the extant cephalopods, and the absence of dedicated learning and memory regions may represent an ancestral condition. It is assumed that the absence of these regions should limit memory storage and recall in nautilus, but this assumption has never been tested. Here we describe the first evidence of learning and memory in chambered nautilus (Nautilus pompilius). Using a Pavlovian conditioning paradigm, we demonstrate that chambered nautilus exhibits temporally separated short- and long-term memory stores, producing a characteristic biphasic memory curve similar to that of cuttlefishes. Short-term memory persisted for less than 1 h post-training, whereas long-term memory was expressed between 6 and 24 h after training. Despite lacking the dedicated neural regions that support learning and memory in all other extant cephalopods, nautilus expressed a similar memory profile to coleoids. Thus the absence of these regions in the nautilus brain does not appear to limit memory expression, as hypothesised. Our results provide valuable insights into the evolution of neural structures supporting memory.

A role for nautilus in studies of the evolution of brain and behavior.

Nautilus is an ancient remnant of a largely extinct cephalopod lineage.1 Its status within its clade is the subject of ongoing debate-its morphology, behavior and neuroanatomy may or may not be representative of an ancestral condition, and therefore its value as a model for ancestral cephalopods is uncertain. While the nautilus brain is simpler than that of more derived cephalopods2 (coleoids), it is plausible that this is a secondary simplification related to ecology, and not a precursor to the vertebrate-like CNS of modern cephalopods. However, the absence of the vertical lobe complex, implicated in learning and memory in coleoids, makes studies of cognition in nautilus particularly interesting from a comparative perspective. Our research on the behavior and sensory biology of Nautilus pompilius gives the first indications of learning and memory in this ancient genus,3 and suggests that even with a far simpler brain containing no clearly defined "memory" center, nautilus performs simple cognitive tasks comparably to its more derived relatives.

How lobsters, crayfishes, and crabs locate sources of odor: current perspectives and future directions.

Olfactory orientation poses many challenges for crustaceans in marine environments. Recent behavioral experiments lead to a new understanding of the role of multiple sensory appendages, whereas application of non-invasive chemical visualization techniques and biomimetic robotics have allowed researchers to correlate the stimulus environment with behavior and to directly test proposed orientation mechanisms in decapod crustaceans.