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 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.

Cephalopod and vertebrate neural-systems are often highlighted as a traditional example of convergent evolution. Their large brains, relative to body size, and complexity of sensory-motor systems and behavioral repertoires offer opportunities for comparative analysis. Despite various attempts, questions on how cephalopod ‘brains’ evolved and to what extent it is possible to identify a vertebrate-equivalence, assuming it exists, remain unanswered. Here, we summarize recent molecular, anatomical and developmental data to explore certain features in the neural organization of cephalopods and vertebrates to investigate to what extent an evolutionary convergence is likely. Furthermore, and based on whole body and brain axes as defined in early-stage embryos using the expression patterns of homeodomain-containing transcription factors and axonal tractography, we describe a critical analysis of cephalopod neural systems showing similarities to the cerebral cortex, thalamus, basal ganglia, midbrain, cerebellum, hypothalamus, brain stem, and spinal cord of vertebrates. Our overall aim is to promote and facilitate further, hypothesis-driven, studies of cephalopod neural systems evolution.

No abstract

The study of molluscan immune systems, and in particular those of bivalve molluscs (clams, oysters, scallops, mussels, etc.) has experienced great growth in recent decades, mainly due to the needs of a rapidly growing aquaculture industry to manage the impacts of disease and the application of -omic tools to this diverse group of invertebrate organisms. Several unique aspects of molluscan immune systems highlighted in this chapter include the importance of feeding behavior and mucosal immunity, the discovery of unique levels of diversity in immune genes, and experimental indication of transgenerational immune priming. The development of comparative functional studies using natural and selectively bred disease-resistant strains, together with the potential but yet to be fully developed application of gene-editing technologies, should provide exciting insights into the functional relevance of immune gene family expansion and molecular diversification in bivalves. Other areas of bivalve immunity that deserve further study include elucidation of the process of hematopoiesis, the molecular characterization of hemocyte subpopulations, and the genetic and molecular mechanisms underlying immune priming. While the most important aspects of the immune system of the largest group of molluscs, gastropods (e.g., snails and slugs), are discussed in detail in Chap. 12, we also briefly outline the most distinctive features of the immune system of another fascinating group of marine molluscs, cephalopods, which include invertebrate animals with extraordinary morphological and behavioral complexity.