Giovanna Ponte

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.

Non-invasive study of Octopus vulgaris arm morphology using ultrasound

SUMMARY Octopus arms are extremely dexterous structures. The special arrangements of the muscle fibers and nerve cord allow a rich variety of complex and fine movements under neural control. Historically, the arm structure has been investigated using traditional comparative morphological ex vivo analysis. Here, we employed ultrasound imaging, for the first time, to explore in vivo the arms of the cephalopod mollusc Octopus vulgaris. Sonographic examination (linear transducer, 18 MHz) was carried out in anesthetized animals along the three anatomical planes: transverse, sagittal and horizontal. Images of the arm were comparable to the corresponding histological sections. We were able, in a non-invasive way, to measure the dimensions of the arm and its internal structures such as muscle bundles and neural components. In addition, we evaluated echo intensity signals as an expression of the difference in the muscular organization of the tissues examined (i.e. transverse versus longitudinal muscles), finding different reflectivity based on different arrangements of fibers and their intimate relationship with other tissues. In contrast to classical preparative procedures, ultrasound imaging can provide rapid, destruction-free access to morphological data from numerous specimens, thus extending the range of techniques available for comparative studies of invertebrate morphology.

Learning and memory in Octopus vulgaris: a case of biological plasticity.

Here we concisely summarize major aspects of the learning capabilities of the cephalopod mollusc Octopus vulgaris, a solitary living marine invertebrate. We aim to provide a backdrop against which neurobiology of these animals can be further interpreted and thus soliciting further interest for one of the most advanced members of invertebrate animals.

Mechanisms of wound closure following acute arm injury in Octopus vulgaris

BackgroundOctopoda utilise their arms for a diverse range of functions, including locomotion, hunting, defence, exploration, reproduction, and grooming. However the natural environment contains numerous threats to the integrity of arms, including predators and prey during capture. Impressively, octopoda are able to close open wounds in an aquatic environment and can fully regenerate arms. The regrowth phase of cephalopod arm regeneration has been grossly described; however, there is little information about the acute local response that occurs following an amputation injury comparable to that which frequently occurs in the wild.MethodsAdult Octopus vulgaris caught in the Bay of Naples were anaesthetised, the distal 10 % of an arm was surgically amputated, and wounded tissue was harvested from animals sacrificed at 2, 6, and 24 h post-amputation. The extent of wound closure was quantified, and the cell and tissue dynamics were observed histologically, by electron microscopy, as well as using ultrasound.ResultsMacroscopic, ultrasonic and ultrastructural analyses showed extensive and significant contraction of the wound margins from the earliest time-point, evidenced by tissue puckering. By 6 h post amputation, the wound was 64.0 ± 17.2 % closed compared to 0 h wound area. Wound edge epithelial cells were also seen to be migrating over the wound bed, thus contributing to tissue repair. Temporary protection of the exposed tip in the form of a cellular, non-mucus plug was observed, and cell death was apparent within two hours of injury. At earlier time-points this was apparent in the skin and deeper muscle layers, but ultimately extended to the nerve cord by 24 h.ConclusionsThis work has revealed that O. vulgaris ecologically relevant amputation wounds are rapidly repaired via numerous mechanisms that are evolutionarily conserved. The findings provide insights into the early processes of repair preparatory to regeneration. The presence of epithelial, chromatophore, vascular, muscle and neural tissue in the arms makes this a particularly interesting system in which to study acute responses to injury and subsequent regeneration.

The use of artificial crabs for testing predatory behavior and health in the octopus.

The willingness of the cephalopod mollusc Octopus vulgaris to attack a live crab is traditionally used as a method to assess the overall health and welfare of octopuses in the laboratory. This method requires placing a crab in the home tank of an animal, measuring the time (latency) taken for the octopus to initiate an attack and withdrawing the crab immediately prior to capture. The same crab is commonly used to assess multiple octopuses as part of daily welfare assessment. Growing concern for the welfare of crustaceans and a review of all laboratory practices for the care and welfare of cephalopods following the inclusion of this taxon in 2010/63/EU prompted a study of the utility of an artificial crab to replace a live crab in the assessment of octopus health. On consecutive days O. vulgaris (N=21) were presented with a live, a dead or an artificial crab, and the latency to attack measured. Despite differences in the predatory performance towards the three different crab alternatives, octopuses readily attacked the artificial (and the dead) crab, showing that they can generalize and respond appropriately towards artificial prey. Researchers should consider using an artificial crab to replace the use of a live crab as part of the routine health assessment of O. vulgaris.

Fostering cephalopod biology research: past and current trends and topics

For the first time, European Union legislation on animal research and testing has extended its remit to include invertebrate species. The class Cephalopoda, represented by more than 700 living species, is the first invertebrate that has been selected in the list of animals considered in the EU Directive 2010/63/EU; indeed, the ‘sole’ representative among more than 30 other living invertebrate animal phyla. The Directive covers ‘all live cephalopods’ used in scientific and training procedures that are likely to cause to animals adverse effects such as ‘pain, suffering, distress or lasting harm’ (European Parliament & Council of the European Union 2010). Directive 2010/63/EU has been currently transposed in 14 (out of 27) Member States (http:// ec.europa.eu/environment/chemicals/lab_animals/transposi tion_en.htm) and will impact research in Europe and beyond. Studies of many invertebrates have assisted in the development of major concepts in neuroscience. Cephalopods and other invertebrate animals have been deployed very successfully in experimental studies of the nervous system and other aspects of biology. ‘Study of cephalopods at marine laboratories has provided material for some of the outstanding discoveries of neuroscience in this century. The giant nerve fibers are the most conspicuous example, but studies of photoreceptors and the memory mechanisms of the brain have been very fruitful, as has work on chromatophores and many other topics’ (Young 1985, p. 153). As pointed out by Professor J.Z. Young, many important discoveries on physiological, cellular and behavioural properties common to the animal kingdom have been achieved through investigations of these diversified organisms. In many instances, the advantages of use of invertebrate as ‘model organisms’ for investigations of nervous systems have been underlined (e.g.: Sattelle and Buckingham 2006; Clarac and Pearlstein 2007). We are committed to facilitating and promoting the sharing of methods and knowledge in a scientific community not restricted to cephalopod workers. We believe that this epochal change in the use of cephalopods in research requires the highest attention and support. As the first of various steps, the no-profit research organization CephRes facilitated meetings and the collection of recent reviews and research publications into coordinated ‘special issues’ in order to depict examples of the current research effort on cephalopod biology. To achieve this goal, a collection of abstracts (Fiorito 2011; EuroCeph 2011 and CephRes 2011) and of 20 contributions collated in a special issue on cephalopod biology (Ponte and Fiorito 2013) have been produced with the help of notable scientific journals. The special issue appearing in Journal of Experimental Marine Biology and Ecology brings together a range of recent studies from diverse areas of cephalopod research. The growing concern to the welfare of invertebrates, and on cephalopods in particular (Andrews 2011a, b; Andrews et al. 2013; Smith et al. 2013), requires a systematic analysis of the impact and guidance on maintenance and use in research of cephalopods. The cephalopod community is working hard, in a coordinated way to keep elevated standards on this aspect, and contribute to the development of This article forms part of a special issue on Cephalopod Biology under the auspices of CephRes-ONLUS (www.cephalopodresearch. org); Guest Editor: Graziano Fiorito.

Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope

The common octopus, or Octopus vulgaris, has the largest nervous system of any invertebrate, and has been shown to possess learning and memory capabilities that in many ways rival those of some vertebrates [1]. Nevertheless, the neural architecture of this cephalopod mollusk differs markedly from that of any vertebrate. Investigating the differences and similarities between the neural architecture—or connectome—of the octopus and mammals, such as the mouse, may lead to deep insights into the computational principles underlying animal cognition. The octopus brain provides some unique advantages for anatomical research, since its axons are generally thick and unmyelinated, allowing traditional staining methods, such as Golgi, to be used effectively. With this in mind, we first imaged the brain using the Knife-Edge Scanning Microscope [2], a custom serial sectioning microscope that can image large blocks of tissue (1 cm3) at sub-micrometer resolution. We imaged large portions of the octopus subesophageal mass (SUB) and the optic lobe (OL) which were stained using Golgi. In order to extract the geometry of the neuronal morphology, we used our Maximum Intensity Projection (MIP)-based tracing algorithm [3]. The imaging results are shown in Figure 1(a-d), and tracing results are shown in 1(e). Although quite preliminary, to our knowledge this is the first time large volumes of the octopus brain have been imaged at sub-micrometer resolution, allowing us to resolve many of the processes that make up the neural network. We expect that this pilot study and the more detailed investigations to follow will allow fruitful comparisons of the neural circuitries of individual octopuses with different ecological life histories, as well as of animals that have been exposed to a variety of neurodegenerative insults. Moreover, such explorations will engender a greater understanding of how functional neural architecture is altered by learning in invertebrates such as the octopus and vertebrates such as the mouse. In sum, this approach should contribute greatly to our understanding of the computational architecture of invertebrates and ultimately provide insights into the differences between invertebrate and vertebrate cognitive capabilities. Figure 1 Octopus subesophageal mass (SUB) and optic lobe (OL) imaged with the KESM (a–d), and tracing results (e). Scale (block width): (a) 1.44 mm, (b) 0.72 mm, (c) 1.44 mm, (d-f) 76.8 μm. Voxel resolution: 0.6 μm x 0.7 μm x 1.0 ...

Salivary Glands in Predatory Mollusks: Evolutionary Considerations

Many marine mollusks attain or increase their predatory efficiency using complex chemical secretions, which are often produced and delivered through specialized anatomical structures of the foregut. The secretions produced in venom glands of Conus snails and allies have been extensively studied, revealing an amazing chemical diversity of small, highly constrained neuropeptides, whose characterization led to significant pharmacological developments. Conversely, salivary glands, the other main secretory structures of molluscan foregut, have been neglected despite their shared occurrence in the two lineages including predatory members: Gastropoda and Cephalopoda. Over the last few years, the interest for the chemistry of salivary mixtures increased based on their potential biomedical applications. Recent investigation with -omics technologies are complementing the classical biochemical descriptions, that date back to the 1950s, highlighting the high level of diversification of salivary secretions in predatory mollusks, and suggesting they can be regarded as a pharmaceutical cornucopia. As with other animal venoms, some of the salivary toxins are reported to target, for example, sodium and/or potassium ion channels or receptors and transporters for neurotransmitters such as, glutamate, serotonin, neurotensin, and noradrenaline, thus manipulating the neuromuscular system of the preys. Other bioactive components possess anticoagulant, anesthetic and hypotensive activities. Here, we overview available knowledge on the salivary glands of key predatory molluscan taxa, gastropods, and cephalopods, summarizing their anatomical, physiological and biochemical complexity in order to facilitate future comparative studies on main evolutionary trends and functional convergence in the acquisition of successful predatory strategies.

The Digestive Tract of Cephalopods: Toward Non-invasive In vivo Monitoring of Its Physiology

Ensuring the health and welfare of animals in research is paramount, and the normal functioning of the digestive tract is essential for both. Here we critically assess non- or minimally-invasive techniques which may be used to assess a cephalopods digestive tract functionality to inform health monitoring. We focus on: (i) predatory response as an indication of appetitive drive; (ii) body weight assessment and interpretation of deviations (e.g., digestive gland weight loss is disproportionate to body weight loss in starvation); (iii) oro-anal transit time requiring novel, standardized techniques to facilitate comparative studies of species and diets; (iv) defecation frequency and analysis of fecal color (diet dependent) and composition (parasites, biomarkers, and cytology); (v) digestive tract endoscopy, but passage of the esophagus through the brain is a technical challenge; (vi) high resolution ultrasound that offers the possibility of imaging the morphology of the digestive tract (e.g., food distribution, indigestible residues, obstruction) and recording contractile activity; (vii) needle biopsy (with ultrasound guidance) as a technique for investigating digestive gland biochemistry and pathology without the death of the animal. These techniques will inform the development of physiologically based assessments of health and the impact of experimental procedures. Although intended for use in the laboratory they are equally applicable to cephalopods in public display and aquaculture.

Cognition and Recognition in the Cephalopod Mollusc Octopus vulgaris : Coordinating Interaction with Environment and Conspecifics

Cephalopods provide numerous examples of behavioral and neural plasticity and richness of the behavioral repertoire that has been claimed in favour of cognitive capabilities. Here we revise the most recent knowledge on octopus cognition and recognition processes. The examination of data and observations available provide the basis for asking new stimulating questions about the cognitive abilities of octopuses and their allies and open novel scenarios for future comparative research.