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Coleoid cephalopods, including the European cuttlefish (Sepia officinalis), possess the remarkable ability to fully regenerate an amputated arm with no apparent fibrosis or loss of function. In model organisms, regeneration usually occurs as the induction of proliferation in differentiated cells. In rare circumstances, regeneration can be the product of naive progenitor cells proliferating and differentiating de novo. In any instance, the immune system is an important factor in the induction of the regenerative response. Although the wound response is well-characterized, little is known about the physiological pathways utilized by cuttlefish to reconstruct a lost arm. In this study, the regenerating arms of juvenile cuttlefish, with or without exposure at the time of injury to sterile bacterial lipopolysaccharide extract to provoke an antipathogenic immune response, were assessed for the transcription of early tissue lineage developmental genes, as well as histological and protein turnover analyses of the resulting regenerative process. The transient upregulation of tissue-specific developmental genes and histological characterization indicated that coleoid arm regeneration is a stepwise process with staged specification of tissues formed de novo, with immune activation potentially affecting the timing but not the result of this process. Together, the data suggest that rather than inducing proliferation of mature cells, developmental pathways are reinstated, and that a pool of naive progenitors at the blastema site forms the basis for this regeneration. Programa Mar2020 [16-02-01-FMP-53] Fundacao para a Ciencia e a TecnologiaPortuguese Foundation for Science and Technology [IF/00576/2014] Natural Sciences and Engineering Research Council of CanadaNatural Sciences and Engineering Research Council of Canada [RGPIN 2018-05160, RGPIN-2018-03884] Vanier Canada Graduate Scholarship

Amongst cephalopods microplastics have been reported only in jumbo squid gut. We investigated microplastics in the digestive system of wild cuttlefish (Sepia officinalis) as they are predators and prey and compared the stomach, caecum/intestine and digestive gland (DG) of wild and cultured animals, exposed to seawater from a comparable source. Fibers were the most common type (approximate to 90% of total count) but were approximate to 2x higher in relation to body weight in wild vs. cultured animals. Fibers were transported to the DG where the count was approximate to 2x higher/g in wild (median 1.85 fibers/g) vs. cultured. In wild-caught animals the DG was the predominant location but in cultured animals the fibers were more evenly distributed in the digestive tract. the potential impact of mi-croplastics on health of cuttlefish is discussed. Cuttlefish represent a previously unrecognized source of micro plastic trophic transfer to fish and finding fibers in cultured animals has implications for aquaculture. Fundacao para a Ciencia e a Tecncologia [IF/00576/2014, UID/Multi/04326/2019] Programa Operacional Mar2020/Portuga12020/FEAMP [SEPIACUL 16-02-01-FMP-53] info:eu-repo/semantics/publishedVersion

The common octopus (Octopus vulgaris) has aroused great interest in recent years as a new species for aquaculture. The current research is focused on developing a formulated feed, although a proper diet management has also promoted rearing success of other commercial cultured species. It is documented that wild animals eat depending on prey availability and most experience fasting in nature. Hence, O. vulgaris subadults were subjected to two different feeding schemes, with a similar semi-moist diet, including either 2 (2FDb, control) or 3 (3FDb) non-consecutive days of fasting per week. Growth, feed efficiency (FE), digestibility and condition were assessed after 56 days of rearing. Both feeding schemes promoted similar growth and digestibility (P>0.05), 100% of survival and higher food-intake after fasting. Interestingly, feed efficiency (FE) was enhanced with the 3 fasting days scheme (58.6% vs. 48.3% for 2FDb scheme; P

8 pages, 3 figures, 1 table, supplemental material https://royalsocietypublishing.org/doi/suppl/10.1098/rsos.210345.-- The data are provided in the electronic supplementary material, Stable isotope compositions of carbon and nitrogen (expressed as δ13C and δ15N) from the European common cuttlefish (Sepia officinalis) were measured in order to evaluate the utility of using these natural tracers throughout the Northeast Atlantic Ocean and Mediterranean Sea (NEAO-MS). Mantle tissue was obtained from S. officinalis collected from 11 sampling locations spanning a wide geographical coverage in the NEAO-MS. Significant differences of both δ13C and δ15N values were found among S. officinalis samples relative to sampling location. δ13C values did not show any discernable spatial trends; however, a distinct pattern of lower δ15N values in the Mediterranean Sea relative to the NEAO existed. Mean δ15N values of S. officinalis in the Mediterranean Sea averaged 2.5‰ lower than conspecifics collected in the NEAO and showed a decreasing eastward trend within the Mediterranean Sea with the lowest values in the most eastern sampling locations. Results suggest δ15N may serve as a useful natural tracer for studies on the population structure of S. officinalis as well as other marine organisms throughout the NEAO-MS, This work was supported by Texas A&M University (R.J.D.W. and J.R.R.) and the McDaniel Charitable Foundation (J.R.R.). A.V.S. was funded by Fundação para a Ciência e a Tecnologia (SFRH/BPD/36100/2007). R.V. was funded by the Spanish Ministry of Science, Innovation and Universities (OCTOSET project, RTI2018-097908-B-I00, MCIU/AEI/FEDER, EU) and the ‘Severo Ochoa Center of Excellence’ accreditation (CEX2019-000928-S) of the Spanish Research Agency [Agencia Española de Investigación (AEI)]

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes. SEPIATECH 31-03-05-FEP-2 Program PROMAR info:eu-repo/semantics/publishedVersion

Bobtail squids (Sepiolidae, Cephalopoda) have recently been growing in popularity in scientific studies due to their symbiotic relationship with light producing bacteria and their corresponding light emitting organs. However, the overall knowledge on the behaviour of sepiolids is based on observations on just a few of the roughly 70 extant species and must still be considered as sparsely. Understanding their behavioural ecology is not only beneficial to further grasp the complex behavioural patterns of cephalopods, it is also vital for establishing a good welfare practice when holding sepiolids in captivity. Hence, the present study characterised several behavioural aspects of the spotty bobtail squid Euprymna parva. Although the burying, hunting and mating behaviour as well as most escape responses of this less investigated sepiolid species greatly resembled those of other observed bobtail squids, differences to sepiolids from other genera or even from the same genus could be identified in the present study. Additionally, the first observation of an up to now undescribed inking behaviour of sepiolids is reported. E. parva was observed to eject a stretch of ink ('ink rope'), approximately 4-5 times the length of the animal, and hold on to it motionless, potentially as a masquerade to resemble a floating seagrass leave. The present study further provides detailed information on daily time and activity budgets as well as the tentacular strike speed during hunting, two up to now barely investigated behavioural aspects of the sepiolid ecology. Okinawa Institute of Science and Technology (OIST) Research Internship ProgramOkinawa Institute of Science & Technology Graduate University Fundacao para a Ciencia e a Tecnologia (FCT)Portuguese Foundation for Science and TechnologyEuropean Commission [IF/00576/2014] FCT Plurennial [UID/Multi/04326/2019] info:eu-repo/semantics/publishedVersion

9 pages, 6 figures, 1 table.-- Data are available as part of the electronic supplementary material https://doi.org/10.6084/m9.figshare.c.5073446, Natural markers (δ13C and δ18O stable isotopes) in the cuttlebones of the European common cuttlefish (Sepia officinalis) were determined for individuals collected across a substantial portion of their range in the Northeast Atlantic Ocean (NEAO) and Mediterranean Sea. Cuttlebone δ13C and δ18O were quantified for core and edge material to characterize geochemical signatures associated with early (juvenile) and recent (sub-adult/adult) life-history periods, respectively. Regional shifts in cuttlebone δ13C and δ18O values were detected across the 12 sites investigated. Individuals collected from sites in the NEAO displayed more enriched δ13C and δ18O values relative to sites in the Mediterranean Sea, with the latter also showing salient differences in both markers among western, central and eastern collection areas. Classification success based on cuttlebone δ13C and δ18O values to four geographical regions (NEAO, western, central and eastern Mediterranean Sea) was relatively high, suggesting that environmental conditions in each region were distinct and produced area-specific geochemical signatures on the cuttlebones of S. officinalis. A modified δ13C and δ18O baseline was developed from sites proximal to the Strait of Gibraltar in both the NEAO and Mediterranean Sea to assess potential mixing through this corridor. Nearly, all (95%) of δ13C and δ18O signatures of S. officinalis collected in the area of the NEAO closest to the Strait of Gibraltar (Gulf of Cadiz) matched the signatures of specimens collected in the western Mediterranean, signifying potential movement and mixing of individuals through this passageway. This study extends the current application of these geochemical markers for assessing the natal origin and population connectivity of this species and potentially other taxa that inhabit this geographical area, This work was supported by the McDaniel Charitable Foundation (J.R.R.), Texas A&M University (J.R.R., R.J.D.W.), Spanish Ministry of Science, Innovation and Universities, OCTOSET Project RTI2018-097908-B-I00, MCIU/AEI/FEDER, EU (R.V.), and Portuguese Foundation for Science and Technology MARE UID/MAR/04292/2013 (R.R.). A.S. was supported through Fundação para a Ciência e a Tecnologia (IF/00576/2014 contract and Plurennial funding to CCMARUID/Multi/04326/2013), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

The aim of the present study was to determine the effect of Artemia metanauplii endogenous fatty acid (FA) metabolism in the actual availability of dietary essential fatty acids (EFA) for Octopus vulgaris paralarvae development. To this end, both Artemia metanauplii inherent radiolabelled FA metabolism and the Octopus vulgaris paralarvae FA metabolism, after being fed with Artemia metanauplii incubated with radiolabelled. FAs, were determined. Metanauplii were incubated in tissue culture plates during 12 h, with 0.3 mu M of a [1-C-14]FA, including either 18:3n-3, 20:4n-6 (ARA), 20:5n-3 (EPA) or 22:6n-3 (DHA), added individually to separate wells as their potassium salts bound to bovine serum albumin. A total of 3600 one-day-old paralarvae were reared up to 24 h in order to ensure the ingestion of a minimum amount of the labelled Artemia. Paralarvae rearing was performed in 4 L cylinder conical tanks at a density of 25 paralarvae L-1. Labelled Artemia metanauplii were added to each paralarvae rearing tank according to a specific [1-C-14]FA treatment. Artemia in vivo metabolism results showed a preferential catabolism of DHA, which was translated into (1) the lower incorporation of this FA into Artemia total lipids (TL) (2) the highest amount of de novo synthesis of shorter chain-length FAs, as a result of the beta-oxidation of the original DHA substrate. The registered amounts of radiolabelled substrates incorporated into O. vulgaris paralarvae TL fed with labelled Artemia metanauplii were extremely low. Nonetheless, certain amount of intact [1-C-14]ARA and [1-C-14]EPA was recovered into octopus paralarvae TL, and particularly into polar lipid classes, suggesting the possibility of using Artemia as a vehicle to provide ARA and EPA to octopus paralarvae without interfering their bioavailability for the de novo synthesis of phospholipids. On the other hand, and despite of the high amount of [1-C-14]18:3n-3 incorporated into Artemia TL, the FAs with the highest esterification rate into Anemia TAG (18:3n-3 and DHA) were also the lowest incorporated into paralarvae TL. Therefore, the present results suggest that O. vulgaris paralarvae may have a potentially low capacity to metabolise dietary TAG, and so, Artemia may not be the most appropriate vehicle to provide DHA to paralarvae. Spanish GovernmentSpanish Government [AGL2010-22120-CO3] Fundacao para a Ciencia e a Tecnologia (FCT)Portuguese Foundation for Science and Technology [SFRH/BD/76863/2011] FCTPortuguese Foundation for Science and Technology [IF/00576/2014]

This chapter reviews the mechanical (physical) and chemical (water quality) related pathologies that have been reported since cephalopods are maintained, reared or cultured in captivity. For the first time, it builds up on the existing knowledge from researchers and aquarists (which are represented as authors of the chapter) in order to provide the most updated and inclusive revision on this theme. It is organized in terms of pathologies that are reported and eventual described for one or more species, which are commonly kept for research and display purposes, and exemplified with photos when possible. It includes pathologies of the mantle, arms, eyes, shell; egg infections, malformations of the shell and eggs; and causes of disease or mortality related with water quality focusing on pH and trace elements.

Taurine is the most abundant amino acid in the blood of the cuttlefish, Sepia officinalis, where levels can exceed 200 mmol L-1. In mammals, intracellular taurine modulates cardiac Ca2+ handling and carbohydrate metabolism at much lower concentrations but it is not clear if it exerts similar actions in cephalopods. Blood Ca2+ levels are high in cephalopods and we hypothesized that taurine would depress cardiac Ca2+ flux and modulate contractility in systemic and branchial hearts of cuttlefish. Heart performance was assessed with an in situ perfused systemic heart preparation and contractility was evaluated using isometrically contracting systemic and branchial heart muscle rings. Stroke volume, cardiac output, and Ca2+ sensitivity were significantly lower in systemic hearts perfused with supplemental taurine (100 mmol L-1) than in controls. In muscle ring preparations, taurine impaired relaxation at high contraction frequencies, an effect abolished by supra-physiological Ca2+ levels. Taurine did not affect oxygen consumption in non-contracting systemic heart muscle, but extracellular glucose utilization was twice that of control preparations. Collectively, our results suggest that extracellular taurine depresses cardiac Ca2+ flux and potentiates glucose utilization in cuttlefish. Variations in taurine levels may represent an important mechanism for regulating cardiovascular function and metabolism in cephalopods. SEPIATECH (31-03-05-FEP-2)

Pollicipes pollicipes (Crustacea; Pedunculata) is a delicacy on the Iberian Peninsula where, in recent years, stock shortages associated with high market value have increased interest in the aquaculture potential of this species. Though broodstock has been maintained in captivity, detailed culture conditions are lacking. The present study investigated the effects of rearing temperature on reproductive conditioning. During a 4-week period, broodstock were subjected to temperature regimes characteristic of stable spring temperatures (spT), increasing spring to summer temperatures (sp–suT) and increasing spring to summer temperatures with daily fluctuations of 1 °C (sp–suT2). Broodstock were monitored for fecundity, egg lamella development and maturation, larval release rate, nauplius size and survival over 24 h. Cultured broodstock were fecund at smaller sizes (15.94 ± 0.23 mm RC) than wild-collected individuals at the beginning of the experiment (17.71 ± 0.65 mm RC). Fecundity increased significantly in all treatments and development of egg lamellae was highest in treatments in which the temperature increased over the experimental period (average 36% of mature egg lamellae in comparison to an initial 0%). Increasing temperature led to greater maturation of lamellae and more frequent spawning peaks. The number of nauplii released per aquarium (average 110 adult individuals) varied according to treatment and time, averaging 4670 ± 506 nauplii day− 1. Due to the low number of larvae released daily, it is suggested that adults might release larvae gradually, as embryos hatch within the mantle cavity. Average release rates increased towards the end of the conditioning period, with releases on peak days ranging from 10,000 to 30,000 nauplii per aquarium. For spT, peak values were observed in week 3, while sp–suT and sp–suT2 showed peaks of release in weeks 2 and 4, when temperatures averaged 20 and 23 °C, respectively. Temperature oscillations led to shorter intervals between peaks of release. In terms of the larvae released, there were neither differences in numbers between treatments (128,147 ± 13,548 nauplii per aquarium over 28 days) nor in size of nauplii (202.89 ± 0.69 μm GW) or 24-h survival (91.56 ± 0.35%). Notwithstanding the need for further optimization, broodstock reproductive conditioning can be accomplished and a continuous supply of larvae obtained using the protocols described herein. Future studies should focus on the impact of food quality and photoperiod on reproductive conditioning, as well as the optimization of larval release induction protocols. Statements of relevance The stalked barnacles (Pollicipes pollicipes) are considered a high-value food delicacy on the Iberian Peninsula, where interest over the culture of this species has risen in recent years. However, very little is known with regard to culture conditions. The paper addresses the effect of temperature on the reproduction in captivity of this new species to culture and establishes the first working protocol for P. pollicipes reproduction and larval collection in culture.

According to the literature, the cuttlefish, Sepia officinalis, possesses a specialized olfactory organ and cells, located in olfactory ventral pits. In this study, the location of olfactory receptor neurons (ORNs) at the cellular level was determined using cellular morphology and immunohistochemistry. An antiserum against PBP3 was used as a marker to identify ORN-like cells in cuttlefish after validation for specificity to cephalopod ORN cells in the common octopus, Octopus vulgaris. The results show that ORN-like cells in S. officinalis were not found in the ventral pits, suckers or the mouth lips. Instead, ORN-like cells were found scattered in the forehead, between the eyes. The absence of ORNlike cells in a pit in S. officinalis and the sharing of four similar types of ORN cells with the squid and octopus lineages suggest that this might be a later innovation in olfaction and is probably associated with the specialized lifestyle of these later evolved cephalopods. Together, this evidence suggests a diversification of ORN cell types in Coleoidea, which did not occur in Nautiloidea, which might have preceded the diversification of the Coleoidea. info:eu-repo/semantics/publishedVersion

High paralarvae mortality is a major bottleneck currently hindering the control over the lifecycle of common octopus (Octopus vulgaris Cuvier, 1797). It is believed that this problem might be related to either zoo-technical and/or nutritional aspects. The present paper is focused on the study of different zoo-technical aspects related to light conditions on the rearing of paralarvae, including the effects of polarization in prey ingestion, the use of a blue filter to simulate natural conditions, and the use of focused light to avoid reflections of the rearing tank’s walls. In the first experiment, O. vulgaris paralarvae ingestion of Artemia sp. and copepods (Tisbe sp.) was assessed under either normal or polarized light. In the second experiment, the effect of a blue filter with natural light or focused artificial light on growth and mortality was assessed over 15 days of rearing. Ingestion rate was not influenced by light polarization. Nonetheless, a significantly higher ingestion of Artemia sp. with respect to copepods was observed. The blue filter promoted the use of natural light conditions in Octopus paralarval culture, while focused light reduced the collision of the paralarvae against the walls. However, no significant differences were found in paralarval growth nor survival, SI

The common cuttlefish (Sepia officinalis), a dominant species in the north-east Atlantic ocean and Mediterranean Sea, is potentially subject to hypoxic conditions due to eutrophication of coastal waters and intensive aquaculture. Here we initiate studies on the biochemical response to an anticipated level of hypoxia. Cuttlefish challenged for 1 h at an oxygen level of 50% dissolved oxygen saturation showed a decrease in oxygen consumption of 37% associated with an 85% increase in ventilation rate. Octopine levels were increased to a small but significant level in mantle, whereas there was no change in gill or heart. There were no changes in mantle free glucose or glycogen levels. Similarly, the hypoxic period did not result in changes in HSP70 or polyubiquinated protein levels in mantle, gill, or heart. As such, it appears that although there was a decrease in metabolic rate there was only a minor increase in anaerobic metabolism as evidenced by octopine accumulation and no biochemical changes that are hallmarks of alterations in protein trafficking. Experiments with isolated preparations of mantle, gill, and heart revealed that pharmacological inhibition of protein synthesis could decrease oxygen consumption by 32 to 42% or Na+/K+ ATPase activity by 24 to 54% dependent upon tissue type. We propose that the decrease in whole animal oxygen consumption was potentially the result of controlled decreases in the energy demanding processes of both protein synthesis and Na+/K+ ATPase activity. Action FA1301 info:eu-repo/semantics/publishedVersion

Studies concerning the perceptual processes of animals are not only interesting, but are fundamental to the understanding of other developments in information processing among non-humans. Carefully used visual illusions have been proven to be an informative tool for understanding visual perception. In this behavioral study, we demonstrate that cuttlefish are responsive to visual cues involving texture gradients. Specifically, 12 out of 14 animals avoided swimming over a solid surface with a gradient picture that to humans resembles an illusionary crevasse, while only 5 out of 14 avoided a non-illusionary texture. Since texture gradients are well-known cues for depth perception in vertebrates, we suggest that these cephalopods were responding to the depth illusion created by the texture density gradient. Density gradients and relative densities are key features in distance perception in vertebrates. Our results suggest that they are fundamental features of vision in general, appearing also in cephalopods. info:eu-repo/semantics/publishedVersion

International audience; The effects of three light intensities (100, 350 and 1200 lux) on cuttlefish hatchling rearing performance was studied in black tanks. A total of 270 cuttlefish with a mean wet weight (MWW) of 0.089 ± 0.012 g were used in the experiment, which was undertaken during the first 50 days after hatching (hatchling stage plus the transition to the juvenile stage). According to results of the present study, light intensity is an important factor for growth and survival consistency in cuttlefish rearing. All three light intensity groups displayed exponential growth. The effect of days, light intensity and their interaction only displayed differences (P < 0.05) between light groups in terms of mean wet weight. The 100 lux light intensity promoted the best absolute values of total biomass and total mortality. We believe that the higher mortality observed in 1200 lux reared cuttlefish during the first 10 days after hatching (DAH) was due to light intensity and individual adaptation to light conditions. Therefore, the 100 lux light intensity, obtained with daylight spectrum bulbs, is recommended for cuttlefish rearing during the first 50 DAH. This light setup promotes higher growth and survival rates and lower energetic costs, which are key aspects to consider in a cuttlefish hatchery.

The present study compared the lipid composition and in vivo capability of Artemia sp. metanauplii (the main live prey used in aquaculture) and Grapsus adscensionis zoeae (as a wild zooplankton model) to metabolise unsaturated fatty acids. The two species were incubated in vivo with 0.3 μM of individual [1-14C]fatty acids (FA) including 18:1n‐9, 18:2n‐6, 18:3n‐3, 20:4n‐6 (ARA), 20:5n‐3 (EPA) and 22:6n‐3 (DHA) bound to bovine serum albumin (BSA). Compared to metanauplii, zoeae contained twice the content of polar lipids (PL) and eight-fold the content of long-chain polyunsaturated fatty acids (LC-PUFA). Artemia sp. metanauplii showed increased short chain fatty acid de novo synthesis from beta-oxidation of [1-14C]LC-PUFA, preferentially DHA. Of the LC-PUFA, DHA showed the highest esterification rate into Artemia sp. triacylglycerols. In contrast, in Grapsus zoeae [1-14C]DHA displayed the highest transformation rate into longer chain-length FAs and was preferentially esterified into PL. EPA and ARA, tended to be more easily incorporated and/or retained than DHA in Artemia sp. Moreover, both EPA and ARA were preferentially esterified into Artemia PL, which theoretically would favour their bioavailability to the larvae. In addition to the inherent better nutritional value of Grapsus zoeae due to their intrinsic lipid composition, the changes taking place after the lipid incorporation, point at two distinct models of lipid metabolism that indicate zoeae as a more suitable prey than Artemia sp. for the feeding of marine animals, This work was partially supported by project OCTOPHYS (AGL2010-22120-CO3) funded by Spanish Government. D.B. Reis (SFRH/BD/76863/2011) wishes to thank to Fundação para a Ciência e a Tecnologia (FCT) for her grant. A.V. Sykes is funded by FCT through Programa Investigador FCT 2014 (IF/00576/2014).

The objective of the present study was to characterise the fatty acid (FA) profiles of the major phospholipids, of Octopus vulgaris and Sepia officinalis hatchlings, namely phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE); and to evaluate the capability of both cephalopod species on dietary phospholipid remodelling. Thus, O. vulgaris and S. officinalis hatchlings were in vivo incubated with 0.3μM of L-∝-1-palmitoyl-2-[1-14C]arachidonyl-PC or L-∝-1-palmitoyl-2-[1-14C]arachidonyl-PE. Octopus and cuttlefish hatchlings phospholipids showed a characteristic FA profiles with PC presenting high contents of 16:0 and 22:6n-3 (DHA); PS having high 18:0, DHA and 20:5n-3 (EPA); PI a high content of saturated FA; and PE showing high contents of DHA and EPA. Interestingly, the highest content of 20:4n-6 (ARA) was found in PE rather than PI. Irrespective of the phospholipid in which [1-14C]ARA was initially bound (either PC or PE), the esterification pattern of [1-14C]ARA in octopus lipids was similar to that found in their tissues with high esterification of this FA into PE. In contrast, in cuttlefish hatchlings [1-14C]ARA was mainly recovered in the same phospholipid that was provided. These results showed a characteristic FA profiles in the major phospholipids of the two species, as well as a contrasting capability to remodel dietary phospholipids, which may suggest a difference in phospholipase activities., SI

This review intends to provide a reflection regarding Directive 2010 ⁄63 ⁄EU, on animal welfare, and its application concerning cephalopod breeding and experimentation in aquaculture research. To do so, we gathered different perspectives of our group members, involving two cephalopod aquaculture researchers, an aquaculture fish production technician and a veterinary professional, the latter with no background on cephalopod research. The inclusion of this class in the animal welfare legislation; the definition of live cephalopods, stress, pain and suffering are revised according to the latest scientific knowledge. Considering the 15 year background that the Centre of Marine Sciences holds on the cultivation of the European cuttlefish, an evaluation of existing production protocols, aquaculture technology related research, ethics and cephalopod welfare are discussed. The application of anaesthesia, analgesia and euthanasia is discussed, bearing in mind the different procedures applied on a daily basis in aquaculture breeding and experimentation and the requirements of the new Directive. Finally, an overview of the above and progress on 3Rs (replacement of animals, reduction in number of animals and refinement of procedures) application to cephalopod welfare is presented.

The culture of common octopus (Octopus vulgaris), one important candidate to the aquaculture diversification, faces significant difficulties, mainly related with an inadequate first development stages diet. A mathematical model integrating disperse information on the nutrient composition throughout the species ontogenic development as well as on the effects of broodstock feeding and diet composition data of O. vulgaris, allowed us to predict the time evolution of paralarvae nutritional composition in terms of protein and lipid fractions and to design an optimal diet composition with the objective to ensure the maximal survival. The optimization routine showed that a diet based on the spider crab (Maja squinado) zoea composition is the most suitable for reaching the best survival rates. Results are verified by comparison with available experimental data. The obtained results and the prospective developments are a good example of how the systemic, quantitative model based approach can be used to analyse and contribute to the understanding of complex biological systems. info:eu-repo/semantics/publishedVersion

The aim of this study was to determine the nutritional value of adult commercial cuttlefish, to develop a Quality Index Method (QIM) scheme, and to evaluate the application of some biochemical methods commonly used for freshness assessment of fish under ice storage. Additionally, shelf-life was to be determined based on both QIM and suitable biochemical methods. The nutritional value of the cuttlefish mantle in the first 24 h and after 13 days was determined. Captured cuttlefish was composed (g/100 g) by 16.60 ± 0.10 g protein, 0.09 ± 0.01 g fat, 79.55 ± 0.14 g moisture and 1.39 ± 0.03 g of ash. After 13 days of ice storage, cuttlefish was composed (g/100 g) of 11.90 ± 0.28 g protein, 0.17 ± 0.09 g fat, 87.04 ± 0.13 g moisture and 0.52 ± 0.01 g of ash. Differences ( p p > 0.05). These results seem to indicate that there is impregnation of the iced water into the mantle tissue promoting protein leaching with the melting ice. TVB-N and TMA-N displayed a similar increasing tendency, peaking beyond EEC regulations proposed maximum between the 9th and 10th days. The developed QIM scheme for cuttlefish was composed of 29 demerit points, divided into 4 attributes and 13 parameters. The calculated quality index (QI) evolved linearly with storage time in ice (QI = 2.68 × days in ice − 0.61, R 2 = 0.9866). Storage time could be estimated with an accuracy of ± 1 day, if five cuttlefish from each sample were included in the QIM assessment. The shelf-life was determined as 8 ± 1 days by both type of methods (QIM and biochemical). However, the suitability of some biochemical methods to assess freshness need to be more thoroughly researched.

The common octopus (Octopus vulgaris, Cuvier 1797) is a promising species for aquaculture diversification, but the massive mortality during the first life stage is the main bottleneck for its commercial production. The aim of the present study was to compare the effects of different live preys (Artemia and crustacean zoeae) and/or Artemia enrichment protocols in the paralarval growth by using a meta-analysis approach. A total of 26 independent assays were used, including data from the bibliography and from experiments carried out by our group. Three comparisons were established: (i) crustacean zoeae vs. Artemia, (ii) different crustacean zoeae species and (iii) Artemia enriched with marine lecithin (rich in polar lipids-PL and docosahexaenoic acid-DHA) vs. previously used Artemia enrichments. The meta-analysis approach allowed a quantitative review of independent studies with reliable conclusions, avoiding the subjectivity inherent to classical reviews. The outputs provided statistical confirmation of the better suitability of crustacean zoeae with respect to Artemia. However, not all crustacean species showed the same results, given that the high variability on Grapsus zoeae hampered finding significant differences with respect to the control treatment (Artemia). Nutrient composition and biometry of the different types of prey are discussed as possible causes of the differences arising from the meta-analysis. Finally, the present results suggest that marine lecithin has a beneficial effect on paralarval growth with respect to previously used enrichments, which could be related to the increase in DHA and PL in Artemia, given the essential role of these lipid components in octopus paralarval physiology., This survey was funded by the Ministerio de Ciencia e Innovación (Spanish Government) under Project OCTOPHYS (Ref. AGL2010‐22120‐C03) and Canary Goverment (Spain) under Project PRESAPUL (PI 2008/162). A.V. Sykes was funded by FCT through Programa Investigador FCT 2014 (IF/00576/2014). D Garrido was financed by Ph.D. grant by Spanish Institute of Oceanography (BOE 3rd November 2011). Most of the authors of this paper participate to the COST network, Action FA1301 (CephsInAction) focused on Cephalopod welfare.

Food limitation is a common challenge for animals. Cephalopods are sensitive to starvation because of high metabolic rates and growth rates related to their "live fast, die young" life history. We investigated how enzymatic capacities of key metabolic pathways are modulated during starvation in the common cuttlefish (Sepia officinalis) to gain insight into the metabolic organization of cephalopods and their strategies for coping with food limitation. In particular, lipids have traditionally been considered unimportant fuels in cephalopods, yet, puzzlingly, many species (including cuttlefish) mobilize the lipid stores in their digestive gland during starvation. Using a comprehensive multi-tissue assay of enzymatic capacities for energy metabolism, we show that, during long-term starvation (12 days), glycolytic capacity for glucose use is decreased in cuttlefish tissues, while capacities for use of lipid-based fuels (fatty acids and ketone bodies) and amino acid fuels are retained or increased. Specifically, the capacity to use the ketone body acetoacetate as fuel is widespread across tissues and gill has a previously unrecognized capacity for fatty acid catabolism, albeit at low rates. The capacity for de novo glucose synthesis (gluconeogenesis), important for glucose homeostasis, likely is restricted to the digestive gland, contrary to previous reports of widespread gluconeogenesis among cephalopod tissues. Short-term starvation (3-5 days) had few effects on enzymatic capacities. Similar to vertebrates, lipid-based fuels, putatively mobilized from fat stores in the digestive gland, appear to be important energy sources for cephalopods, especially during starvation when glycolytic capacity is decreased perhaps to conserve available glucose. SEPIATECH (31-03-05-FEP-2) info:eu-repo/semantics/publishedVersion

The transition of Sepia officinalis culture to industrial large scale has been hampered due to bottlenecks related to the limited knowledge on nutritional physiology of the species. Determination of the endogenous ability of S. officinalis hatchlings to metabolise unsaturated fatty acids (FA) may provide new insight on the capability of hatchlings to biosynthesise different FA, as well as lipid classes containing essential fatty acids (EFA). In the present study, cuttlefish hatchlings were incubated with [1-14C]FA including C18 FA (18:1n-9, 18:2n-6, 18:3n-3) and long-chain polyunsaturated fatty acids (LC-PUFA) (20:4n-6 (ARA), 20:5n-3 (EPA) or 22:6n-3 (DHA)), which were added individually as potassium salts bound to bovine serum albumin. As a result, it was possible to investigate the in vivo FA metabolism of S. officinalis hatchlings by following the incorporation of specific [1-14C]FA, which points to the suitability of this methodology to study lipid metabolism of newly hatched cephalopods. The majority of radioactivity incorporated was recovered esterified into polar lipids (PL). A pattern was detected, where [1-14C]DHA, [1-14C]C18 FA and their metabolic products were preferentially esterified into phosphatidylcholine, whereas [1-14C]ARA and [1-14C]EPA were mainly esterified into phosphatidylethanolamine. [1-14C]C18 FA were the most transformed FA with several metabolites produced by elongation and possible desaturation being obtained. As a contrary the radioactivity incorporated into hatchling total lipid (TL) from supplemented [1-14C]LC-PUFA only one elongation product was recovered from the three substrates, except for [1-14C]ARA, where an unidentified product was also detected. The present in vivo results indicated that S. officinalis hatchlings may have capability for the first steps in the biosynthesis of ARA and EPA from 18:2n-6 and 18:3n-3, respectively, including the existence of a desaturase potentially involved. Nonetheless, considering the low desaturation rates detected, this process may not be sufficient to cover EFA demands during development of this species. Therefore, dietary ARA and EPA, as well as DHA, should be supplied during the hatchling stage of Sepia. While designing an inert diet, which ensures normal growth and development of this species during the hatchling stage, the C18 FA and LC-PUFA levels and ratios should be considered, since the esterification pattern detected in the present study suggested competition between these FA for esterification into specific lipid classes. Moreover, considering the observed esterification pattern of LC-PUFA into different lipid classes, it is likely that the DHA/EPA/ARA ratio, rather than DHA/EPA or EPA/ARA ratios, would be of great importance for S. officinalis hatchling development., SI

A moving object is considered conspicuous because of the movement itself. Once moving from one background to another, even dynamic camouflage experts such as cephalopods, should sacrifice their extraordinary camouflage. Therefore, minimizing detection at this stage is crucial and highly beneficial. In this study we describe a background-matching mechanism during movement, which aids the cuttlefish to downplay its presence throughout movement. In situ behavioural experiments using video and image analysis, revealed a delayed, sigmoidal, colour-changing mechanism during movement of Sepia officinalis across a uniform black and grey backgrounds, which we describe below. This is a fist and important step in understanding dynamic camouflage during movement, while the new behavioural mechanism may be incorporated and applied to any dynamic camouflaging animal or man-made system on the move.

A biometric, nutritional and sensory analysis of raw and cooked mussels comparing Mytilus sp. from the north-west coast of Portugal and Spain (Minho and Galicia, respectively) and the new offshore production site of Armona (Algarve, south Portugal) was carried out. In addition, multiple factorial analysis was performed to explore potential relationships between sensory attributes and nutritional content properties between the different mussels. Results showed that, at similar times of sale, biometrics of mussels from Armona and Vigo were similar and bigger than the remaining. Nonetheless, despite some similarities in proximate composition, mussels presented differences in lipid classes, fatty acid content and free amino acids profiles. These differences were not fully reflected in the sensory assessment by the panel, which were able to distinguish different production sites in raw specimens but displayed problems in discrimination these in cooked mussels. Some nutritional components were related to specific sensory sensations. INTERREG 0251_ECOAQUA_5_E info:eu-repo/semantics/publishedVersion

Three feeding experiments, using live mysid shrimp, grass shrimp or fish fry as prey for 1-, 30- and 60-day-old cuttlefish were conducted to determine the efficiency of each dietary source in relation to cuttlefish size and age. Additionally, a fourth experiment using fish fry and grass shrimp, but previously frozen, was also conducted. The results showed that when 1-day-old cuttlefish were fed mysids, grass shrimp or fish for 4 weeks, mysids were the best prey, but only during the first week. From this moment until the end of the experiment, the best growth rate was when cuttlefish were fed grass shrimp. Cuttlefish fed fish fry showed the poorest growth rate throughout the experiment. Similarly, cuttlefish aged 30 or 60 days fed grass shrimp or fish fry had the best growth rates when fed grass shrimp. When cuttlefish were fed live fish, survival increased with size of cuttlefish (73.3%, 91.7% and 100% for 1, 30 and 60 days cuttlefish, respectively). In the fourth experiment, using frozen diets, overall acceptance of each diet (feeding rates) was the same for fish and shrimp. However, lower growth was obtained when cuttlefish were fed fish compared to grass shrimp. This lower growth was due to a lower food conversion (28% vs. 41%). Since cephalopod paralarvae and juvenile most likely need prey rich in polyunsaturated fatty acids (PUFA), phospholipids and cholesterol, and a moderate content in neutral lipids, we have analyzed the biochemical compositions of the different prey to evaluate the influence of this factor on growth and survival. info:eu-repo/semantics/publishedVersion

The effects of feeding live or frozen grass shrimp (Palaemonetes varians) to the cuttlefish, Sepia officinalis, were determined in two experiments. During Experiment I, two populations of 30 cuttlefish (aged 90 days old) were fed either live or frozen grass shrimp. Cuttlefish fed live shrimp grew larger, matured earlier, had a shorter life cycle (255 days) than the ones fed frozen shrimp (282 days), and had lower mortality. Females from the group fed frozen shrimp matured a month later but were significantly larger, 130.9 ± 38.5 g, compared to 74.2 ± 16.0 g, laid larger eggs, 0.47 ± 0.11 g, compared to 0.28 ± 0.10 g, and had higher individual fecundity (411 eggs female−1, compared to 150 eggs female−1). Newly born hatchlings from both groups had similar weights. During Experiment II, six replicates of 15 cuttlefish (50 days old) were used, three for each of the two diets tested. The exact same amount of live or frozen shrimp was provided to both populations twice a day. No differences in growth and feeding rates or food conversions were found at the end of the experiment. During the first week, cuttlefish fed frozen shrimp grew larger, and had higher conversion rates, compared to the ones fed live shrimp. Mortality was higher for the group fed live shrimp (36.6%) in Experiment II, mainly occurring during the last week. Mortality for cuttlefish fed frozen shrimp in Experiment II was 2.2%. Results obtained here indicate that freezing the grass shrimp only had a negative effect on the survival of S. officinalis in Experiment I.

The effects of culture density on growth and survival of juvenile cuttlefish were tested. Groups of 1, 3 and 5 hatchlings were placed in small containers with bottom surface of 80 cm2, obtaining individual densities of 125, 375 and 625 cuttlefish m−2, respectively. Additionally, groups of 5 hatchlings were placed in containers with 2 different bottom areas (80 and 240 cm2), providing culture densities of 625 and 42 cuttlefish m−2, respectively. A total of 120 hatchlings were used and experiments lasted for 40 days. No differences were found in growth between any of the densities tested throughout the experiment until 35 days old. After this, cuttlefish placed in isolation grew significantly larger. A second experiment was conducted in a flow through system, using two rectangular tanks with bottom surface of 0.5 m2. Two groups of 25 cuttlefish hatchlings were used in this experiment, which lasted for 40 days. Both groups were fed live juvenile shrimp (Crangon crangon) during the first 5 days. Afterwards, one group was fed live fish fry of different species, while the other continued to be fed shrimp. After day 10 and until the end of the experiment, hatchlings fed shrimp grew significantly larger than those fed fish fry. Survival of hatchlings fed shrimp or fish fry after 40 days was of 100% and 68%, respectively. Total protein content of both prey types was similar. Therefore, the higher polar lipid content, especially due to the higher phosphatidylcholine and phosphatidylethanolamine levels observed in the shrimp, compared to fish fry could possibly be one of the major factor to explain the significantly higher growth rates for S. officinalis juveniles fed shrimp. Also, the percentage of polar lipids in the shrimp (47.4%) was closer to the one of juvenile cuttlefish (38.1%) than the composition of polar lipids in fish fry (10.4%). This could also be an important factor to explain the poor growth and survival obtained when feeding fish fry to the cuttlefish.

In this study, two new alternative preys: Grapsus adscensionis zoeae (as sole prey) and Palaemon elegans zoeae (in cofeeding with Artemia sp.), as well as, Artemia sp. juveniles were used as feed for octopus paralarvae, as a way to understand its lipid requirements. Total lipid (TL) content, lipid class (LC) and fatty acid (FA) profiles of preys, octopus hatchlings and 9-day-old paralarvae were analysed. Growth and survival of the paralarvae were also determined. Regardless the prey provided, a notable shift in the lipid profile of paralarvae was registered after 9 days of rearing. The highest index of growth rate (IGR) recorded when decapod crustacean zoeae were supplied might have some relation with levels of 20:4n-6 (ARA) and DHA/EPA ratio observed. In this sense, Grapsus adscensionis zoeae leaded to a higher content of ARA and a lower content of EPA, which may indicate a possible competition between these two FA. For that a balanced EPA/ARA ratio might be significant in this species nutrition without disconsidering DHA levels as an essential fatty acid. Finally, the changes observed in paralarvae FA profile might not only be related to prey FA profile, but also with changes occurring in the lipid classes contents., SI

We are presently culturing the 4th generation of thecuttlefish, Sepia officinalis in our laboratory. A firstgeneration (F1) was grown from eggs collected from the wild (Ria Formosa–South Portugal) during the summer, at mean temperatures of 27°C ± 3°. In the present study, a second generation(F2), originated from eggs laid in the laboratory by females from F1 wascultured between the start of autumn and the end of spring, at meantemperaturesof 15 °C ± 4 °C. The life cycle ofcuttlefish from F2 was compared to F1. Populations of 30 cuttlefish were usedineach experiment. Cuttlefish were grown from one day old until the cycle wascompleted (when the last female in each population had died). Cuttlefish fromF2cultured at much lower temperatures had a longer life cycle, of almost 9 months(260 days) compared to cuttlefish from F1, which completed their cycle in lessthan 6 months (165 days). Cuttlefish from F2 grew significantly larger (U =0.00; p < 0.01) with mean weights of 343.3 ± 80.5 g and248 ± 33.1 g for males and females, respectively, comparedtoF1 (199.6 ± 40 g and 143.3 ± 30.9 g formales and females, respectively). Females from F2 had higher fecundity (225eggsfemale−1) compared to females from F1 (144 eggs perfemale−1), produced bigger eggs (t = 45.60752; p < 0.0001),weighing 0.74 ± 0.18 g, compared to 0.46 ± 0.11 fromF1,and bigger hatchlings (t = 7,144783; p < 0.0001), weighing 0.10 ±0.02g, compared to 0.09 ± 0.02 g for the summerpopulation.

Cephalopods are considered as an alternate group for aquaculture species diversification [1,2], not only because they are a good food source (highly appreciated in some worldwide markets, e.g., Portugal, Spain, Italy and Asia) and have the potential to quickly reach a market size, but also because they are considered animal models in several fields of research [3–5], and it is expected that such models are progressively obtained from bioteriums instead of being captured from nature. Despite the recent advances in culture protocols of several cephalopod species [2], there is still little information available about the biological rhythms of cephalopods in captivity. In fact, to the best of our knowledge, there are only 4 published papers concerning this theme, and these are focused on octopus species [6–9]. This issue assumes increasing importance with the enforcement of EU welfare legislation (Directive 2010/63/EU), and this information is critical for the future update of Annex III (housing conditions) for cephalopods of that Directive, and for a future update of the Guidelines for the Care and Welfare of Cephalopods in Research [10]. If it is considered that the European cuttlefish Sepia officinalis (Linnaeus, 1758) may have, at the least, two different sleep‐like states [11], the importance of this study becomes even more evident. info:eu-repo/semantics/publishedVersion

Individual growth rates, feeding rates (%BWd-1) and food conversions for cuttlefish (S. officinalis) hatchlings and juveniles were determined during this study. A flow-through system was used. Water temperature reached 30 degrees C during the hottest part of the day, gradually decreasing to 25 degrees C during the night; salinity varied between 37 +/- 3 ppt and lights were kept on for 14 h day(-1). Hatchlings were placed in separate compartments with a water volume of 1.2 L. Juvenile cuttlefish (from 0.5 to 25 g) were placed in bigger baskets, with a water volume of 5.2 L. Water flow was 120 L h(-1). The biggest cuttlefish used in these experiments (> than 25 g) were gathered in groups of five and placed in circular tanks (water volume of 250-300 L). Thus, results obtained in this case are means and not individual data. During the first 10, 20, 30 and 40 days, mean growth rates (of all individuals sampled by age group) decreased consistently (11.8 +/- 4.1, 9.8 +/- 1.8, 8.1 +/- 2.2 and 7.3 +/- 0.7%BW-1 respectively); in similar fashion, mean feeding rates decreased with age group (33.7 +/- 13.5, 22.0 +/- 7.9, 17.3 +/- 3.9 and 16.7%BWd-1 respectively). Mean food conversions varied between 3.6 and 2.5 between the age groups. When grouping results by weight class, similar patterns occur, as growth and feeding rates decrease consistently as cuttlefish grow bigger. Highest mean growth and feeding rates are obtained by hatchlings (< 0.1 g) with 12.4 +/- 4.5 and 35.3 +/- 15.1%BWd-1, respectively, while the lowest growth and feeding rates were recorded for the largest animals, between 15 and 25 g (3.4 +/- 1.1 and 10.8 +/- 4.1%BWd-1 respectively). For these weight classes, mean food conversions varied between 2.7 +/- 0.9 and 3.8 +/- 2.8.

Twoexperiments were conducted to determine the effects ofArtemia sp. or mysids on growth and survival ofS. officinalis hatchlings, and their effect throughout thelife cycle. For experiment I, for the first 20 days, one group was fed adultArtemia sp. and the other was fed mysid shrimp(Paramysis nouvelli). Eggs laid by females in both groupswere counted and weighed, and hatchlings were weighed, to determine differencesin both groups. For experiment II, during the first 10 days, one group was fedArtemia sp. and the other was fed mysids (P.nouveli). After the period of differentiated feeding, the 2 groupsinexperiment I were fed grass shrimp (Paleomonetes varians)to 70 days old, and dead crabs (Carcinus maenas)afterwards. Cuttlefish in experiment II were fed grass shrimp from day 10 untilthe end of the experiment. For both experiments, hatchlings fed mysids grewsignificantly bigger (p < 0.01) and survival was higher. For experiment I,eggs laid by females fed mysids and the hatchlings born from these eggs werebigger (p < 0.001) compared to the group fed Artemiasp.initially. Individual fecundity was slightly higher for females in the groupfedArtemia sp. (163 eggs female−1) than forthe group fed mysids (144 eggs female−1). Egg laying startedatthe age of 125 days and lasted 45 days in both groups. Time between first egglaying day and first hatchlings to be born was 21 days. The last female to die(after spawning) in both groups was 167 days (less than 6 months old).

11 pages, This chapter presents an overall perspective on the current status of cephalopod culture, its bottlenecks and future challenges. It focuses on the species that have received more research effort and consequently accumulated more scientific literature during the present century, namely Sepia officinalis, Sepioteuthis lessoniana, Octopus maya and Octopus vulgaris. Knowledge regarding physiology, metabolism and nutrition of different species is still lacking. Two main challenges are identified: the development of a sustainable artificial diet and the control of reproduction. Understanding cephalopod physiology and nutrition will probably be the biggest challenge in developing the large-scale culture of this group of molluscs on a medium to long term. In addition, zootechnical parameters need future research and improvement. The performance of an ethical experimentation with cephalopods is strongly encouraged and any zootechnical development should be performed and adapted accordingly. The potential of cephalopod culture extends far beyond its use for research and human consumption and probably it will be translated in a remarkable production in the coming years

24 páginas, 4 tablas.-- Graziano Fiorito ... et al.-- This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited., 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, D.M. has been supported by POR Campania FSE 2007–2013, Project MODO-Model Organism.

This chapter reviews the history of cephalopod culture since the 1960s until 2000, compiling the most important contributions in each decade and identifying key research laboratories and researchers. The literature found is vast in cephalopod species, methodologies and technology. Hence, this chapter focuses mainly on those species with an established aquaculture potential. It includes a description of the evolving seawater systems, broodstock acclimatisation to captivity and rearing/culture methodologies of different life stages.

The high mortalities observed during Octopus vulgaris paralarvae culture have been associated with a nutritional imbalance, with long-chain polyunsaturated fatty acids (LC-PUFA) appearing to have a critical role. In order to determine the in vivo capability of O. vulgaris hatchlings to incorporate and metabolise unsaturated fatty acids (FA), hatchlings were incubated in flat-bottom 6-well tissue culture plates at a density of 90 hatchlings/well in 10mL of seawater (36‰). Incubations were performed with gentle stirring at 21°C for 6h with 0.2μCi (0.3μM) of [1-14C]-labelled FA including 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6 (ARA), 20:5n-3 (EPA) or 22:6n-3 (DHA), which were added directly to the seawater as their potassium salts bound to bovine serum albumin (BSA). A control treatment without [1-14C]FA was also assessed. O. vulgaris hatchlings not only possessed the ability to incorporate FA bound to BSA, but also to esterify them into phospholipid, with marked specificity. [1-14C]DHA and [1-14C]C18 FA substrates were mainly esterified into phosphatidylcholine, while [1-14C]ARA and [1-14C]EPA were esterified into phosphatidylethanolamine. The majority of radioactivity from [1-14C]FA incorporated into hatchling total lipid was recovered as unmodified FA with elongation being the only metabolism detected. Of the FA investigated, [1-14C]ARA was the most efficiently incorporated into hatchling lipids, but it was also the least modified FA. The fact that no desaturation activity was recorded towards the FA tested in this experiment may indicate that the nutritional requirements of O. vulgaris hatchlings in terms of FA are highly specific and LC-PUFA must be considered essential dietary nutrients. © 2014 Elsevier B.V., This work was partially supported by project OCTOPHYS. Ref. AGL 2010-22120-CO3 funded by the Spanish Government. D.B. Reis (SFRH/BD/76863/2011) and A.V. Sykes (SFRH/BPD/36100/2007) wish to thank Fundação para a Ciência e a Tecnologia for their grants.

The common octopus (Octopus vulgaris), a promising species for aquaculture, spawns easily under captivity from mature females (usually above 1 kg wet weight). Octopus juveniles and adults are collected from nature to obtain eggs and paralarvae for aquaculture development trials. In July 2011, a very small female (150 g wet weight but with an age estimation of 300 days-old) spawned almost 77,000 paralarvae. Malformations of paralarvae were noticed in the first spawning days, namely the absence of arms. Despite not being lethal, these abnormalities might be derived from the physiological condition of the breeding specimen (the female’s lower weight to the estimated amount of living days), which might be eventually related to nutritional unbalances or genetic parameters that were transferred to the eggs., Sí

16 pages, This chapter reviews the welfare and diseases that have been reported since cephalopods are maintained, reared or cultured in captivity. Although cephalopod welfare is only going to be assured in terms of the European Union (EU) legislation from January 2013, it has long been enforced in other regions or countries all over the world. Pathologies registered under captive conditions derive, most of the times, from bad welfare practices. A revision of cephalopods’ immune system and the most important pathologies are presented, which are divided into viral, bacterial, fungal and parasitic pathogenic agents as well as chemical and mechanical damages. In addition, information regarding healing, antibiotics application and surgery is provided. Welfare under research and commercial culture conditions is discussed in terms of the use of anaesthesia and euthanasia agents and their assessment in terms of effectiveness. Further research on the different aspects considered is suggested

The suitability of early weaning cuttlefish usingfrozen grass shrimp, and its effects on growth andmortality was determined in a 50-day experiment.Three food groups were used. One group (C) wasfed with live prey until 25th DAH and weanedwith frozen prey until the end of the experiment.In the other two groups, weaning started at thefifth DAH (W5) and first DAH (W1), also withfrozen diet. Significant differences were foundbetween C and the remaining treatments (P

The objective of this research was to determine the effects of using tanks with different bottom areas/volumes on the growth and reproduction of the European cuttlefish, Sepia officinalis. One hundred and eighty-four juvenile cuttlefish (46.1 ± 20.9 g) were used to test replicates of 9000 L (6.67 m 2 ), 750 L (1.54 m 2 ) and 250 L (0.79 m 2 – control). Growth and reproduction data were registered. In addition, egg proximate composition differences were assessed by collecting egg samples from each tank. Regarding growth, no statistical differences between tank types were found (P > 0.05), but total absolute mortality was lower in 9000 L tanks. As for reproduction, differences were found (P < 0.05) between individual tanks for mean egg (MEW), male (MW♂) and hatchling (MHW) weights; and egg batch number. One of the 9000 L tanks displayed the highest overall and individual fecundity (16 593 and 1383 eggs respectively) and egg viability (~72%) ever obtained in our facilities. Differences (P < 0.05) regarding egg proximate composition of different tanks were noted. Significant (P < 0.05) correlations were established between the duration of reproduction stage and batch, MEW and MW♀, egg viability and MHW, and MHW and egg ash content. The present results might be related to sex ratios, differences in weight between ♀ and ♂, and/or derived from breeders condition.

Anaesthetics are frequently used to prevent injuries, stress and to promote welfare while handling animals. The efficiency of five anaesthetics for short-term handling of the European cuttlefish ( Sepia officinalis ) was tested in order to determine the best agent and its concentration. Ethanol (10.0, 20.0 and 30.0 mL.L − 1 ), clove oil (0.05, 0.15 mL.L − 1 ), hypothermia (8 °C), magnesium chloride (hexahydrated; 20.0, 27.0 g.L − 1 ) and AQUI-S® (10.9 μl.L − 1 ) were the selected anaesthetics. This choice was preceded by a preliminary trial, with additional agents, where 2-phenoxyethanol (10, 15, 20, 30 mL.L − 1 ) and MS-222® (30, 40 mg.L − 1 ) were used. Due to the lack of reliable results from these two agents, they were discarded. Anaesthetic procedure was performed in 85 cuttlefish juveniles with a mean wet weight of 45.69 ± 12.01 g, on a 5 L container, under hyper-oxygenated seawater (~ 200%), plus the dose of a given anaesthetic agent. After achieving induction, cuttlefish were handled for 180 s, which consisted in a weighing procedure according to a developed CCMAR protocol. Recovery from anaesthesia was performed in a tank with flow-through hyper-oxygenated water. All these procedures were video recorded and used to, a posteriori, obtain the time frames of induction, handling and recovery, as well as for the behaviour analysis during each stage. For the analysis of results and taking into account the best welfare practice, we established the following time period criteria to determine the optimum anaesthetic dose: induction Despite the multitude of new anaesthetics developed for marine vertebrates, with this work we verified that MgCl 2 , at 27 g.L − 1 , is still the anaesthetic agent to be used in short-term handling of cuttlefish. Relative to other agents used, MgCl 2 proved superior in ease of application, price, availability, and its minimal distressing effect on individuals. However, it's of utmost importance to increase the knowledge regarding this species physiology related to the field of animal welfare. The development of tools for the identification of possible biomolecular indicators of stress in cuttlefish and the confirmation of similar physiology to vertebrates will allow a correct verification of stress induction and reduction, when using an anaesthetic agent and during the handling process.

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K depolarization was found to be very high (0.04 pmol AChs -1 mg -1 protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Chmin -1 mg -1 protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe. Cuttlefish, like other coleoid cephalopods, has been the subject of many neurobiological studies. Although the functional anatomy of the cuttlefish nervous system has been well illustrated, including investigation by electrical stimu

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3) on choline uptake and acetylcholine release only resembled those of ouabain when rat synaptosomes were assayed. Therefore, important differences were found between the species regarding the cholinotoxic action of aluminum. The variability of (Na(+)/K(+))ATPase sensitivity to aluminum of cholinergic neurons might contribute to their differential susceptibility to this neurotoxic agent. QLK1-CT2002-00172 info:eu-repo/semantics/publishedVersion