Growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax) fed an environmentally and economically sustainable low marine protein diet in sea cages.

The large use of fish meal/fish oil in carnivorous fish feeds is the main concern regarding environmental sustainability of aquaculture. Here, we evaluated the effects of an innovative diet, designed to be (1) environmentally sustainable by lowering the marine protein content while being (2) cost effective by using sustainable alternative raw materials with acceptable cost and produced on an industrial scale, on growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax), a key species of the Mediterranean marine aquaculture, reared in sea cages. Results show that the specific growth rate of fish fed the low marine protein diet was significantly lower than those fed conventional diet (0.67% vs 0.69%). Fatty acid profile of fillets from fish fed a low marine protein diet presented significant lower n-6 and higher n-3 content when compared to conventional ones. Then, a significant increase in the abundance of Vibrio and reduction of Photobacterium were found in the gut of fish fed with the low marine protein diet but effects on sea bass health needs further investigation. Finally, no major health and welfare alterations for fish fed the low marine protein diet were observed, combined with a potential slight benefit related to humoral immunity. Overall, these results suggest that despite the low marine protein diet moderately affects growth performance, it nevertheless may enhance environmental and economic sustainability of the sea bass aquaculture.

Life-cycle traits in the demosponge Hymeniacidon perlevis in a land-based fish farm.

Background: The demosponge Hymeniacidon perlevis is characterized by wide geographic distribution and great adaptability to numerous and highly variable climatic and hydrological conditions. Indeed, the species can colonize many different environments, including several unusual ones, such as concrete drainage conduits of a marine land-based fish farm plant. This research aimed to enhance existing knowledge on the reproductive cycle and growth performance of H. perlevis while also evaluating the impact of a controlled supply of trophic resources, wastewater flow and constant water temperature on these biological traits. Methods: Specimens included in this one-year study inhabited drainage conduits of a land-based fish farm. The approach included measurements of sponge biomass and occurrence and abundance of reproductive elements across different seasons and environmental parameters, such as fish biomass, trophic resources, and wastewater flow. Sponge growth and reproductive elements, including oocytes, spermatic cysts, and embryos, were measured monthly in sponges positioned in the drainage conduit, thus with different trophic resources but with constant water temperature. Finally, we used generalized additive models to describe variables that contribute the most to the growth of sponges. Results: Growth performance showed marked variations during the study period. The highest increase in sponge volume was observed from August/September to January/March. The volume of sponges was principally determined by the reduction of reared fish biomass and the increase of pellet amount and wastewater flow. Sponge specimens exhibited an active state during the entire study, as proven by the occurrence of recruits. However, sexual elements were only sporadically observed, thus not permitting the recognition of a true sexual cycle. Discussion: The results of the present study confirmed that H. perlevis exhibits high flexibility and adaptability to the differential, and somewhat extreme, environmental conditions. Indeed, this species can live, grow and reproduce in the drainage conduits of the fish farm, where the species face constant darkness, water temperature and continuous nutritional supply. In such conditions, H. perlevis display an active state during the entire year, while avoiding stages of decline and long dormancy usually observed in wild populations. It seems plausible that stable environmental conditions induce an almost continuous sexual phase, probably under the control of endogenous factors. No asexual elements were detected, although it was impossible to exclude the contribution of asexual reproduction in the origin of the newly settled sponges, which were repeatedly detected throughout the study. The growth performance seemed linked to the fish farm conditions, thus providing useful indications on the best maintenance conditions for H. perlevis in land-based integrated multitrophic systems, where the species could be used for wastewater treatment.

Movement patterns analysis as a tool in Fully Protected Areas design: Influence of relocations on travelled distances of Palinurus elephas (Fabr. 1787) in Sardinian FPAs (central-western Mediterranean).

Effectiveness of restocking programs in Marine Fully Protected Areas' (FPAs) can be highly affected by the movement behavior of the species to protect. We analyzed the data of 744 Palinurus elephas specimens tagged, relocated inside 12 FPAs, established in the seas surrounding Sardinia (central-western Mediterranean), and then recaptured. The overall aims were to characterize the individuals' movements after the relocation and to discuss strengths and weaknesses of the current FPAs' design. Almost 80% of tagged individuals travelled progressively increasing distances, as a function of the release distance, up to a maximum value of 11,500 m. Then, for larger release distances, individuals' mobility appeared reduced and more chaotic. A similar trend was found analyzing the distances travelled with respect to the FPAs with the highest number of recaptured individuals and to the size. These results allow to establish a threshold limit of the release distance, to take into account when designing FPAs, as a useful tool to keep a portion of the individuals into FPAs' borders, in order to both preserve the species and guarantee a spillover of individuals in the commercial areas.

Mapping the Energetic Costs of Free-Swimming Gilthead Sea Bream (Sparus aurata), a Key Species in European Marine Aquaculture.

Measurement of metabolic rates provides a valuable proxy for the energetic costs of different living activities. However, such measurements are not easy to perform in free-swimming fish. Therefore, mapping acceleration from accelerometer tags with oxygen consumption rates (MO2) is a promising method to counter these limitations and could represent a tool for remotely estimating MO2 in aquaculture environments. In this study, we monitored the swimming performance and MO2 of 79 gilthead sea bream (Sparus aurata; weight range, 219-971 g) during a critical swimming test. Among all the fish challenged, 27 were implanted with electromyography (EMG) electrodes, and 27 were implanted with accelerometer tags to monitor the activation pattern of the red/white muscles during swimming. Additionally, we correlated the acceleration recorded by the tag with the MO2. Overall, we found no significant differences in swimming performance, metabolic traits, and swimming efficiency between the tagged and untagged fish. The acceleration recorded by the tag was successfully correlated with MO2. Additionally, through EMG analyses, we determined the activities of the red and white muscles, which are indicative of the contributions of aerobic and anaerobic metabolisms until reaching critical swimming speed. By obtaining insights into both aerobic and anaerobic metabolisms, sensor mapping with physiological data may be useful for the purposes of aquaculture health/welfare remote monitoring of the gilthead sea bream, a key species in European marine aquaculture.

Calibrating Accelerometer Tags with Oxygen Consumption Rate of Rainbow Trout (Oncorhynchus mykiss) and Their Use in Aquaculture Facility: A Case Study.

Metabolic rates are linked to the energetic costs of different activities of an animal's life. However, measuring the metabolic rate in free-swimming fish remains challenging due to the lack of possibilities to perform these direct measurements in the field. Thus, the calibration of acoustic transmitters with the oxygen consumption rate (MO2) could be promising to counter these limitations. In this study, rainbow trout (Oncorhynchus mykiss Walbaum, 1792; n = 40) were challenged in a critical swimming test (Ucrit) to (1) obtain insights about the aerobic and anaerobic metabolism throughout electromyograms; and (2) calibrate acoustic transmitters' signal with the MO2 to be later used as a proxy of energetic costs. After this calibration, the fish (n = 12) were implanted with the transmitter and were followed during ~50 days in an aquaculture facility, as a case study, to evaluate the potential of such calibration. Accelerometer data gathered from tags over a long time period were converted to estimate the MO2. The MO2 values indicated that all fish were reared under conditions that did not impact their health and welfare. In addition, a diurnal pattern with higher MO2 was observed for the majority of implanted trout. In conclusion, this study provides (1) biological information about the muscular activation pattern of both red and white muscle; and (2) useful tools to estimate the energetic costs in free-ranging rainbow trout. The use of acoustic transmitters calibrated with MO2, as a proxy of energy expenditure, could be promising for welfare assessment in the aquaculture industry.

New insights into life-history traits of Mediterranean Electric rays (Torpediniformes: Torpedinidae) as a contribution to their conservation.

Despite being commonly recognized by the general public, knowledge about the biology and life-history of electric rays is still scarce. Globally, according to the International Union for Conservation of Nature, more of the 80% of the species belonging to the Torpedinidae family are, indeed, classified as Data Deficient, so as, in most of the cases, no proper management measures are available for the conservation of these species. For this reason, the main goal of this study is to provide new information on life-history traits of three species inhabiting the Mediterranean Sea. Age and growth were estimated through direct annuli count of 146 vertebral centra sections of Torpedo marmorata. Females' growth pattern was found to be better described by a S-shaped function (Gompertz L∞ = 622mm TL; k = 0.15; IP=2.75 years), while males' by a logarithmic one (von Bertalanffy L∞ = 485mm TL; k = 0.14; t0 = -2.28). The oldest female and male were aged 17 (560mm TL) and 11 (432mm TL), respectively. Females (L50=379mm TL and A50=5.5 years) appeared to mature later than males (L50=272mm TL and A50=3.8 years), also attaining bigger sizes. Despite the observation of late-pregnant females being found only in autumn and regressing ones in autumn and winter, the presence of juveniles during the whole year may indicate a longer reproductive period. The stomach content analysis identified T. marmorata as a top predator (TROPH=4.38) with a very narrow trophic niche (Bi=0.22), extremely specialized in feeding on bony fishes. In addition, this work provides new and updated information on T. torpedo and the rare Tetronarce nobiliana growth, reproductive features and diet. Therefore, offering new essential data to assess the population conditions of all Torpedinidae species that inhabit the Mediterranean Sea, will lead to effective conservation plans.

Development of molecular and histological methods to evaluate stress oxidative biomarkers in sea bass (Dicentrarchus labrax).

In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.

Behavioral and physiological responses to stocking density in sea bream (Sparus aurata): Do coping styles matter?

Stocking density is considered a stress factor for fish and is therefore one of the numerous concerns about fish welfare in an aquaculture context. Stress coping styles (SCS) are defined as a coherent set of individual physiological and behavioral differences in stress responses that are consistent across time and context and appear to be promising for improving fish welfare in aquaculture. The aim of the present study was to describe the physiological and zootechnical performances of gilthead sea bream (Sparus aurata) at different stocking densities (low density, LD: 15 kg/m3 and high density, HD: 30 kg/m3), depending on individual SCS. To do so, the fish SCS were first screened by measuring boldness (prior to the experiment). Three consecutive samplings were performed over the experiment to measure several blood parameters, including hematocrit (Hct), red blood cell count (RBCC), hemoglobin (Hb), cortisol, adrenalin, noradrenalin, glucose, lactate, and lysozyme, to infer the consequence of the SCS profile on the welfare condition in response to stocking density. Finally, swimming activity was recorded in a subsample of individuals (9 BOLD and 9 SHY individuals per density), and BOLD individuals displayed higher swimming activity than SHY ones at HD, while the opposite pattern was observed at LD. According to principal component analysis, physiological parameters are linked to the SCS profile, mostly at the beginning of the experiment, while density effects on physiology remain during the entire experiment duration. In conclusion, regarding all the variables observed, fish SCS appeared to be promising criteria to select the most adaptive individuals relating to rearing conditions and therefore improve welfare.

On the presence of Dipturus nidarosiensis (Storm, 1881) in the Central Mediterranean area.

The Norwegian skate Dipturus nidarosiensis (Storm, 1881) has only recently been recorded in the western Mediterranean Sea along the coast of southern Sardinia, off Algeria and the Alboran Sea. The present study confirmed the presence of the species in the Central Mediterranean Sea by identifying morphometric, morphological features and molecular markers. Biological sampling was conducted from 2010 to 2016 on eight specimens collected through commercial landings, offshore observer programmes and scientific surveys in Adriatic and Ionian waters at depths between 320 and 720 m. The total lengths of the specimens (juveniles and adults) ranged from 268 to 1,422 mm, and their body weights ranged from 44.5 to 12,540.0 g. They showed morphometric features that corresponded to those of Norwegian skates in the Northeast Atlantic and the Western Mediterranean. In previous analyses, molecular data were obtained by mitochondrial COI sequences. The haplotype network showed the occurrence of a common haplotype (Hap_1) shared by the individuals from areas in the North Atlantic, Sardinian, Algerian and Spanish Mediterranean Sea areas but not South Africa. The occurrence of individuals in different stages of life (i.e., juveniles, sub-adults and adults) and sexual development (immature and mature) suggested the presence of a species with a permanent reproductive allocation in the deep waters of the Mediterranean, which was exposed to a low level of fishing exploitation. Indeed, the deep depth distribution of the species could be the reason for the absence of information about this species in onshore or offshore fishery data collection programmes and scientific surveys.

Basic knowledge of social hierarchies and physiological profile of reared sea bass Dicentrarchus labrax (L.).

The effects of social hierarchies (dominant/subordinate individuals), such as aggressiveness, feeding order, and territoriality, are some of the characteristics used for describing fish behaviour. Social hierarchy patterns are still poorly understood in European-reared sea bass (Dicentrarchus labrax). In this work, we examine the social interactions among captive fish integrating behavioural and physiological profiles. Groups of three fish with EMG (electromyogram) radio transmitters were monitored for two weeks via video recording. Plasma levels of cortisol, glucose, lactate and lysozyme as well as haematological parameters such as haemoglobin, haematocrit and RBCC (red blood cell count) were measured at the beginning and end of the experiments. Behaviour and muscle activity were monitored daily. The results highlighted that the social hierarchic order was established after one to two days, and it was maintained throughout the experimental period. Dominant and subordinate fish (ß and γ) showed significant differences in muscle activity, hormonal profile (cortisol), aspecific immunity (lysozyme), carbohydrate metabolism (lactate) and behavioural patterns (food order and aggressiveness). This holistic approach helps to provide insights into the physiological status of the subordinate (ß and γ) and dominant individuals. These data have wide implications for aquaculture practice.

A holistic approach to the age validation of Mullus barbatus L., 1758 in the Southern Adriatic Sea (Central Mediterranean).

The growth of Mullus barbatus has been widely studied using different methods, but no previous study has focused on age validation. The uncertainty in estimating the age of the red mullet by otolith reading is linked to the number of false-growth increments laid down before the annulus. The capture of red mullets in the early life stage allowed us to estimate their size at the metamorphosis from the pelagic to the demersal phase. The comparison between the metamorphosis size and the back-calculated length of the first growth increment clarified the position of the false growth increment on the otolith. Moreover, the analyses of the otolith marginal increments in adult and juvenile specimens allowed us to define the deposition patterns of their annuli. The modal components of the length-frequency distribution analysis (LFDA) were identified in the winter survey (ELEFAN and Bhattacharya methods), and they did not show significant differences from the length back-calculation of the annuli. Moreover, no significant differences were found between the growth curves calculated by otolith reading (back-calculation and direct otolith reading) and the LFDA. The agreement between the length-frequency results and the otolith age estimation either corroborated or indirectly validated the growth pattern estimated in the otoliths of the red mullet, mainly when the direct validation methods (e.g. mark-recapture, captivity, radiochemical) were difficult to implement, like the case of this species. The comparison of the results of the present work to previous Mediterranean studies showed agreement with the slow growth pattern.

Behavioural indicators of welfare in farmed fish.

Behaviour represents a reaction to the environment as fish perceive it and is therefore a key element of fish welfare. This review summarises the main findings on how behavioural changes have been used to assess welfare in farmed fish, using both functional and feeling-based approaches. Changes in foraging behaviour, ventilatory activity, aggression, individual and group swimming behaviour, stereotypic and abnormal behaviour have been linked with acute and chronic stressors in aquaculture and can therefore be regarded as likely indicators of poor welfare. On the contrary, measurements of exploratory behaviour, feed anticipatory activity and reward-related operant behaviour are beginning to be considered as indicators of positive emotions and welfare in fish. Despite the lack of scientific agreement about the existence of sentience in fish, the possibility that they are capable of both positive and negative emotions may contribute to the development of new strategies (e.g. environmental enrichment) to promote good welfare. Numerous studies that use behavioural indicators of welfare show that behavioural changes can be interpreted as either good or poor welfare depending on the fish species. It is therefore essential to understand the species-specific biology before drawing any conclusions in relation to welfare. In addition, different individuals within the same species may exhibit divergent coping strategies towards stressors, and what is tolerated by some individuals may be detrimental to others. Therefore, the assessment of welfare in a few individuals may not represent the average welfare of a group and vice versa. This underlines the need to develop on-farm, operational behavioural welfare indicators that can be easily used to assess not only the individual welfare but also the welfare of the whole group (e.g. spatial distribution). With the ongoing development of video technology and image processing, the on-farm surveillance of behaviour may in the near future represent a low-cost, noninvasive tool to assess the welfare of farmed fish.