Living naked: first case of lack of skin-related structures in an elasmobranch, the blackmouth catshark (Galeus melastomus).

As far as is known, in this paper the first case of lacking of skin-related structures (epidermis, stratum laxum, dermal denticles and teeth) in a free-swimming elasmobranch, the blackmouth catshark, Galeus melastomus, is reported. The individual was caught by trawl in Sardinian waters (central-western Mediterranean) in July 2019 at a depth of 500 m. Although this kind of morphological abnormality is potentially fatal, the observations suggested that the specimen was in good health and well developed.

Ammonium Recovery and Biogas Upgrading in a Tubular Micro-Pilot Microbial Electrolysis Cell (MEC).

Here, a 12-liter tubular microbial electrolysis cell (MEC) was developed as a post treatment unit for simultaneous biogas upgrading and ammonium recovery from the liquid effluent of an anaerobic digestion process. The MEC configuration adopted a cation exchange membrane to separate the inner anodic chamber and the external cathodic chamber, which were filled with graphite granules. The cathodic chamber performed the CO2 removal through the bioelectromethanogenesis reaction and alkalinity generation while the anodic oxidation of a synthetic fermentate partially sustained the energy demand of the process. Three different nitrogen load rates (73, 365, and 2229 mg N/Ld) were applied to the inner anodic chamber to test the performances of the whole process in terms of COD (Chemical Oxygen Demand) removal, CO2 removal, and nitrogen recovery. By maintaining the organic load rate at 2.55 g COD/Ld and the anodic chamber polarization at +0.2 V vs. SHE (Standard Hydrogen Electrode), the increase of the nitrogen load rate promoted the ammonium migration and recovery, i.e., the percentage of current counterbalanced by the ammonium migration increased from 1% to 100% by increasing the nitrogen load rate by 30-fold. The CO2 removal slightly increased during the three periods, and permitted the removal of 65% of the influent CO2, which corresponded to an average removal of 2.2 g CO2/Ld. During the operation with the higher nitrogen load rate, the MEC energy consumption, which was simultaneously used for the different operations, was lower than the selected benchmark technologies, i.e., 0.47 kW/N·m3 for CO2 removal and 0.88 kW·h/kg COD for COD oxidation were consumed by the MEC while the ammonium nitrogen recovery consumed 2.3 kW·h/kg N.

Sexual selection and the evolution of sperm morphology in sharks.

Post-copulatory sexual selection, and sperm competition in particular, is a powerful selective force shaping the evolution of sperm morphology. Although mounting evidence suggests that post-copulatory sexual selection influences the evolution of sperm morphology among species, recent evidence also suggests that sperm competition influences variation in sperm morphology at the intraspecific level. However, contradictory empirical results and limited taxonomic scope have led to difficulty in assessing the generality of sperm morphological responses to variation in the strength of sperm competition. Here, we use phylogenetically controlled analyses to explore the effects of sperm competition on sperm morphology and variance in sharks, a basal vertebrate group characterized by wide variation in rates of multiple mating by females, and consequently sperm competition risk. Our analyses reveal that shark species experiencing greater levels of sperm competition produce sperm with longer flagella and that sperm flagellum length is less variable in species under higher sperm competition risk. In contrast, neither the length of the sperm head and midpiece nor variation in sperm head and midpiece length was associated with sperm competition risk. Our findings demonstrate that selection influences both the inter- and intraspecific variation in sperm morphology and suggest that the flagellum is an important target of sexual selection in sharks. These findings provide important insight into patterns of selection on the ejaculate in a basal vertebrate lineage.

Ontogenetic changes in the tyrosine hydroxylase immunoreactive preoptic area in the small-spotted catshark Scyliorhinus canicula (L., 1758) females: catecholaminergic involvement in sexual maturation.

Introduction: The catecholaminergic component of the brain-pituitary-gonadal axis, which mediates the influence of external and internal stimuli on the central nervous system and gonad development in vertebrates, is largely unexplored in Chondrichthyes. We considered Scyliorhinus canicula (L., 1758) females as a model for this vertebrate's class, to assess the involvement of the catecholaminergic system of the brain in its reproduction. Along the S. canicula reproductive cycle, we characterized and evaluated differences in somata morphometry and the number of putative catecholaminergic neurons in two brain nuclei: the periventricular preoptic nucleus, hypothesized to be a positive control for ovarian development, and the suprachiasmatic nucleus, examined as a negative control. Materials and methods: 16 S. canicula wild females were sampled and grouped in maturity stages (immature, maturing, mature, and mature egg-laying). The ovary was histologically processed for the qualitative description of maturity stages. Anti-tyrosine hydroxylase immunofluorescence was performed on the diencephalic brain sections. The immunoreactive somata were investigated for morphometry and counted using the optical fractionator method, throughout the confocal microscopy. Results and discussions: Qualitative and quantitative research confirmed two separate populations of immunoreactive neurons. The modifications detected in the preoptic nucleus revealed that somata were more numerous, significantly smaller in size, and more excitable during the maturing phase but decreased, becoming slightly bigger and less excitable in the egg-laying stage. This may indicate that the catecholaminergic preoptic nucleus is involved in the control of reproduction, regulating both the onset of puberty and the imminent spawning. In contrast, somata in the suprachiasmatic nucleus grew in size and underwent turnover in morphometry, increasing the total number from the immature-virgin to maturing stage, with similar values in the more advanced maturity stages. These changes were not linked to a reproductive role. These findings provide new valuable information on Chondrichthyes, suggesting the existence of an additional brain system implicated in the integration of internal and environmental cues for reproduction.

Ontogenetic Shifts in Body Morphology of Demersal Sharks' Species (Order: Squaliformes) Inhabiting the Western-Central Mediterranean Sea, with Implications for Their Bio-Ecological Role.

Several elasmobranch species undergo shifts in body proportions during their ontogenetic growth. Such morphological changes could reflect variation in diet, locomotion, or, more broadly, in the species' interactions with their environment. However, to date, only a few studies have been conducted on this topic, and most of them focused on particular body regions. In the present study, the ontogenetic growth of five different demersal shark species was investigated by using both traditional linear morphometry of the entire body and shape analysis of the caudal fin. A total of 449 sharks were analysed: 95 little gulper sharks, 80 longnose spurdogs, 103 kitefin sharks, 124 velvet belly lanternsharks, and 47 angular roughsharks. From each specimen, 36 linear morphometric measurements were taken. While a first canonical analysis of principal coordinates ruled out the possibility of different growth patterns between males and females, the same analysis statistically discriminated between small and large individuals in every species based on their morphology. A Similarity Percentage analysis revealed that the most important measurements in distinguishing these two groups were those related to body lengths, indicating that large individuals are more elongated than small individuals. The shape analysis of caudal fins revealed allometric growth during ontogenetic development, with adult individuals having a wider fin (discriminant analysis, p < 0.05). These findings could be related to changes in predatory skills, supporting the hypothesis of a shift in the ecological role that these sharks play in their environment, thus providing new essential information for their conservation.

Antibodies targeting the European lobster (Palinurus elephas) vitellogenin developed by mRNA isolation and in-silico-designed antigenic peptides.

Vitellogenin is an essential protein involved in ovary maturation in many animals. Detection of this protein correlated with reproductive capacity may be important if carried out on marine organisms such as the red spiny lobster Palinurus elephas, a crustacean that is an economically important crop from wild fish catches. Moreover, in recent years, vitellogenin has assumed an important role as a possible biomarker of marine environmental pollution, as its expression levels can be influenced by the presence of similar estrogen pollutants and can affect the reproductive sphere of marine organisms such as crustaceans. The P. elephas vitellogenin protein and its coding gene have never been isolated, so there is little information about its presence in this lobster. The aim of the present study was to develop a molecular strategy to create, for the first time, an antibody for the detection and quantization of vitellogenin in P. elephas.

Investigating the Ovarian Microstructure in the Genera Helicolenus and Scorpaena (Teleostei, Sub-Order Scorpaenoidei) with Implications for Ovarian Dynamics and Spawning.

The sub-order Scorpenoidei appears to be particularly interesting due to the presence of intermediate stages between oviparity and viviparity in several species. The present study aims to describe the ovarian morphology, using a histological and histochemical approach, in four ovuliparous species belonging to Scorpaena genus compared with a zygoparous species, H. dactylopterus, focusing also on the assessment of the ovarian dynamics in the populations of such species in Sardinia waters (central-western Mediterranean). Ovarian sections of all species were examined using light microscopy. All species showed a specialized ovary, cystovarian type II-3, strictly related to the production of gelatinous matrices surrounding the eggs. Some microscopic peculiarities in the oogenesis process were found: thin zona pellucida, small and low cortical alveoli, and a specialized ovarian wall during the spawning period. All species analyzed were batch-spawners with an asynchronous ovarian organization. A continuous recruitment of oocytes and the occurrence of de novo vitellogenesis was also observed. During the spawning period, low atresia intensity was detected, while a marked increase in this intensity found in the ovaries at the end of spawning season. Our observations may support an indeterminate fecundity type for these species.

The Use of Reproductive Indicators for Conservation Purposes: The Case Study of Palinurus elephas in Two Fully Protected Areas and Their Surrounding Zones (Central-Western Mediterranean).

In 1990s, the European spiny lobster Palinurus elephas, one of the most commercially important species in the Mediterranean, exhibited a population decline. For this reason, fully protected areas (FPAs) appeared effective in re-establishing natural populations and supporting fishery-management objectives. Here, the reproductive parameters of P. elephas populations in two different FPAs (Su Pallosu and Buggerru, central-western Mediterranean), where a restocking programme was carried out, and in their surrounding commercial zones, were investigated from quantitative and qualitative perspectives. The comparison of fecundity between females collected inside and outside FPAs did not show statistical differences as well as the vitellogenin concentration, which did not vary among eggs of different size classes of females caught inside and outside the FPAs, indicating the same reproductive potential. The study demonstrated a benefit of overexploited populations in terms of enhancement of egg production overtime (15 years for Su Pallosu and 6 years for Buggerru) with a mean egg production 4.25-5.5 times higher at the end of the study than that observed at the beginning of the study. The main driver of eggs production appeared to be size, with larger lobsters more present inside the FPAs than outside. Given these results, the dominant contribution of the two studied FPAs to the regional lobster reproduction is remarkable.

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.

Scattered accumulation hotspots of macro-litter on the seafloor: Insights for mitigation actions.

Marine litter is an ever-increasing problem that demands immediate reduction plans and mitigation actions that should act synergically to efficiently meet ambitious goals. Since the seafloor has been recognized as the major sink for marine debris, the study of litter accumulation dynamics represent a fundamental tool to evaluate possible removal actions. We analysed a 7 years (2013-2019) standardized data series collected along Sardinian fishing grounds through MEDiterranean International Trawl Survey, for which estimates of density and weight of seafloor macro-litter were calculated for over 707 hauls. Results show the absence of any temporal trend in seafloor macro-litter density and weight, but rather indicate a spatial and bathymetric segregation of different litter categories. Our data showed how different sources and physical features of macro-litter items (i.e., plastic and fishing gear, rubber, glass, metals, and cloth) led to spatially segregated accumulation hotspots. We also point out here how the identification of seafloor macro-litter hotspots using aggregated data that include plastic items could obscure the identification of other segregated but yet relevant hotspots of other macro-litter categories accumulated in the marine environment. These hotspots often occurred at shallower depths and closer to coastlines, thus representing potential spots where eventual future litter removal action could be prioritized.

A Taxonomic Survey of Female Oviducal Glands in Chondrichthyes: A Comparative Overview of Microanatomy in the Two Reproductive Modes.

Oviducal glands (OGs) are distinct expanded regions of the anterior portion of the oviduct, commonly found in chondrichthyans, which play a key role in the production of the egg in-vestments and in the female sperm storage (FSS). The FSS phenomenon has implications for understanding the reproductive ecology and management of exploited populations, but little information is available on its taxonomic extent. For the first time, mature OGs from three lecithotrophic oviparous and four yolk-sac viviparous species, all considered at risk from the fishing impacts in the central western Mediterranean Sea, were examined using light microscopy. The OG microanatomy, whose morphology is generally conserved in all species, shows differences within the two reproductive modalities. Oviparous species show a more developed baffle zone in respect to viviparous ones because of the production of different egg envelopes produced. Among oviparous species, Raja polystigma and Chimaera monstrosa show presence of sperm, but not sperm storage as observed, instead, in Galeus melastomus and in all the viviparous sharks, which preserve sperm inside of specialized structures in the terminal zone.

Bio-Ecological Features Update on Eleven Rare Cartilaginous Fish in the Central-Western Mediterranean Sea as a Contribution for Their Conservation.

Cartilaginous fish are commonly recognized as key species in marine ecosystems for their fundamental ecological role as top predators. Nevertheless, effective management plans for cartilaginous fish are still missing, due to the lack of knowledge on their abundance, distribution or even life-history. In this regard, this paper aims at providing new information on the life-history traits, such as age, maturity, reproductive period, in addition to diet characteristics of eleven rare cartilaginous fish inhabiting the Central-Western Mediterranean Sea belonging to the orders Chimaeriformes (Chimaera monstrosa), Hexanchiformes (Heptranchias perlo and Hexanchus griseus), Myliobatiformes (Aetomylaeus bovinus and Myliobatis aquila), Rajiformes (Dipturus nidarosiensis and Leucoraja circularis), Squaliformes (Centrophorus uyato, Dalatias licha and Oxynotus centrina) and Torpediniformes (Tetronarce nobiliana), useful for their assessment and for future management actions. Particularly, the present paper provides for the first time the age estimation of D. nidarosienis and L. circularis which were both found capable of becoming older than ten years. In addition, the present study updates the sizes of first maturity of C. uyato and D. licha, which appeared to be capable of reproducing earlier than what was previously hypothesized, representing very valuable information for a better understanding of these rare species populations status and, eventually, their conservation. On the basis of the stomach content analysis, it was possible to identify five different predator groups.

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.

Scattered accumulation hotspots of macro-litter on the seafloor: Insights for mitigation actions.

Marine litter is an ever-increasing problem that demands immediate reduction plans and mitigation actions that should act synergically to efficiently meet ambitious goals. Since the seafloor has been recognized as the major sink for marine debris, the study of litter accumulation dynamics represents a fundamental tool to evaluate future removal actions. We analysed a 7 years (2013-2019) standardized data series collected along Sardinian fishing grounds through MEDiterranean International Trawl Survey, for which estimates of density and weight of seafloor macro-litter were calculated over 707 hauls. Results show the absence of any temporal trend in seafloor macro-litter density and weight, but rather indicate a spatial and bathymetric segregation of different litter categories. Our data showed how different sources and physical features of macro-litter items (i.e., plastic and fishing gears, rubber, glass, metal and textile) led to spatially segregated accumulation hotspots. These hotspots often occurred at shallower depths and closer to coastlines, representing spots where future litter removal action could be prioritized. We also point out here how the identification of seafloor macro-litter hotspots using aggregated data that include plastic items could indeed hide the identification of hotspots of other less abundant but yet detrimental macro-litter categories accumulated in the marine environment.

Chromosome Mapping of 5S Ribosomal Genes in Indo-Pacific and Atlantic Muraenidae: Comparative Analysis by Dual Colour Fluorescence In Situ Hybridisation.

The Muraenidae is one of the largest and most complex anguilliform families. Despite their abundance and important ecological roles, morays are little studied, especially cytogenetically, and both their phylogenetic relationships and the taxonomy of their genera are controversial. With the aim of extending the karyology of this fish group, the chromosomal mapping of the 5S ribosomal gene family was performed on seven species belonging to the genera Muraena and Gymnothorax from both the Atlantic and Pacific oceans. Fluorescence in situ hybridisation (FISH) experiments were realized using species-specific 5S rDNA probes; in addition, two-colour FISH was performed to investigate the possible association with the 45S ribosomal gene family. Multiple 5S rDNA clusters, located either in species-specific or in possibly homoeologous chromosomes, were found. Either a syntenic or different chromosomal location of the two ribosomal genes was detected. Our results revealed variability in the number and location of 5S rDNA clusters and confirmed a substantial conservation of the number and location of the 45S rDNA.

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.

Shelf-life and labels: A cheap dating tool for seafloor macro litter? Insights from MEDITS surveys in Sardinian sea.

The global plastic economy demands to reduce the flow of plastic into oceans and promote remedial actions for already accumulated seafloor litter. In this perspective, baseline levels of contamination and tools for dating litter items in order to assess the efficacy of those actions, are thus needed. In this note we discuss the utility of introducing the acquisition of shelf-life and labels features from litter items into already established standardized protocols such as the one proposed by MEDiterranean International Trawl Survey (MEDITS). Our investigation was conducted on 612 high resolution images of litter items collected in each haul, which was retrieved during 6 years of surveys (2013-2018) around Sardinian sea (central western Mediterranean). While for the majority of items (89%) expiration date or labels were not present or legible, over 50% of dated items were likely dumped at sea from a period <5 years. In this perspective, this sort of data could represent a useful tool for monitoring the effectiveness of input reduction actions that implicitly rely on the gradual reduction of recently dumped items.

European spiny lobster recovery from overfishing enhanced through active restocking in Fully Protected Areas.

Fully protected areas (FPAs) help preserving biodiversity and reversing the global decline of fishery resources. Stocks of the European spiny lobster Palinurus elephas (Fabr. 1787), among the most precious gourmet seafood worldwide, are currently facing a dramatic decline. Previous attempts of recovery based on fishery restrictions or active post-larval restocking in marine reserves provided unsuccessful outcomes. Here we present results of a 5-year restocking program carried through a Collaborative Fishery Research (CFR) project, in three ad-hoc established FPAs replenished using below-legal size wild juveniles. Results showed that Catch per Unit Effort (CPUE) in terms of both density and biomass burst (by ca. 300-700%) just 2 years since FPAs establishment, regardless of location. We also report tangible spillover effects (ca. 30-50% increase in density and biomass CPUE outside the FPAs) by the end of the program. Data from a 15-years lasting monitoring of a pilot FPA established in 1998, where the restocking protocol was conducted and protection kept in force once restocking ceased, demonstrated the persistence in time of restocking' benefits. We foster that creation of FPAs assisted with local restocking under oriented CFR programs can represent an option for the recovery of European spiny lobster stocks from overfishing.

Oviducal gland microstructure of Raja miraletus and Dipturus oxyrinchus (Elasmobranchii, Rajidae).

We studied the morphology and histology of the oviducal gland (OG) in the brown ray (Raja miraletus) and the long-nosed skate (Dipturus oxyrinchus) to understand its functional role in the reproductive strategy of these species. The external morphology of the gland was similar in both species, with lateral extensions like those found in other members of the Rajidae. Microscopic analysis showed a similar internal organization in both species. Immature and developing glands did not react to histochemical techniques. On reaching maturity, the OG had the largest width due to an increase in the production of secretory materials. In both species, the club zone of the gland showed a strong reaction to Periodic acid-Schiff (PAS) and alcian blue (AB) stains, indicating production of neutral and sulfated acid mucins. The secretory material produced by the papillary zone varied greatly between the two species. Both displayed tubular glands similar to those observed in the club zone, but in D. oxyrinchus the region near the lumen was intensely PAS+, whereas the last row of tubules of the brown ray stained intensely for a mixture of neutral and sulfated mucins. The baffle zone was the most conspicuous and extensive segment of all OGs, and it did not react to PAS/AB. The terminal zone, which is responsible for production of hair filaments, differed between the two species in terms of composition and organization of serous and mucous glands. This difference probably is related to the different substrates in which they release the egg capsules. Individual sperm detected in the brown ray baffle lamellae could be the result of a recent mating, whereas their presence in the deep recesses of the baffle and in the terminal zone of the long-nosed skate might indicate sperm storage.