Shark Microbiome Analysis Demonstrates Unique Microbial Communities in Two Distinct Mediterranean Sea Shark Species.

Our knowledge regarding the role of the microbiome in fish health has been steadily increasing in the last decade, especially for species of commercial interest. Conversely, relatively few studies focus on the microbiomes of wild fish, especially apex predators like sharks, due to lower economic interest and greater difficulty in obtaining samples. Studies investigating microbiome differences between diverse anatomical locations of sharks are limited, and the majority of the available studies are focused on the microbial diversity present on shark teeth, with the aim of preventing infections due to bites of these animals or evaluating the presence of certain pathogens in healthy or diseased specimens. Here, we investigated the skin, mouth, gills, and cloaca microbiomes of five individuals of two phylogenetically distant species of sharks (Prionace glauca and Somniosus rostratus) to obtain a better understanding of the diversity regarding the microbiomes of these animals, how they change throughout different body parts, and how much they are influenced and determined by the ecology and evolutionary relationship between host and microbiome. To confirm the taxonomy of the sharks under study, we barcoded the specimens by sequencing the mtDNA COI from a biopsy of their skin. Microbial diversity based on the 16S rRNA gene reveals that partially overlapping microbiomes inhabit different body parts of each shark species, while the communities are distinct between the two species. Our results suggest that sharks' microbiome species-specific differences are controlled by the ecology of the shark species. This is the first study comparatively analyzing the microbiome diversity of different anatomical locations in two shark species of the Mediterranean Sea.

Rhodobacteraceae dominate the core microbiome of the sea star Odontaster validus (Koehler, 1906) in two opposite geographical sectors of the Antarctic Ocean.

Microbiota plays essential roles in the health, physiology, and in adaptation of marine multi-cellular organisms to their environment. In Antarctica, marine organisms have a wide range of unique physiological functions and adaptive strategies, useful for coping with extremely cold conditions. However, the role of microbiota associated with Antarctic organisms in such adaptive strategies is underexplored. In the present study, we investigated the diversity and putative functions of the microbiome of the sea star Odontaster validus, one of the main keystone species of the Antarctic benthic ecosystems. We compared the whole-body bacterial microbiome of sea stars from different sites of the Antarctic Peninsula and Ross Sea, two areas located in two opposite geographical sectors of the Antarctic continent. The taxonomic composition of O. validus microbiomes changed both between and within the two Antarctic sectors, suggesting that environmental and biological factors acting both at large and local scales may influence microbiome diversity. Despite this, one bacterial family (Rhodobacteraceae) was shared among all sea star individuals from the two geographical sectors, representing up to 95% of the microbial core, and suggesting a key functional role of this taxon in holobiont metabolism and well-being. In addition, the genus Roseobacter belonging to this family was also present in the surrounding sediment, implying a potential horizontal acquisition of dominant bacterial core taxa via host-selection processes from the environment.

Marine Robotics for Deep-Sea Specimen Collection: A Taxonomy of Underwater Manipulative Actions.

In order to develop a gripping system or control strategy that improves scientific sampling procedures, knowledge of the process and the consequent definition of requirements is fundamental. Nevertheless, factors influencing sampling procedures have not been extensively described, and selected strategies mostly depend on pilots' and researchers' experience. We interviewed 17 researchers and remotely operated vehicle (ROV) technical operators, through a formal questionnaire or in-person interviews, to collect evidence of sampling procedures based on their direct field experience. We methodologically analyzed sampling procedures to extract single basic actions (called atomic manipulations). Available equipment, environment and species-specific features strongly influenced the manipulative choices. We identified a list of functional and technical requirements for the development of novel end-effectors for marine sampling. Our results indicate that the unstructured and highly variable deep-sea environment requires a versatile system, capable of robust interactions with hard surfaces such as pushing or scraping, precise tuning of gripping force for tasks such as pulling delicate organisms away from hard and soft substrates, and rigid holding, as well as a mechanism for rapidly switching among external tools.

Marine Robotics for Deep-Sea Specimen Collection: A Systematic Review of Underwater Grippers.

The collection of delicate deep-sea specimens of biological interest with remotely operated vehicle (ROV) industrial grippers and tools is a long and expensive procedure. Industrial grippers were originally designed for heavy manipulation tasks, while sampling specimens requires dexterity and precision. We describe the grippers and tools commonly used in underwater sampling for scientific purposes, systematically review the state of the art of research in underwater gripping technologies, and identify design trends. We discuss the possibility of executing typical manipulations of sampling procedures with commonly used grippers and research prototypes. Our results indicate that commonly used grippers ensure that the basic actions either of gripping or caging are possible, and their functionality is extended by holding proper tools. Moreover, the approach of the research status seems to have changed its focus in recent years: from the demonstration of the validity of a specific technology (actuation, transmission, sensing) for marine applications, to the solution of specific needs of underwater manipulation. Finally, we summarize the environmental and operational requirements that should be considered in the design of an underwater gripper.

Research Trends and Future Perspectives in Marine Biomimicking Robotics.

Mechatronic and soft robotics are taking inspiration from the animal kingdom to create new high-performance robots. Here, we focused on marine biomimetic research and used innovative bibliographic statistics tools, to highlight established and emerging knowledge domains. A total of 6980 scientific publications retrieved from the Scopus database (1950-2020), evidencing a sharp research increase in 2003-2004. Clustering analysis of countries collaborations showed two major Asian-North America and European clusters. Three significant areas appeared: (i) energy provision, whose advancement mainly relies on microbial fuel cells, (ii) biomaterials for not yet fully operational soft-robotic solutions; and finally (iii), design and control, chiefly oriented to locomotor designs. In this scenario, marine biomimicking robotics still lacks solutions for the long-lasting energy provision, which presently hinders operation autonomy. In the research environment, identifying natural processes by which living organisms obtain energy is thus urgent to sustain energy-demanding tasks while, at the same time, the natural designs must increasingly inform to optimize energy consumption.

Don't catch me if you can - Using cabled observatories as multidisciplinary platforms for marine fish community monitoring: An in situ case study combining Underwater Video and environmental DNA data.

Cabled observatories are marine infrastructures equipped with biogeochemical and oceanographic sensors as well as High-Definition video and audio equipment, hence providing unprecedented opportunities to study marine biotic and abiotic components. Additionally, non-invasive monitoring approaches such as environmental DNA (eDNA) metabarcoding have further enhanced the ability to characterize marine life. Although the use of non-invasive tools beholds great potential for the sustainable monitoring of biodiversity and declining natural resources, such techniques are rarely used in parallel and understanding their limitations is challenging. Thus, this study combined Underwater Video (UV) with eDNA metabarcoding data to produce marine fish community profiles over a 2 months period in situ at a cabled observatory in the northeast Atlantic (SmartBay Ireland). By combining both approaches, an increased number of fish could be identified to the species level (total of 22 species), including ecologically and economically important species such as Atlantic cod, whiting, mackerel and monkfish. The eDNA approach alone successfully identified a higher number of species (59%) compared to the UV approach (18%), whereby 23% of species were detected by both methods. The parallel implementation of point collection eDNA and time series UV data not only confirmed expectations of the corroborative effect of using multiple disciplines in fish community composition, but also enabled the assessment of limitations intrinsic to each technique including the identification of false-negative detections in one sampling technology relative to the other. This work showcased the usefulness of cabled observatories as key platforms for in situ empirical assessment of both challenges and prospects of novel technologies in aid to future monitoring of marine life.

First record of the pencil cardinal Epigonus denticulatus (Perciformes: Epigonidae) in the Azores archipelago.

The pencil cardinal Epigonus denticulatus is a small deep-water fish inhabiting continental slopes usually between 300 and 600 m depth. We report the first record of E. denticulatus in the Azores archipelago, where one specimen was found floating by fisherman off Faial island. Meristic and morphometric characters are in accordance with those reported for the species and molecular analyses further supported species identity. The record of E. denticulatus as a native species in the Azores increases the number of Epigonus species in the region to a total of three.

First record of the opal chimaera, Chimaera opalescens (Holocephali: Chimaeridae) and revision of the occurrence of the rabbitfish Chimaera monstrosa in the Azores waters.

The presence of the opal chimaera, Chimaera opalescens, is reported for the first time in the deep waters of the Azores, with the capture of four specimens by fishermen and the video recording of an additional five individuals. Species identification was supported by the 646 bp sequenced fragment of the mitochondrial gene cytochrome oxidase subunit I. Because C. opalescens is a recently recognised species that had been recurrently misidentified as rabbitfish, Chimaera monstrosa, the historical data of C. monstrosa in the Azores were reviewed to assess the possible presence of both Chimaera species in the region. Although several authors have reported the occurrence of C. monstrosa in the Azorean waters since the 1800s, the majority of these are based on only three specimens caught during the late 1800s. The investigation performed using literature and examination of the museum specimens still available concluded that the most likely scenario is that C. monstrosa is absent from the Azores and past records of that species in the region are most likely misidentifications of C. opalescens.

Locomotory behaviour of the intertidal marble crab (Pachygrapsus marmoratus) supports the underwater spring-loaded inverted pendulum as a fundamental model for punting in animals.

In aquatic pedestrian locomotion the dynamics of terrestrial and aquatic environments are coupled. Here we study terrestrial running and aquatic punting locomotion of the marine-living crab Pachygrapsus marmoratus. We detected both active and passive phases of running and punting through the observation of crab locomotory behaviour in standardized settings and by three-dimensional kinematic analysis of its dynamic gaits using high-speed video cameras. Variations in different stride parameters were studied and compared. The comparison was done based on the dimensionless parameter the Froude number (Fr) to account for the effect of buoyancy and size variability among the crabs. The underwater spring-loaded inverted pendulum (USLIP) model better fitted the dynamics of aquatic punting. USLIP takes account of the damping effect of the aquatic environment, a variable not considered by the spring-loaded inverted pendulum (SLIP) model in reduced gravity. Our results highlight the underlying principles of aquatic terrestrial locomotion by comparing it with terrestrial locomotion. Comparing punting with running, we show and increased stride period, decreased duty cycle and orientation of the carapace more inclined with the horizontal plane, indicating the significance of fluid forces on the dynamics due to the aquatic environment. Moreover, we discovered periodicity in punting locomotion of crabs and two different gaits, namely, long-flight punting and short-flight punting, distinguished by both footfall patterns and kinematic parameters. The generic fundamental model which belongs to all animals performing both terrestrial and aquatic legged locomotion has implications for control strategies, evolution and translation to robotic artefacts.

Towards Naples Ecological REsearch for Augmented Observatories (NEREA): The NEREA-Fix Module, a Stand-Alone Platform for Long-Term Deep-Sea Ecosystem Monitoring.

Deep-sea ecological monitoring is increasingly recognized as indispensable for the comprehension of the largest biome on Earth, but at the same time it is subjected to growing human impacts for the exploitation of biotic and abiotic resources. Here, we present the Naples Ecological REsearch (NEREA) stand-alone observatory concept (NEREA-fix), an integrated observatory with a modular, adaptive structure, characterized by a multiparametric video-platform to be deployed in the Dohrn canyon (Gulf of Naples, Tyrrhenian Sea) at ca. 650 m depth. The observatory integrates a seabed platform with optoacoustic and oceanographic/geochemical sensors connected to a surface transmission buoy, plus a mooring line (also equipped with depth-staged environmental sensors). This reinforced high-frequency and long-lasting ecological monitoring will integrate the historical data conducted over 40 years for the Long-Term Ecological Research (LTER) at the station "Mare Chiara", and ongoing vessel-assisted plankton (and future environmental DNA-eDNA) sampling. NEREA aims at expanding the observational capacity in a key area of the Mediterranean Sea, representing a first step towards the establishment of a bentho-pelagic network to enforce an end-to-end transdisciplinary approach for the monitoring of marine ecosystems across a wide range of animal sizes (from bacteria to megafauna).

New High-Tech Flexible Networks for the Monitoring of Deep-Sea Ecosystems.

Increasing interest in the acquisition of biotic and abiotic resources from within the deep sea (e.g., fisheries, oil-gas extraction, and mining) urgently imposes the development of novel monitoring technologies, beyond the traditional vessel-assisted, time-consuming, high-cost sampling surveys. The implementation of permanent networks of seabed and water-column-cabled (fixed) and docked mobile platforms is presently enforced, to cooperatively measure biological features and environmental (physicochemical) parameters. Video and acoustic (i.e., optoacoustic) imaging are becoming central approaches for studying benthic fauna (e.g., quantifying species presence, behavior, and trophic interactions) in a remote, continuous, and prolonged fashion. Imaging is also being complemented by in situ environmental-DNA sequencing technologies, allowing the traceability of a wide range of organisms (including prokaryotes) beyond the reach of optoacoustic tools. Here, we describe the different fixed and mobile platforms of those benthic and pelagic monitoring networks, proposing at the same time an innovative roadmap for the automated computing of hierarchical ecological information on deep-sea ecosystems (i.e., from single species' abundance and life traits to community composition, and overall biodiversity).

Multi-marker metabarcoding approach to study mesozooplankton at basin scale.

Zooplankton plays a pivotal role in marine ecosystems and the characterisation of its biodiversity still represents a challenge for marine ecologists. In this study, mesozooplankton composition from 46 samples collected in summer along the western Adriatic Sea, was retrieved by DNA metabarcoding analysis. For the first time, the highly variable fragments of the mtDNA COI and the V9 region of 18S rRNA genes were used in a combined matrix to compile an inventory of mesozooplankton at basin scale. The number of sequences retrieved after quality filtering were 824,148 and 223,273 for COI and 18S (V9), respectively. The taxonomical assignment against reference sequences, using 95% (for COI) and 97% (for 18S) similarity thresholds, recovered 234 taxa. NMDS plots and cluster analysis divided coastal from offshore samples and the most representative species of these clusters were distributed according to the dominant surface current pattern of the Adriatic for the summer period. For selected sampling sites, mesozooplankton species were also identified under a stereo microscope providing insights on the strength and weakness of the two approaches. In addition, DNA metabarcoding was shown to be helpful for the monitoring of non-indigenous marine metazoans and spawning areas of commercial fish species. We defined pros and cons of applying this approach at basin scale and the benefits of combining the datasets from two genetic markers.

The role of the Strait of Gibraltar in shaping the genetic structure of the Mediterranean Grenadier, Coryphaenoides mediterraneus, between the Atlantic and Mediterranean Sea.

Population genetic studies of species inhabiting the deepest parts of the oceans are still scarce and only until recently we started to understand how oceanographic processes and topography affect dispersal and gene flow patterns. The aim of this study was to investigate the spatial population genetic structure of the bathyal bony fish Coryphaenoides mediterraneus, with a focus on the Atlantic-Mediterranean transition. We used nine nuclear microsatellites and the mitochondrial cytochrome c oxidase I gene from 6 different sampling areas. No population genetic structure was found within Mediterranean with both marker types (mean ΦST = 0.0960, FST = -0.0003, for both P > 0.05). However, within the Atlantic a contrasting pattern of genetic structure was found for the mtDNA and nuclear markers (mean ΦST = 0.2479, P < 0.001; FST = -0.0001, P > 0.05). When comparing samples from Atlantic and Mediterranean they exhibited high and significant levels of genetic divergence (mean ΦST = 0.7171, FST = 0.0245, for both P < 0.001) regardless the genetic marker used. Furthermore, no shared haplotypes were found between Atlantic and Mediterranean populations. These results suggest very limited genetic exchange between Atlantic and Mediterranean populations of C. mediterraneus, likely due to the shallow bathymetry of the Strait of Gibraltar acting as a barrier to gene flow. This physical barrier not only prevents the direct interactions between the deep-living adults, but also must prevent interchange of pelagic early life stages between the two basins. According to Bayesian simulations it is likely that Atlantic and Mediterranean populations of C. mediterraneus were separated during the late Pleistocene, which is congruent with results for other deep-sea fish from the same region.

Substitutions in the Glycogenin-1 Gene Are Associated with the Evolution of Endothermy in Sharks and Tunas.

Despite 400-450 million years of independent evolution, a strong phenotypic convergence has occurred between two groups of fish: tunas and lamnid sharks. This convergence is characterized by centralization of red muscle, a distinctive swimming style (stiffened body powered through tail movements) and elevated body temperature (endothermy). Furthermore, both groups demonstrate elevated white muscle metabolic capacities. All these traits are unusual in fish and more likely evolved to support their fast-swimming, pelagic, predatory behavior. Here, we tested the hypothesis that their convergent evolution was driven by selection on a set of metabolic genes. We sequenced white muscle transcriptomes of six tuna, one mackerel, and three shark species, and supplemented this data set with previously published RNA-seq data. Using 26 species in total (including 7,032 tuna genes plus 1,719 shark genes), we constructed phylogenetic trees and carried out maximum-likelihood analyses of gene selection. We inferred several genes relating to metabolism to be under selection. We also found that the same one gene, glycogenin-1, evolved under positive selection independently in tunas and lamnid sharks, providing evidence of convergent selective pressures at gene level possibly underlying shared physiology.

The Pillars of Hercules as a bathymetric barrier to gene flow promoting isolation in a global deep-sea shark (Centroscymnus coelolepis).

Knowledge of the mechanisms limiting connectivity and gene flow in deep-sea ecosystems is scarce, especially for deep-sea sharks. The Portuguese dogfish (Centroscymnus coelolepis) is a globally distributed and near threatened deep-sea shark. C. coelolepis population structure was studied using 11 nuclear microsatellite markers and a 497-bp fragment from the mtDNA control region. High levels of genetic homogeneity across the Atlantic (Φ(ST) = -0.0091, F(ST) = 0.0024, P > 0.05) were found suggesting one large population unit at this basin. The low levels of genetic divergence between Atlantic and Australia (Φ(ST) = 0.0744, P < 0.01; F(ST) = 0.0015, P > 0.05) further suggested that this species may be able to maintain some degree of genetic connectivity even across ocean basins. In contrast, sharks from the Mediterranean Sea exhibited marked genetic differentiation from all other localities studied (Φ(ST) = 0.3808, F(ST) = 0.1149, P < 0.001). This finding suggests that the shallow depth of the Strait of Gibraltar acts as a barrier to dispersal and that isolation and genetic drift may have had an important role shaping the Mediterranean shark population over time. Analyses of life history traits allowed the direct comparison among regions providing a complete characterization of this shark's populations. Sharks from the Mediterranean had markedly smaller adult body size and size at maturity compared to Atlantic and Pacific individuals. Together, these results suggest the existence of an isolated and unique population of C. coelolepis inhabiting the Mediterranean that most likely became separated from the Atlantic in the late Pleistocene.

Transcriptome of the Deep-Sea Black Scabbardfish, Aphanopus carbo (Perciformes: Trichiuridae): Tissue-Specific Expression Patterns and Candidate Genes Associated to Depth Adaptation.

Deep-sea fishes provide a unique opportunity to study the physiology and evolutionary adaptation to extreme environments. We carried out a high throughput sequencing analysis on a 454 GS-FLX titanium plate using unnormalized cDNA libraries from six tissues of A. carbo. Assemblage and annotations were performed by Newbler and InterPro/Pfam analyses, respectively. The assembly of 544,491 high quality reads provided 8,319 contigs, 55.6% of which retrieved blast hits against the NCBI nonredundant database or were annotated with ESTscan. Comparison of functional genes at both the protein sequences and protein stability levels, associated with adaptations to depth, revealed similarities between A. carbo and other bathypelagic fishes. A selection of putative genes was standardized to evaluate the correlation between number of contigs and their normalized expression, as determined by qPCR amplification. The screening of the libraries contributed to the identification of new EST simple-sequence repeats (SSRs) and to the design of primer pairs suitable for population genetic studies as well as for tagging and mapping of genes. The characterization of the deep-sea fish A. carbo first transcriptome is expected to provide abundant resources for genetic, evolutionary, and ecological studies of this species and the basis for further investigation of depth-related adaptation processes in fishes.

Ancient divergence in the trans-oceanic deep-sea shark Centroscymnus crepidater.

Unravelling the genetic structure and phylogeographic patterns of deep-sea sharks is particularly challenging given the inherent difficulty in obtaining samples. The deep-sea shark Centroscymnus crepidater is a medium-sized benthopelagic species that exhibits a circumglobal distribution occurring both in the Atlantic and Indo-Pacific Oceans. Contrary to the wealth of phylogeographic studies focused on coastal sharks, the genetic structure of bathyal species remains largely unexplored. We used a fragment of the mitochondrial DNA control region, and microsatellite data, to examine genetic structure in C. crepidater collected from the Atlantic Ocean, Tasman Sea, and southern Pacific Ocean (Chatham Rise). Two deeply divergent (3.1%) mtDNA clades were recovered, with one clade including both Atlantic and Pacific specimens, and the other composed of Atlantic samples with a single specimen from the Pacific (Chatham Rise). Bayesian analyses estimated this splitting in the Miocene at about 15 million years ago. The ancestral C. crepidater lineage was probably widely distributed in the Atlantic and Indo-Pacific Oceans. The oceanic cooling observed during the Miocene due to an Antarctic glaciation and the Tethys closure caused changes in environmental conditions that presumably restricted gene flow between basins. Fluctuations in food resources in the Southern Ocean might have promoted the dispersal of C. crepidater throughout the northern Atlantic where habitat conditions were more suitable during the Miocene. The significant genetic structure revealed by microsatellite data suggests the existence of present-day barriers to gene flow between the Atlantic and Pacific populations most likely due to the influence of the Agulhas Current retroflection on prey movements.

Bathymetric barriers promoting genetic structure in the deepwater demersal fish tusk (Brosme brosme).

Population structuring in the North Atlantic deepwater demersal fish tusk (Brosme brosme) was studied with microsatellite DNA analyses. Screening eight samples from across the range of the species for seven loci revealed low but significant genetic heterogeneity (F(ST) = 0.0014). Spatial genetic variability was only weakly related to geographical (Euclidean) distance between study sites or separation of study sites along the path of major ocean currents. Instead, we found a significant effect of habitat, indicated by significant differentiation between relatively closely spaced sites: Rockall, which is surrounded by very deep water (>1000 m), and the Mid-Atlantic Ridge, which is separated from the European slope by a deep ocean basin, were differentiated from relatively homogeneous sites across the Nordic Seas. Limited adult migration across bathymetric barriers in combination with limited intersite exchange of pelagic eggs and larvae due to site-specific circulatory retention or poor survival during drift phases across deep basins may be reducing gene flow. We regard these limitations to gene flow as the most likely mechanisms for the observed population structure in this demersal species. The results underscore the importance of habitat boundaries in marine species.

Unexpected panmixia in a long-lived, deep-sea fish with well-defined spawning habitat and relatively low fecundity.

The marine environment presents particular challenges for our understanding of the factors that determine gene flow and consequent population structure. For marine fish, various aspects of life history have been considered important in an environment with few physical barriers, but dominated by current patterns, often varying with depth. These factors include the abundance and longevity of larval stages, typically more susceptible to movement along current paths. It also includes adult body size, fecundity and longevity with 'r-selected' species typically thought capable of greater gene flow and consequent panmixia. Here we investigate the population genetics of the orange roughy (Hoplostethus atlanticus), a clearly 'K-selected' species with habitat dependence on sea mounts for spawning, relatively large body size, a brief larval stage and relatively low fecundity. We used 14 polymorphic microsatellite loci to test the hypothesis that these characteristics will result in philopatry and genetic structure in the Atlantic Ocean. We discuss possible evolutionary mechanisms that could explain the results, which show the opposite pattern, with effective panmixia across thousands of kilometres in the North Atlantic.

Rapid polymerase chain reaction-restriction fragment length polymorphism method for discrimination of the two Atlantic cryptic deep-sea species of scabbardfish.

The present investigation provides an efficient diagnostic method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis to discriminate between two cryptic species of scabbardfish, Aphanopus carbo and A. intermedius, with commercial relevance in several European fish markets. Two DNA fragments from the mtDNA, including control region and partial cytochrome oxidase subunit I genes of about 1100 bp and 700 bp, respectively, were isolated by PCR amplification. Digestion of the amplicon including the control region with HaeII and the amplicon including the COI gene with Sau3AI restriction enzymes allowed an unequivocal discrimination between the two scabbardfish species. This PCR-RFLP method allowed a clear and rapid discrimination of the trichiurid species studied.