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Next generation sequencing (NGS) technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin) and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric) and mammal (ciliary) classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic behaviour differences between the two species. The results confirm (i) the ability of these brittle stars to perceive light using opsin-based photoreception, (ii) suggest the co-occurrence of both rhabdomeric and ciliary photoreceptors, and (iii) emphasise the complexity of light perception in this echinoderm class.

The vast majority of species within the lanternshark genus Etmopterus harbour complex luminescent markings on their flanks, whose functional significance has long remained obscure. Recent studies, however, suggest these enigmatic photophore aggregations to play a role in intraspecific communication. Using visual modelling based on in vivo luminescence measurements from a common lanternshark species, we show that etmopterid flank markings can potentially work as a medium range signal for intraspecific detection/recognition. In addition, using molecular phylogenetic analyses, we demonstrate that the Etmopterus clade exhibits a greater than expected species richness given its age. This is not the case for other bioluminescent shark clades with no (or only few) species with flank markings. Our results therefore suggest that etmopterid flank markings may provide a way for reproductive isolation and hence may have facilitated speciation in the deep-sea.

Shipwrecks are almost the only subtidal substrata available for epifaunal colonization along the Belgian coastal waters and have never been scientifically prospected up to now. Two shipwrecks have been investigated during the summers of 2001 and 2002. De-visu observations and examination of 14 scraped surfaces of 0.0625 m2 allowed the identification of a total of 121 macrofauna species. Species richness cumulative curves provide an estimated number of 150–280 species. Both shipwrecks are dominated by Cnidarians. The tube-dwelling amphipod Jassa herdmani was also particularly abundant. Striking differences were observed between shipwrecks. Different faunal assemblages were observed on vertical and horizontal surfaces. Species richness could not been correlated with diversity indices. Samples with high species diversity were observed when the Hydrozoan Tubularia indivisa was dominant. On the contrary, when the Anthozoan Metridium senile was dominant, samples showed a very low species richness. Finally, the Poriferan Dysidea fragilis has to be considered as a new species for the Belgian fauna.

Counterilluminating animals use ventral photogenic organs (photophores) to mimic the residual downwelling light and cloak their silhouette from upward-looking predators. To cope with variable conditions of pelagic light environments they typically adjust their luminescence intensity. Here, we found evidence that bioluminescent sharks instead emit a constant light output and move up and down in the water column to remain cryptic at iso-luminance depth. We observed, across 21 globally distributed shark species, a correlation between capture depth and the proportion of a ventral area occupied by photophores. This information further allowed us, using visual modelling, to provide an adaptive explanation for shark photophore pattern diversity: in species facing moderate predation risk from below, counterilluminating photophores were partially co-opted for bioluminescent signalling, leading to complex patterns. In addition to increase our understanding of pelagic ecosystems our study emphasizes the importance of bioluminescence as a speciation driver.

The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (?max) fall within the range 484–491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep-sea sharks. In particular, bioluminescent sharks possess higher rod densities, which might provide them with improved temporal resolution particularly useful for bioluminescent communication during social interactions.

Tomopteris helgolandica Greeff 1879 (Tomopteridae) is a transparent holoplanktonic polychaete that can emit a bright light. In this study, we investigated the emission pattern and control of this deep-sea worm's luminescence. Potassium chloride depolarisation applied on anaesthetized specimens triggered a maximal yellow light emission from specific parapodial sites, suggesting that a nervous control pathway was involved. Pharmacological screening revealed a sensitivity to carbachol, which was confirmed by a dose–light response associated with a change in the light emission pattern, where physiological carbachol concentrations induced flashes and higher concentrations induced glows. The light response induced by its hydrolysable agonist, acetylcholine, was significantly weaker but was facilitated by eserine pretreatment. In addition, a specific inhibitory effect of tubocurarine was observed on carbachol-induced emission. Lastly, KCl- and carbachol-induced light responses were significantly reduced when preparations were pre-incubated in Ca2+-free artificial seawater or in different calcium channel blockers (verapamil, diltiazem) and calmodulin inhibitor (trifluoperazine) solutions. All of these results strongly suggest that T. helgolandica produces its light flashes via activation of nicotinic cholinergic receptors and a calcium-dependent intracellular mechanism involving L-type calcium channels.

Transparency is a common passive cryptic adaptation in pelagic environment.[1] In fact, by reducing light reflection and light scattering, biological transparency makes organism invisible. Paradoxally, many gelatinous zooplankton species are able to produce visible light.[2] So, what ecological advantage(s) bioluminescence could provide to such organisms? The model studied here is Tomopteris helgolandica, a transparent planktonic worm collected by trawling between 250 and 300 m depth in fjords near Bergen (Norway). The specimens were picked out and maintained during few days in sea water containers placed in a dark cold room. The bioluminescence potential was measured on pieces of three pairs of parapods from anaesthetized organisms. Each preparation was stocked in cold artificial sea water up to chemical induction of bioluminescence response.Available data about tomopterids bioluminescence were limited to anecdotic observations and morphological descriptions, poorly documented.[3] In way of having a global view of its bioluminescent potential, different aspects of the light emission process are currently explored, including neural basis of physiological control. After a screening of major pharmacological components, investigation has been oriented in cholinergic control hypothesis. It has been confirmed by dose-dependent emission of light in response to carbachol stimulation (Fig. 1). Inhibitory effect of tubocurarine on light emission suggested that nicotinic receptors were involved in the signal transmission pathway leading to light emission (Fig. 2). These results constitute the first data about neural control mechanisms in bioluminescence of a pelagic worm.Two annelid families have been previously studied and both concerned benthic species.[4] Only pharmacological investigations have been conducted so far but these results will be completed by electrophysiological and immunohistochemical approaches.

The smalleye pygmy shark (Squaliolus aliae) is a dwarf pelagic shark from the Dalatiidae family that harbours thousands of tiny photophores. In this work, we studied the organisation and physiological control of these photogenic organs. Results show that they are mainly situated on the ventral side of the shark, forming a homogeneous ventral photogenic area that appears well suited for counterillumination, a well-known camouflage technique of pelagic organisms. Isolated ventral skin patches containing photophores did not respond to classical neurotransmitters and nitric oxide but produced light after melatonin (MT) application. Prolactin and a-melanocyte-stimulating hormone inhibited this hormonally induced luminescence as well as the spontaneous luminescence from the photogenic tissue. The action of MT seems to be mediated by binding to the MT2 receptor subtype, as the MT2 receptor agonist 4P-PDOT inhibited the luminescence induced by this hormone. Binding to this receptor probably decreases the intracellular cAMP concentration because forskolin inhibited spontaneous and MT-induced luminescence. In addition, a GABA inhibitory tonus seems to be present in the photogenic tissue as well, as GABA inhibited MT-induced luminescence and the application of bicuculline provoked luminescence from S. aliae photophores. Similarly to what has been found in Etmopteridae, the other luminous shark family, the main target of the luminescence control appears to be the melanophores covering the photocytes. Results suggest that bioluminescence first appeared in Dalatiidae when they adopted a pelagic style at the Cretaceous/Tertiary boundary, and was modified by Etmopteridae when they started to colonize deep-water niches and rely on this light for intraspecific behaviours.

Social organization is an important attribute of the animal society. We describe the social structure of a bottlenose dolphins population living in Panama City, a seaside resort located on the north-west coast of Florida. Study was conducted with 46 individuals. Dolphins are associated on average half weight index of 0.11. Preferred long-term associations are observed. The proportion of the non-zero association indices suggests that some dolphins seem to avoid others. Associations between and within sex-classes were investigated using only dolphins of known sex and observed at least 4 times. Highly significant differences are found in associations between and within sex-classes (Mantel test, t = 3.7987; P = 1); indeed, male associations are stronger than between inter-sexual associations or between females only. Sociogram of males reveals a complex network with strong associations between pairs or trios that reach up to 0.97, whereas female associations are lower than males. The cluster analysis shows no clear division in the social organization of bottlenose dolphins in Panama City, except for dyads, triads and their multiple networks. The population structure seems to be temporally stable over the study and constant companionships are observed in the dolphin population in Panama City.

Photophores (photogenic organs) of the lantern shark Etmopterus spinax are under hormonal control, with prolactin (PRL) and melatonin (MT) triggering the light emission. Differential sensitivity to these hormones in adult individuals suggests, however, that the luminescence of this shark is controlled by an additional mechanism. In this study, different techniques were used to investigate a potential modulator of E. spinax luminescence – nitric oxide (NO). NO synthase (NOS)-like immunoreactivity (IR) was found in the photocytes (photogenic cells) of the photophores. In addition, acetylated tubulin IR also supported the presence of nerves running through the photogenic tissue and innervating different structural elements of the photophores: photocytes, pigmented cells from the iris-like structure and lens cells. Pharmacological experiments confirmed a modulatory action of NO on the hormonally induced luminescence: NO donors sodium nitroprusside (SNP) and hydroxylamine decreased the time to reach the maximum amplitude (TLmax) of MT-induced luminescence while these substances decreased the maximum amplitude of PRL-induced luminescence (and also the TLmax in the case of SNP). The small impact of the NOS inhibitor l-NAME on hormonally induced luminescence suggests that NO is only produced on demand. The cGMP analogue 8BrcGMP mimicked the effects of NO donors suggesting that the effects of NO are mediated by cGMP.

Lantern sharks are small deep-sea sharks that harbour complex species-specific luminescent photophore patterns. The luminescent pattern of one of these sharks, Etmopterus spinax, is made up of nine luminous zones. Previous experiments revealed that in the largest of these zones (ventral zone), photophores are under hormonal control, light being triggered by both melatonin (MT) and prolactin (PRL). In this study, we analysed the luminescent responses to MT and PRL in five other luminous zones from 12 female and eight male E. spinax specimens. The results showed that all luminous zones respond to both hormones, with each zone having its own kinetic parameters (maximum light intensity, Lmax; total light emitted, Ltot; time from stimulation to Lmax, TLmax), which confirms the multifunctional character of this shark's luminescence. Ltot and Lmax were found to be directly dependent on the photophore density (PD) of the luminous zone, while TLmax varied independently from PD. In addition, we demonstrate a sexual dimorphism in the luminescent response to PRL, with male specimens having smaller Ltot and TLmax in the luminous zones from the pelvic region. As this region also harbours the sexual organs of this species, this strongly suggests a role for the luminescence from these zones in reproduction.

Bioluminescence is known to be of great ecological importance to a luminous organism but extremely few studies investigate the ontogeny of luminous capabilities. The photogenic pattern of the velvet belly lantern shark Etmopterus spinax was investigated over ontogeny (14.0–52.5 cm total length) to determine the scaling of the surface area and the photophore density of different luminous zones as well as the ecological consequences of ontogenetic variations in bioluminescence efficiency. According to the luminous zone considered, different scaling patterns were found for the surface areas while the photophore densities of all zones scale with negative allometry, even though photophore insertion occurs. No sexual differences in these relationships were found. Luminous zones can be placed in two morphologically different groups: the “coverage” and the “isolated” zones. While counter-illumination is certainly the function of the former, the latter are probably involved in intraspecific behaviours. Due to the discrepancy between luminous capabilities of these two luminous zone categories, there is an ontogenetic increase in the luminescence heterogeneity of the luminous pattern as it was shown by luminescence modelling and confirmed by direct observations of spontaneous luminescence in living sharks. This heterogeneity certainly represents a trade-off between an efficient ventral camouflage and a strong identification tool for intraspecific behaviours such as coordinate hunting, which would be particularly useful when E. spinax become fish eaters (>19 cm total length), and for sexual recognition in mature individuals.

The main aim of the project is to document the fauna found on five shipwrecks in the Belgian part of the North Sea (map). While we have a fairly good understanding of the fauna of soft bottoms of our part of the North Sea, the fauna of these artificial hard substrates is largely unknown. Study of these habitats will allow us to understand species distribution patterns, and allow us to predict which species to expect on other artificial hard substrates, such as sokkels of wind mills. It is a two year research project carried out in the framework of the SPSD-II research action of the Belgian Federal Science Policy Office.

The development of luminous structures and the acquisition of luminescence competence during the ontogeny of the velvet belly lantern shark Etmopterus spinax, a deep-sea squalid species, were investigated. The sequential appearance of nine different luminous zones during shark embryogenesis were established, and a new terminology for them given. These zones form the complex luminous pattern observed in free-swimming animals. The organogenesis of photophores (photogenic organs) from the different luminous zones was followed, and photophore maturation was marked by the appearance of green fluorescent vesicles inside the photocytes (photogenic cells). Peroxide-induced light emissions as well as spontaneous luminescence analysis indicated that the ability of E. spinax to produce light was linked to the presence of these fluorescent vesicles and occured prior to birth. The size of photogenic organs, as well as the percentage of ventral body surface area occupied by the luminous pattern and covered by photophores increased sharply during embryogenesis but remained relatively stable in free-swimming animals. All these results strongly suggest camouflage by counter-illumination in juvenile E. spinax. (C) 2008 The Authors Journal compilation (C) 2008 The Fisheries Society of the British Isles

We have analyzed the composition, diversity, density and biomass of a temporal series of samples taken in a Tubularia indivisa community, which dominates a shipwreck in the North Sea waters (N 51°23',730-E 02°29',790, 17 nautical miles from the coast, 30 m depth). This shipwreck has structures emerging up to 8 m above the seabed. Water temperature ranged from 4.2°C in March to 20.3°C in August. Salinity showed few variations around 33.9 psu. Bottom tidal currents followed a semi-diurnal cycle and were preferentially NE oriented with 84% of them in the range 0.25-0.75 m s-1. The mean value for total suspended matter was 6.2 mg l-1 with large variations on a monthly scale. The species richness of samples varied from 15 in October to 42 in August with a mean value of 33 species. Diversity indices were higher during autumn and winter because of the strong dominance of a few crustacean species during the warmer months. The total density of individuals ranged from 6,500 ind m-2 in October to 445,800 ind m-2 in July, most of these individuals belonging to the amphipod species Jassa herdmani. The biomass of the T. indivisa community varied from 9 g AFDW m-2 in October to 1,106 g AFDW m-2 in July, with T. indivisa itself constituting between 59 and 82% of the total biomass. The biomass of T. indivisa was positively correlated with species richness and with the density of 23% of the species identified on this community, suggesting that T. indivisa plays an important structural role in this habitat. This was further confirmed by the number of species associated with T. indivisa which was generally superior to 55% of the sorted species. Multivariate analysis indicated strong differences between spring/summer-autumn/winter assemblages mostly but not solely due to the abundance patterns of species. These findings support the conclusion that shipwrecks in Belgian waters allow the development of assemblages dominated by a high biomass of T. indivisa which in turn provides shelter for high densities and biomass of epizoites. These assemblages will further show large monthly variations in densities and composition due to large variation in T. indivisa biomass under an apparent repetitive annual cycle.

We investigated the cover, community structure and abiotic environment of nine shipwrecks lying at increasing distance from the Belgian coast. Results indicated that all shipwrecks were strongly dominated by cnidarians in terms of biomass and by amphipods in terms of abundances. Based on their epifaunal composition, three groups of shipwrecks could be determined. Metridium senile dominated a species poor community of the coastal sites. On the same sites, a Tubularia larynx community with a more species-rich assemblage was also developing. The T. larynx community had a lower biomass value (102 g AFDW m-2) and significantly lower species richness compared to the other sites. The coastal sites were characterized by periodic salinity decreases, large seasonal temperature fluctuation, high total suspended matter load and reduced current velocity. Channel water masses influence the offshore sites causing a more stable temperature and salinity environment, less turbid waters and high current speed. Tubularia indivisa dominated this community, with an average biomass of 229 g AFDW m-2. Intermediate sites were also dominated by T. indivisa, but a higher biomass (424 g AFDW m-2) was observed. They showed intermediate results for the abiotic parameters and fast current velocities. Hypotheses for the observed variation in community structures are discussed in the light of the abiotic characterization of the shipwrecks.

Hard bottom substrates at sea allow the development of communities that are often rich in terms of species diversity. Non-biogenic structures such as shipwrecks are an integral part of these substrates, even if they have an anthropogenic origin and the species assemblages they harbor could be for that reason qualified as 'exotic'. There are 200 recent shipwrecks on the Belgian Continental Shelf (BCS), which represent a large fraction of the hard substrate available locally; their presence has an additional interest if we know that the major part of the English Channel and Southern Bight of the North Sea consists almost exclusively of soft sediments. Five shipwrecks on the BCS will be studied in order to assess the meio- and macrofaunal diversity using direct observations and scuba sampling techniques. The soft sediments close to shipwrecks will also be studied to serve as model for areas relatively undisturbed by fisheries (untrawled). Added to this, the influence of shipwrecks on local hydrodynamics and sediment transport will favor the colonization by fragile epibenthic species and as a consequence increase habitat complexity. For each site, standard abiotic parameters and current vectors will be measured and modeled. The information will be centralized in a database and disseminated through a web site devoted to the biodiversity of the BCS. The results will be relevant to the management of the BCS; the anthropogenic hard substrates of shipwrecks can serve as a model for what will happen with the installation of offshore windmills. A brochure will increase public awareness of the importance of marine diversity, and increase public support for marine protected areas.

Influence of temperature on the bioluminescence in the ophiuroid Amphiura filiformis was experimentally tested. Amputated arms from individuals acclimated at 6° or 14° were incubated at five different temperatures ranging from 6° to 14° for 2 hours before KCl stimulation. Light response comparisons do not reveal any difference neither for the two temperatures of acclimation nor for the five temperatures of incubation. Therefore, the temperature does not affect the studies parameters of the light emission.