Atlantic Oceanic Squids in the "Grey Speciation Zone".

Cryptic species complexes represent an important challenge for the adequate characterization of Earth's biodiversity. Oceanic organisms tend to have greater unrecognized cryptic biodiversity since the marine realm was often considered to lack hard barriers to genetic exchange. Here, we tested the effect of several Atlantic and Mediterranean oceanic barriers on 16 morphospecies of oceanic squids of the orders Oegopsida and Bathyteuthida using three mitochondrial and one nuclear molecular marker and five species delimitation methods. Number of species recognized within each morphospecies differed among different markers and analyses, but we found strong evidence of cryptic biodiversity in at least four of the studied species (Chtenopteryx sicula, Chtenopteryx canariensis, Ancistrocheirus lesueurii, and Galiteuthis armata). There were highly geographically structured units within Helicocranchia navossae that could either represent recently diverged species or population structure. Although the species studied here can be considered relatively passive with respect to oceanic currents, cryptic speciation patterns showed few signs of being related to oceanic currents. We hypothesize that the bathymetry of the egg masses and duration of the paralarval stage might influence the geographic distribution of oceanic squids. Because the results of different markers and different species delimitation methods are inconsistent and because molecular data encompassing broad geographic sampling areas for oceanic squids are scarce and finding morphological diagnostic characters for early life stages is difficult, it is challenging to assess the species boundaries for many of these species. Thus, we consider many to be in the "grey speciation zone." As many oceanic squids have cosmopolitan distributions, new studies combining genomic and morphological information from specimens collected worldwide are needed to correctly assess the actual oceanic squid biodiversity.

Regional patterns of δ 13C and δ 15N for European common cuttlefish (Sepia officinalis) throughout the Northeast Atlantic Ocean and Mediterranean Sea.

Stable isotope compositions of carbon and nitrogen (expressed as δ 13C and δ 15N) from the European common cuttlefish (Sepia officinalis) were measured in order to evaluate the utility of using these natural tracers throughout the Northeast Atlantic Ocean and Mediterranean Sea (NEAO-MS). Mantle tissue was obtained from S. officinalis collected from 11 sampling locations spanning a wide geographical coverage in the NEAO-MS. Significant differences of both δ 13C and δ 15N values were found among S. officinalis samples relative to sampling location. δ 13C values did not show any discernable spatial trends; however, a distinct pattern of lower δ 15N values in the Mediterranean Sea relative to the NEAO existed. Mean δ 15N values of S. officinalis in the Mediterranean Sea averaged 2.5‰ lower than conspecifics collected in the NEAO and showed a decreasing eastward trend within the Mediterranean Sea with the lowest values in the most eastern sampling locations. Results suggest δ 15N may serve as a useful natural tracer for studies on the population structure of S. officinalis as well as other marine organisms throughout the NEAO-MS.

Potentially handicapped but otherwise functional: Malformations in prey capture tools show no impacts on octopus life.

Larval mortality is a keystone ecological factor for many benthic octopus since it mostly occurs before their settlement in the sea bottom as benthic juveniles. The literature had revealed that records of adult animals with morphological abnormalities (teratologies) are fewer in species with complex life cycle than in those with direct development. This is a direct consequence of the morphological, physiological, and development challenges that the transition from the larval to the adult morphology represents. During a routine fishing sample, we found an immature female horned octopus with additional buccal structures in two suckers of its ventral arms, likely rendering these suckers as inefficient. Based on the literature about the natural history of octopus, we provide evidence that these abnormalities were present at the moment of hatch. We evaluated the impact of the teratologies by comparing the shape of the buccal beaks and the trophic niche of the individual with five normal conspecifics. Although the beaks showed a different shape than normal individuals, the trophic niche was similar. Surprisingly, the teratological condition of the individual likely had no severe impacts on its life, even though it likely represents a handicap for its survival during its planktonic life. We also comment on other previous records from the literature of teratological adult octopus to highlight the amazing adaptive capacity of octopus to deal with challenging morphologies.

Predatory flying squids are detritivores during their early planktonic life.

Cephalopods are primarily active predators throughout life. Flying squids (family Ommastrephidae) represents the most widely distributed and ecologically important family of cephalopods. While the diets of adult flying squids have been extensively studied, the first feeding diet of early paralarvae remains a mystery. The morphology of this ontogenetic stage notably differs from other cephalopod paralarvae, suggesting a different feeding strategy. Here, a combination of Laser Capture Microdissection (LCM) and DNA metabarcoding of wild-collected paralarvae gut contents for eukaryotic 18S v9 and prokaryotic 16S rRNA was applied, covering almost every life domain. The gut contents were mainly composed by fungus, plants, algae and animals of marine and terrestrial origin, as well as eukaryotic and prokaryotic microorganisms commonly found in fecal pellets and particulate organic matter. This assemblage of gut contents is consistent with a diet based on detritus. The ontogenetic shift of diet from detritivore suspension feeding to active predation represents a unique life strategy among cephalopods and allows ommastrephid squids to take advantage of an almost ubiquitous and accessible food resource during their early stages. LCM was successfully applied for the first time to tiny, wild-collected marine organisms, proving its utility in combination with DNA metabarcoding for dietary studies.

Cephalopods as Predators: A Short Journey among Behavioral Flexibilities, Adaptions, and Feeding Habits.

The diversity of cephalopod species and the differences in morphology and the habitats in which they live, illustrates the ability of this class of molluscs to adapt to all marine environments, demonstrating a wide spectrum of patterns to search, detect, select, capture, handle, and kill prey. Photo-, mechano-, and chemoreceptors provide tools for the acquisition of information about their potential preys. The use of vision to detect prey and high attack speed seem to be a predominant pattern in cephalopod species distributed in the photic zone, whereas in the deep-sea, the development of mechanoreceptor structures and the presence of long and filamentous arms are more abundant. Ambushing, luring, stalking and pursuit, speculative hunting and hunting in disguise, among others are known modes of hunting in cephalopods. Cannibalism and scavenger behavior is also known for some species and the development of current culture techniques offer evidence of their ability to feed on inert and artificial foods. Feeding requirements and prey choice change throughout development and in some species, strong ontogenetic changes in body form seem associated with changes in their diet and feeding strategies, although this is poorly understood in planktonic and larval stages. Feeding behavior is altered during senescence and particularly in brooding octopus females. Cephalopods are able to feed from a variety of food sources, from detritus to birds. Their particular requirements of lipids and copper may help to explain why marine crustaceans, rich in these components, are common prey in all cephalopod diets. The expected variation in climate change and ocean acidification and their effects on chemoreception and prey detection capacities in cephalopods are unknown and needs future research.

Early Mode of Life and Hatchling Size in Cephalopod Molluscs: Influence on the Species Distributional Ranges.

Cephalopods (nautiluses, cuttlefishes, squids and octopuses) exhibit direct development and display two major developmental modes: planktonic and benthic. Planktonic hatchlings are small and go through some degree of morphological changes during the planktonic phase, which can last from days to months, with ocean currents enhancing their dispersal capacity. Benthic hatchlings are usually large, miniature-like adults and have comparatively reduced dispersal potential. We examined the relationship between early developmental mode, hatchling size and species latitudinal distribution range of 110 species hatched in the laboratory, which represent 13% of the total number of live cephalopod species described to date. Results showed that species with planktonic hatchlings reach broader distributional ranges in comparison with species with benthic hatchlings. In addition, squids and octopods follow an inverse relationship between hatchling size and species latitudinal distribution. In both groups, species with smaller hatchlings have broader latitudinal distribution ranges. Thus, squid and octopod species with larger hatchlings have latitudinal distributions of comparatively minor extension. This pattern also emerges when all species are grouped by genus (n = 41), but was not detected for cuttlefishes, a group composed mainly of species with large and benthic hatchlings. However, when hatchling size was compared to adult size, it was observed that the smaller the hatchlings, the broader the latitudinal distributional range of the species for cuttlefishes, squids and octopuses. This was also valid for all cephalopod species with benthic hatchlings pooled together. Hatchling size and associated developmental mode and dispersal potential seem to be main influential factors in determining the distributional range of cephalopods.

Cephalopod culture: current status of main biological models and research priorities.

A recent revival in using cephalopods as experimental animals has rekindled interest in their biology and life cycles, information with direct applications also in the rapidly growing ornamental aquarium species trade and in commercial aquaculture production for human consumption. Cephalopods have high rates of growth and food conversion, which for aquaculture translates into short culture cycles, high ratios of production to biomass and high cost-effectiveness. However, at present, only small-scale culture is possible and only for a few species: the cuttlefish Sepia officinalis, the loliginid squid Sepioteuthis lessoniana and the octopuses Octopus maya and O. vulgaris. These four species are the focus of this chapter, the aims of which are as follows: (1) to provide an overview of the culture requirements of cephalopods, (2) to highlight the physical and nutritional requirements at each phase of the life cycle regarded as essential for successful full-scale culture and (3) to identify current limitations and the topics on which further research is required. Knowledge of cephalopod culture methods is advanced, but commercialization is still constrained by the highly selective feeding habits of cephalopods and their requirement for large quantities of high-quality (preferably live) feed, particularly in the early stages of development. Future research should focus on problems related to the consistent production of viable numbers of juveniles, the resolution of which requires a better understanding of nutrition at all phases of the life cycle and better broodstock management, particularly regarding developments in genetic selection, control of reproduction and quality of eggs and offspring.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Cryobiology of cephalopod (Illex coindetii) spermatophores.

Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4 °C for 5 min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30 min. In a third experiment, we tested two cryopreservation protocols: LN2 vapor or -80 °C freezer, using a 15% cryoprotectant and 15 or 30 min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5-10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs.

Radioisotopes demonstrate the contrasting bioaccumulation capacities of heavy metals in embryonic stages of cephalopod species.

Cephalopods play a key role in many marine trophic food webs and also constitute alternative fishery resources in the context of the ongoing decline in finfish stocks. Most coastal cephalopod species of commercial importance migrate into shallow waters during the breeding season to lay their eggs, and are consequently subjected to coastal contamination. Eggs of common cuttlefish Sepia officinalis, European squid Loligo vulgaris, common octopus Octopus vulgaris and the sepiolid Rossia macrosoma were exposed during embryonic development to dissolved (110m)Ag, (109)Cd, (60)Co, (54)Mn and (65)Zn in order to determine their metal accumulation efficiencies and distribution among different egg compartments. Cuttlefish eggs, in which hard shells enclose the embryos, showed the lowest concentration factor (CF) values despite a longer duration of exposure. In contrast, octopus eggs, which are only protected by the chorionic membrane, accumulated the most metal. Uptake appears to be linked to the selective retention properties of the egg envelopes with respect to each element. The study also demonstrated that the octopus embryo accumulated (110m)Ag directly from the dissolved phase and also indirectly through assimilation of the contaminated yolk. These results raise questions regarding the potential contrasting vulnerability of early life stages of cephalopods to the metallic contamination of coastal waters.

The biodiversity of the Mediterranean Sea: estimates, patterns, and threats.

The Mediterranean Sea is a marine biodiversity hot spot. Here we combined an extensive literature analysis with expert opinions to update publicly available estimates of major taxa in this marine ecosystem and to revise and update several species lists. We also assessed overall spatial and temporal patterns of species diversity and identified major changes and threats. Our results listed approximately 17,000 marine species occurring in the Mediterranean Sea. However, our estimates of marine diversity are still incomplete as yet-undescribed species will be added in the future. Diversity for microbes is substantially underestimated, and the deep-sea areas and portions of the southern and eastern region are still poorly known. In addition, the invasion of alien species is a crucial factor that will continue to change the biodiversity of the Mediterranean, mainly in its eastern basin that can spread rapidly northwards and westwards due to the warming of the Mediterranean Sea. Spatial patterns showed a general decrease in biodiversity from northwestern to southeastern regions following a gradient of production, with some exceptions and caution due to gaps in our knowledge of the biota along the southern and eastern rims. Biodiversity was also generally higher in coastal areas and continental shelves, and decreases with depth. Temporal trends indicated that overexploitation and habitat loss have been the main human drivers of historical changes in biodiversity. At present, habitat loss and degradation, followed by fishing impacts, pollution, climate change, eutrophication, and the establishment of alien species are the most important threats and affect the greatest number of taxonomic groups. All these impacts are expected to grow in importance in the future, especially climate change and habitat degradation. The spatial identification of hot spots highlighted the ecological importance of most of the western Mediterranean shelves (and in particular, the Strait of Gibraltar and the adjacent Alboran Sea), western African coast, the Adriatic, and the Aegean Sea, which show high concentrations of endangered, threatened, or vulnerable species. The Levantine Basin, severely impacted by the invasion of species, is endangered as well. This abstract has been translated to other languages (File S1).

Utilidad de la teofilina en el Manejo del Asma Agudo en emergencia

El análisis de los resultados, muestra que ambas poblaciones fueron comparativamente similares en la medición del Peak Flow basal (antes de instaurarse el tratamiento), lo que posibilita poder hacer comparaciones entre el tratamiento en ambos grupos. El análisis de evolución dentro de los grupos (análisis pareado en T de student),muestra que en los pacientes qe siguieron el protocolo "A" desde el inicio del tratamiento broncodilatador hasta el final del mismo, fueron comparables a los pacientes que siguieron el protocolo "B". Esto demuestra que el agregado de teofilina al tratamiento broncodilatador en asma aguda leve, moderada y seera no proporciona mayor broncodilatación que la sola utiliación de beta agonistas y corticoides. Concluimos por tanto que ambos tratamiento son efectivos en el tratamiento del asma aguda en emergencia.