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In recent years, flathead grey mullets (Mugil cephalus) cultured in Eilat (Israel) have been highly affected by Vibrio harveyi, showing neurological signs such as uncoordinated circular swimming followed by high mortality rates. Despite the advances in and different approaches to control vibriosis associated with Vibrio harveyi, including commercial vaccines, most of them have not succeeded in long-term protection. In this study, we evaluated the effectiveness, long-term protection, and antibody production of three vaccine preparations: heat-killed bacteria (HKB), membrane proteins denaturation (BME PROT), and internal proteins (INT PROT) developed specifically against Vibrio harveyi for grey mullets. Our results show that fish immunized with heat-killed bacteria emulsified with adjuvant presented the most effective and long-lasting protection against the bacterium, and a cross-protection against other bacteria from the harveyi clade. The effectiveness of each immunization treatment correlated with the levels of specific antibody production against Vibrio harveyi in the serum of the immunized fish.
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Enfermedades de los Peces , Smegmamorpha , Vibriosis , Vibrio , Animales , Vacunas Bacterianas , Vibriosis/prevención & control , Vibriosis/veterinaria , InmunizaciónRESUMEN
Dietary influence on the microbiome in algivorous sea urchins such as Tripneustes gratilla elatensis suggests a bacterial contribution to the digestion of fiber-rich seaweed. An ecological insight into the spatial arrangement in the gut bacterial community will improve our knowledge of host-microbe relations concerning the involved taxa, their metabolic repertoire, and the niches of activity. Toward this goal, we investigated the bacterial communities in the esophagus, stomach, and intestine of Ulva-fed sea urchins through 16S rRNA amplicon sequencing, followed by the prediction of their functional genes. We revealed communities with distinct features, especially those in the esophagus and intestine. The esophageal community was less diverse and was poor in food digestive or fermentation genes. In contrast, bacteria that can contribute to the digestion of the dietary Ulva were common in the stomach and intestine and consisted of genes for carbohydrate decomposition, fermentation, synthesis of short-chain fatty acids, and various ways of N and S metabolism. Bacteroidetes and Firmicutes were found as the main phyla in the gut and are presumably also necessary in food digestion. The abundant sulfate-reducing bacteria in the stomach and intestine from the genera Desulfotalea, Desulfitispora, and Defluviitalea may aid in removing the excess sulfate from the decomposition of the algal polysaccharides. Although these sea urchins were fed with Ulva, genes for the degradation of polysaccharides of other algae and plants were present in this sea urchin gut microbiome. We conclude that the succession of microbial communities along the gut obtained supports the hypothesis on bacterial contribution to food digestion. IMPORTANCE Alga grazing by the sea urchin Tripneustes gratilla elatensis is vital for nutrient recycling and constructing new reefs. This research was driven by the need to expand the knowledge of bacteria that may aid this host in alga digestion and their phylogeny, roles, and activity niches. We hypothesized alterations in the bacterial compositional structure along the gut and their association with the potential contribution to food digestion. The current spatial insight into the sea urchin's gut microbiome ecology is novel and reveals how distinct bacterial communities are when distant from each other in this organ. It points to keynote bacteria with genes that may aid the host in the digestion of the complex sulfated polysaccharides in dietary Ulva by removing the released sulfates and fermentation to provide energy. The gut bacteria's genomic arsenal may also help to gain energy from diets of other algae and plants.
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Bacterias , Erizos de Mar , Animales , ARN Ribosómico 16S/genética , Bacterias/genética , Erizos de Mar/genética , Alimentos Marinos , Digestión , SulfatosRESUMEN
Halotolerant bacteria capable of starch hydrolysis by their amylases will benefit various industries, specifically since the hydrolytic activity of current industrial amylases is inhibited or even absent in salt-rich or alkaline environments. Seeking novel enzymes, we analyzed the entire genome content of a marine bacterium isolated from the gut of sea urchins to compare it against other bacterial genomes. Conditions underlying α-amylase activity were examined in vitro at various salinities (0 to 4%) and temperatures (25°C to 37°C). Genomic analyses revealed the isolated bacterium as a new species of Alkalihalobacillus. Comparative analysis of the contents of carbohydrate-active enzymes revealed various α-amylases, each with its respective carbohydrate-binding module for starch hydrolysis. Functional analysis identified the hydrolysis of starch and the maltooligosaccharides maltose and dextrin into d- and UDP-glucose. The fastest growth and α-amylase production occurred at 3% salinity at a temperature of 30°C. The Alkalihalobacillus sp. consists of exclusive contents of α-amylases and other enzymes that may be valuable in the hydrolysis of the algal polysaccharides cellulose and laminarin. IMPORTANCE Toward the discovery of novel carbohydrate-active enzymes that may be useful in the hydrolysis of starch, we examined a halotolerant bacterial isolate of Alkalihalobacillus sp. regarding its genomic content and conditions underlying the production of active α-amylases. The production of α-amylases was measured in bacterial cultures at relatively high temperature (37°C) and salinity (4%). The Alkalihalobacillus sp. revealed an exclusive content of amylases and other carbohydrate-active enzymes compared to other relevant bacteria. These enzymes may be valuable for the hydrolysis of algal polysaccharides. The enzymatic cascade of the Alkalihalobacillus sp. for starch metabolism allows polysaccharide degradation into monosugars while preventing the accumulation of intermediate inhibitors of maltose or dextrin.
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Maltosa , Almidón , Amilasas , Dextrinas , Concentración de Iones de Hidrógeno , Hidrólisis , Polisacáridos , Almidón/química , Almidón/metabolismo , Temperatura , alfa-Amilasas/química , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Shipwrecks attract divers. Therefore, decommissioned marine vehicles are used as artificial reefs for recreational diving tourism around the world. We examined divers' attitudes towards shipwrecks and their function as an ecological enhancers. The study was carried out in the Karaburun Peninsula, Turkey, where two passenger ships were sunk as artificial wreck reefs to attract diving tourism. Using a questionnaire survey accompanied by face-to-face interviews, of both certified divers and non-certified discovery divers we collected information about divers reefs preferences, perception of artificial reefs as well as their socio-demographic characteristics, dive experiences etc. Differences in divers' wreck (historical or artificial) and reef preferences (natural reefs or artificial wrecks) were analyzed with the Mann-Whitney U test. Overall, divers held the position that artificial wreck reefs provide ecological benefits on fish diversity and stocks (p < 0.05), and experienced divers supported this position more that novice ones. However, experience and number of annual dives did not affect this notion. Non-certified respondents were mostly neutral towards the ecologic and management statements. However, the majority of divers (84%) preferred to dive on natural reefs rather than on shipwrecks. Similarly, most of them (83%) preferred diving to historical shipwrecks which sank in wars or accidents, and not on ships intentionally sunk as attractions. These findings, which are in line with previous literature, put a question mark on the growing practice of sinking nearly any ship to create diving attractions.
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Antozoos , Buceo , Animales , Conservación de los Recursos Naturales , Peces , TurquíaRESUMEN
Aggregated larval co-settlement has been documented in myriad marine invertebrate taxa, shaping adult population structures. Still, kinship settlement patterns in brooding corals have not been studied in detail, especially under scenarios of enhanced larval assemblies. Employing two sets of ex-situ experiments, planulae staining for kinship resolution and a computer random settlement simulation, we show that larval settlement of the coral Stylophora pistillata, a brooding species in the Gulf of Aqaba/Eilat, is mostly affected by the number of larval donors, and that larvae tend to aggregate (up to 50% tissue-contacts; distances <3 mm), compared to 3% predicted in a computer simulation, all without a kinship-bias. Field surveys on juvenile colonies revealed a similar clustering pattern. Although aggregated settlement inevitably carries disadvantages such as intraspecific competition, it may be bracketed in adult colonies with benefits such as enhanced fertilization and chimerism-related ecological advantages, including augmented colony size and survivorship. These improved life-history traits of brooding coral species that aggregate could be harnessed as applied ecological engineering tools in reef restoration acts.
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Antozoos , Animales , Simulación por Computador , Arrecifes de Coral , LarvaRESUMEN
Flathead grey mullet Mugil cephalus is an important species in the aquaculture industry in the Mediterranean basin and throughout the world. During the last 10 yr, M. cephalus breeding stocks, larvae, and juveniles cultured in Eilat (Israel) have shown neurological signs such as uncoordinated circular swimming, while also presenting oral hemorrhages. Death follows days after the onset of the clinical signs, and mortality rates may reach 80% in some cases, causing high economical losses. Bacteriology isolations from different organs, including the brain, and a Koch's postulate experiment, confirmed Vibrio harveyi as the causative agent. Histological analyses showed the presence of the bacterium in different organs. However, in the brain, the bacterium was observed only within blood vessels and meninges. In some samples, mild to severe brain tissue damage was seen. In order to understand the virulence and lethality of V. harveyi, a median lethal dose was calculated, and the result was 106 colony-forming units fish-1. To the best of our knowledge, this is the first report that describes V. harveyi isolated from the brain of M. cephalus and validates it as an etiological agent causing neurological signs in this fish species.
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Smegmamorpha , Vibrio , Animales , Peces , MorbilidadRESUMEN
BACKGROUND: Algivorous sea urchins can obtain energy from a diet of a single algal species, which may result in consequent changes in their gut microbe assemblies and association networks. METHODS: To ascertain whether such changes are led by specific microbes or limited to a specific region in the gut, we compared the microbial assembly in the three major gut regions of the sea urchin Tripneustes gratilla elatensis when fed a mono-specific algal diet of either Ulva fasciata or Gracilaria conferta, or an algal-free diet. DNA extracts from 5 to 7 individuals from each diet treatment were used for Illumina MiSeq based 16S rRNA gene sequencing (V3-V4 region). Niche breadth of each microbe in the assembly was calculated for identification of core, generalist, specialist, or unique microbes. Network analyzers were used to measure the connectivity of the entire assembly and of each of the microbes within it and whether it altered with a given diet or gut region. Lastly, the predicted metabolic functions of key microbes in the gut were analyzed to evaluate their potential contribution to decomposition of dietary algal polysaccharides. RESULTS: Sea urchins fed with U. fasciata grew faster and their gut microbiome network was rich in bacterial associations (edges) and networking clusters. Bacteroidetes was the keystone microbe phylum in the gut, with core, generalist, and specialist representatives. A few microbes of this phylum were central hub nodes that maintained community connectivity, while others were driver microbes that led the rewiring of the assembly network based on diet type through changes in their associations and centrality. Niche breadth agreed with microbes' richness in genes for carbohydrate active enzymes and correlated Bacteroidetes specialists to decomposition of specific polysaccharides in the algal diets. CONCLUSIONS: The dense and well-connected microbial network in the gut of Ulva-fed sea urchins, together with animal's rapid growth, may suggest that this alga was most nutritious among the experimental diets. Our findings expand the knowledge on the gut microbial assembly in T. gratilla elatensis and strengthen the correlation between microbes' generalism or specialism in terms of occurrence in different niches and their metabolic arsenal which may aid host nutrition.
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Chimerism is a coalescence of conspecific genotypes. Although common in nature, fundamental knowledge, such as the spatial distribution of the genotypes within chimeras, is lacking. Hence, we investigated the spatial distribution of conspecific genotypes within the brooding coral Stylophora pistillata, a common species throughout the Indo-Pacific and Red Sea. From eight gravid colonies, we collected planula larvae that settled in aggregates, forming 2-3 partner chimeras. Coral chimeras grew in situ for up to 25 months. Nine chimeras (8 kin, 1 non-related genotypes) were sectioned into 7-17 fragments (6-26 polyps/fragment), and genotyped using eight microsatellite loci. The discrimination power of each microsatellite-locus was evaluated with 330 'artificial chimeras,' made by mixing DNA from three different S. pistillata genotypes in pairwise combinations. In 68% of 'artificial chimeras,' the second genotype was detected if it constituted 5-30% of the chimera. Analyses of S. pistillata chimeras revealed that: (a) chimerism is a long-term state; (b) conspecifics were intermixed (not separate from one another); (c) disproportionate distribution of the conspecifics occurred; (d) cryptic chimerism (chimerism not detected via a given microsatellite) existed, alluding to the underestimation of chimerism in nature. Mixed chimerism may affect ecological/physiological outcomes for a chimera, especially in clonal organisms, and challenges the concept of individuality, affecting our understanding of the unit of selection.
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Antozoos/genética , Quimerismo , Repeticiones de Microsatélite , Animales , Antozoos/crecimiento & desarrollo , Evolución Molecular , GenotipoRESUMEN
Lacking an external shell and a rigid endoskeleton, octopuses exhibit a remarkable flexibility in their movements. Bipedal locomotion is perhaps the most iconic example in this regard. Until recently, this peculiar mode of locomotion had been observed only in two species of tropical octopuses: Amphioctopus marginatus and Abdopus aculeatus. Yet, recent evidence indicates that bipedal walking is also part of the behavioral repertoire of the common octopus, Octopus vulgaris. Here we report a further observation of a defense behavior that encompasses both postural and locomotory elements of bipedal locomotion in this cephalopod. By highlighting differences and similarities with the other recently published report, we provide preliminary considerations with regard to bipedal locomotion in the common octopus.
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In La Manche (English Channel) the level of turbidity changes, not only seasonally and daily in seawater but also along the coast. As a consequence, vision in marine species is limited when based only on contrast-intensity. It is hypothesized that polarization sensitivity (PS) may help individuals detect preys and predators in turbid environments. In the cuttlefish, Sepia officinalis, to date, all behavioral studies have been conducted on animals reared in clear water. But the cuttlefish sensory system is adapted to a range of turbid environments. Our hypothesis was that rearing cuttlefish in clear water may affect the development of their visual system, and potentially affect their visually guided behaviors. To test this, newly-hatched cuttlefish, from eggs laid by females brought in from the wild, were reared for 1 month under three different conditions: clear water (C group), low turbidity (0.1 g / l of clay, 50-80 NTU, LT group) and high turbidity (0.5 g / l of clay, 300-400 NTU, HT group). The visual capacities of cuttlefish were tested with an optomotor apparatus at 7 days and at 1 month post-hatching. Optomotor responses of juveniles were measured by using three screen patterns (black and white stripes, linearly polarized stripes set at different orientations, and a uniform gray screen). Optomotor responses of juveniles suggest that exposure to turbid water improves the development of their PS when tested in clear water (especially in LT group) but not when tested in turbid water. We suggest that the use of slightly turbid water in rearing systems may improve the development of vision in young cuttlefish with no detrimental effect to their survival rate. Future research will consider water turbidity as a possible factor for the improvement of cuttlefish well-being in artificial rearing systems.
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BACKGROUND: Most studies on architectural rules in corals have focused on the branch and the colony level, unveiling a variety of allometric rules. Working on the branching coral Stylophora pistillata, here we further extend the astogenic directives of this species at the polyp level, to reveal allometric and morphometric rules dictating polyps' arrangement. RESULTS: We identified a basic morphometric landscape as a six-polyp circlet developed around a founder polyp, with established distances between polyps (six equilateral triangles), reflecting a strong genetic-based background vs high plasticity on the population level. Testing these rules in regular and chimeric S. pistillata colonies, we revealed similar morphometric/allometric rules developed via a single astogenic pathway. In regular colonies, this pathway was driven by the presence/absence of intra-circlet budding polyps, while in chimeras, by the distances between the two founder polyps. In addition, we identified the intra-circlet budding as the origin of first branching, if BPC distances are kept <1.09 ± 0.25 mm. CONCLUSIONS: The emerged allometric/morphometric rules indicate the existence of a positional information paradigm for polyps' landscape distribution, where each polyp creates its own positional field of morphogen gradients through six inductive sites, thus forming six positional fields for the development of the archetypal "six-polyp crown".
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Antozoos/crecimiento & desarrollo , Animales , Arrecifes de CoralRESUMEN
Artificial reefs are increasingly being used around the globe to attract recreational divers, for both environmental and commercial reasons. This paper examines artificial coral reefs as recreational ecosystem services (RES) by evaluating their attractiveness and effectiveness and by examining divers' attitudes toward them. An online survey targeted at divers in Israel (n = 263) indicated that 35% of the dives in Eilat (a resort city on the shore of the Red Sea) take place at artificial reefs. A second study monitored divers' behavior around the Tamar artificial reef, one of the most popular submerged artificial reefs in Eilat, and juxtaposed it with divers' activities around two adjacent natural reefs. Findings show that the average diver density at the artificial reef was higher than at the two nearby natural knolls and that the artificial reef effectively diverts divers from natural knolls. A third study that examined the attitudes towards natural vs. artificial reefs found that the artificial reefs are considered more appropriate for training, but that divers feel less relaxed around them. By utilizing the RES approach as a framework, the study offers a comprehensive methodology that brings together the aesthetic, behavioral, and attitudinal aspects in terms of which artificial reefs can be evaluated.
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Conservación de los Recursos Naturales , Arrecifes de Coral , Animales , Antozoos , Buceo , Ecosistema , Humanos , Océano Índico , IsraelRESUMEN
While octopuses are mostly benthic animals, and squid prefer the open waters, cuttlefish present a special intermediate stage. Although their body structure resembles that of a squid, in many cases their behavior is mostly benthic. To test cuttlefish's preference in the use of space, we trained juvenile Sepia gibba and Sepia officinalis cuttlefish to reach a shelter at the opposite side of a tank. Afterwards, rock barriers were placed between the starting point and the shelter. In one experiment, direct paths were available both through the sand and over the rocks. In a second experiment the direct path was blocked by small rocks requiring a short detour to by-pass. In the third experiment instead, the only direct path available was over the rocks; or else to reach the goal via an exclusively horizontal path a longer detour would have to be selected. We showed that cuttlefish prefer to move horizontally when a direct route or a short detour path is available close to the ground; however when faced with significant obstacles they can and would preferentially choose a more direct path requiring a vertical movement over a longer exclusively horizontal path. Therefore, cuttlefish appear to be predominantly benthic dwellers that prefer to stay near the bottom. Nonetheless, they do view and utilize the vertical space in their daily movements where it plays a role in night foraging, obstacles negotiation and movement in their home-range.
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Polarized light detection has been documented in only a small number of fish species. The benefit of polarization vision for fish is not fully understood, nor is the transduction mechanism that underlies it. Past studies proposed that one possible advantage of polarization vision is that it enhances the contrast of zooplankton targets by breaking their transparency. Here, we used an optomotor apparatus to test the responses of the planktivorous Hardyhead silverside fish Atherinomorus forskalii (Atherinidae) to vertical unpolarized (intensity) and polarized gratings. We also tested and compared the spatial and temporal resolutions of A. forskalii in the intensity and polarization domains. A. forskalii responded to the polarization pattern, but only under illumination that included ultraviolet-blue (λ>380nm) wavelengths. The spatial resolution of A. forskalii was measured as a minimum separable angle of 0.57° (a 1-mm prey viewed from 100-mm distance). The temporal resolution to unpolarized vs. polarized gratings was constant, at 33 and 10Hz respectively at most of the stripe widths tested. At the smallest stripe width tested (1mm=the minimal separable angle), which correlates with the size of prey typically consumed by these fish, the temporal resolution to the polarized grating increased to 42Hz. We conclude that A. forskalii is polarization sensitive, may use polarization vision to improve detection of its planktonic prey, and that polarization may be perceived by the fish via a separate visual pathway than intensity.
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Sensibilidad de Contraste/fisiología , Reconocimiento Visual de Modelos/fisiología , Células Fotorreceptoras/fisiología , Conducta Predatoria/fisiología , Smegmamorpha/fisiología , Percepción Visual/fisiología , Zooplancton , Animales , Conducta Animal , Luz , Océanos y Mares , Fenómenos Fisiológicos Oculares , Dispersión de Radiación , Especificidad de la EspecieRESUMEN
The world is three-dimensional; hence, even surface-bound animals need to learn vertical spatial information. Separate encoding of vertical and horizontal spatial information seems to be the common strategy regardless of the locomotory style of animals. However, a difference seems to exist in the way freely moving species, such as fish, learn and integrate spatial information as opposed to surface-bound species, which prioritize the horizontal dimension and encode it with a higher resolution. Thus, the locomotory style of an animal may shape how spatial information is learned and prioritized. An alternative hypothesis relates the preference for vertical information to the ability to sense hydrostatic pressure, a prominent cue unique to this dimension. Cuttlefish are mostly benthic animals, but they can move freely in a volume. Therefore, they present an optimal model to examine these hypotheses. We tested whether cuttlefish could separately recall the vertical and horizontal components of a learned two-dimensional target, and whether they have a preference for vertical or horizontal information. Sepia officinalis cuttlefish were trained to select one of two visual cues set along a 45 deg diagonal. The animals were then tested with the two visual cues arranged in a horizontal, vertical or opposite 45 deg configuration. We found that cuttlefish use vertical and horizontal spatial cues separately, and that they prefer vertical information to horizontal information. We propose that, as in fish, the availability of hydrostatic pressure, combined with the ecological value of vertical movements, determines the importance of vertical information.
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Conservation and environmental management are principal countermeasures to the degradation of marine ecosystems and their services. However, in many cases, current practices are insufficient to reverse ecosystem declines. We suggest that restoration ecology, the science underlying the concepts and tools needed to restore ecosystems, must be recognized as an integral element for marine conservation and environmental management. Marine restoration ecology is a young scientific discipline, often with gaps between its application and the supporting science. Bridging these gaps is essential to using restoration as an effective management tool and reversing the decline of marine ecosystems and their services. Ecological restoration should address objectives that include improved ecosystem services, and it therefore should encompass social-ecological elements rather than focusing solely on ecological parameters. We recommend using existing management frameworks to identify clear restoration targets, to apply quantitative tools for assessment, and to make the re-establishment of ecosystem services a criterion for success.
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Camouflage is common throughout the phylogenetic tree and is largely used to minimize detection by predator or prey. Cephalopods, and in particular Sepia officinalis cuttlefish, are common models for camouflage studies. Predator avoidance behavior is particularly important in this group of soft-bodied animals that lack significant physical defenses. While previous studies have suggested that immobile cephalopods selectively camouflage to objects in their immediate surroundings, the camouflage characteristics of cuttlefish during movement are largely unknown. In a heterogenic environment, the visual background and substrate feature changes quickly as the animal swim across it, wherein substrate patch is a distinctive and high contrast patch of substrate in the animal's trajectory. In the current study, we examine the effect of substrate patch size on cuttlefish camouflage, and specifically the minimal size of an object for eliciting intensity matching response while moving. Our results indicated that substrate patch size has a positive effect on animal's reflectance change, and that the threshold patch size resulting in camouflage response falls between 10 and 19 cm (width). These observations suggest that the animal's length (7.2-12.3 cm mantle length in our case) serves as a possible threshold filter below which objects are considered irrelevant for camouflage, reducing the frequency of reflectance changes-which may lead to detection. Accordingly, we have constructed a computational model capturing the main features of the observed camouflaging behavior, provided for cephalopod camouflage during movement.
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A moving object is considered conspicuous because of the movement itself. When moving from one background to another, even dynamic camouflage experts such as cephalopods should sacrifice their extraordinary camouflage. Therefore, minimizing detection at this stage is crucial and highly beneficial. In this study, we describe a background-matching mechanism during movement, which aids the cuttlefish to downplay its presence throughout movement. In situ behavioural experiments using video and image analysis, revealed a delayed, sigmoidal, colour-changing mechanism during movement of Sepia officinalis across uniform black and grey backgrounds. This is a first important step in understanding dynamic camouflage during movement, and this new behavioural mechanism may be incorporated and applied to any dynamic camouflaging animal or man-made system on the move.
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Conducta Animal , Sepia/fisiología , Adaptación Fisiológica , Animales , Color , Movimiento , Pigmentación de la PielRESUMEN
This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of â¼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.