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The aim of this work is to investigate the effect of a temperature increase on the behaviour of adult zebrafish (Danio rerio) maintained for 21 days at 34 °C (treatment) and 26 °C (control). The temperatures chosen are within the vital range of zebrafish and correspond to temperatures that this species encounters in the natural environment. Previous results showed that the same treatment affects the brain proteome and the behaviour of adult zebrafish by producing alterations in the proteins involved in neurotransmitter release and synaptic function and impairing fish exploratory behaviour. In this study, we have investigated the performance of treated and control zebrafish during environmental exploration by using four behavioural tests (novel tank diving, light and dark preference, social preference and mirror biting) that are paradigms for assessing the state of anxiety, boldness, social preference and aggressive behaviour, respectively. The results showed that heat treatment reduces anxiety and increases the boldness of zebrafish, which spent more time in potentially dangerous areas of the tank such as the top and the uncovered bright area and at a distance from the social group, thus decreasing protection for the zebrafish. These data suggest that the increase in ambient temperature may compromise zebrafish survival rate in the natural environment.

Brain-derived neurotrophic factor (BDNF) plays a pivotal role in neuronal growth and differentiation, neuronal plasticity, learning, and memory. Using CRISPR/Cas9 technology, we generated a vital Bdnf null mutant line in zebrafish and carried out its molecular and behavioral characterization. Although no defects are evident on a morphological inspection, 66% of coding genes and 37% of microRNAs turned out to be differentially expressed in

The number of farmed fish in the world has increased considerably. Aquaculture is a growing industry that will in the future provide a large portion of fishery products. Moreover, in recent years, the number of teleost fish used as animal models for scientific research in both biomedical and ecological fields has increased. Therefore, it is increasingly important to implement measures designed to enhance the welfare of these animals. Currently, a number of European rules exist as requirements for the establishment, care and accommodation of fish maintained for human purposes. As far as (teleost) fish are concerned, the fact that the number of extant species is much greater than that of all other vertebrates must be considered. Of further importance is that each species has its own specific physical and chemical requirements. These factors make it difficult to provide generalized recommendations or requirements for all fish species. An adequate knowledge is required of the physiology and ecology of each species bred. This paper integrates and discusses, in a single synthesis, the current issues related to fish welfare, considering that teleosts are target species for both aquaculture and experimental models in biological and biomedical research. We first focus on the practical aspects, which must be considered when assessing fish welfare in both research and aquaculture contexts. Next, we address husbandry and the care of fish housed in research laboratories and aquaculture facilities in relation to their physiological and behavioural requirements, as well as in reference to the suggestions provided by European regulations. Finally, to evaluate precisely which parameters described by Directive 2010/63/EU are reported in scientific papers, we analysed 82 articles published by European researchers in 2014 and 2015. This review found that there is a general lack of information related to the optimal environmental conditions that should be provided for the range of species covered by this directive.

This study investigated the effect of 4-d acute thermal treatments at 18 °C, 26 °C (control) and 34 °C on the nervous system of adult zebrafish (Danio rerio) using a multidisciplinary approach based on behavioural tests and brain proteomic analysis. The behavioural variations induced by thermal treatment were investigated using five different tests, the novel tank diving, light and dark preference, social preference, mirror biting, and Y-Maze tests, which are standard paradigms specifically tailored for zebrafish to assess their anxiety-like behaviour, boldness, social preference, aggressiveness, and explorative behaviour, respectively. Proteomic data revealed that several proteins involved in energy metabolism, messenger RNA translation, protein synthesis, folding and degradation, cytoskeleton organisation and synaptic vesiculation are regulated differently at extreme temperatures. The results showed that anxiety-like behaviours increase in zebrafish at 18 °C compared to those at 26 °C or 34 °C, whereas anxiety-related protein signalling pathways are downregulated. Moreover, treatments at both 18 °C and 34 °C affect the exploratory behaviour that appears not to be modulated by past experiences, suggesting the impairment of fish cognitive abilities. This study is the continuation of our previous work on the effect of 21-d chronic treatment at the same constant temperature level and will enable the comparison of acute and chronic treatment effects on the nervous system function in adult zebrafish.

Fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) is counted as an organic manganese (Mn)-derived compound. The toxic effects of Mn (alone and complexed) on dopaminergic (DA) neurotransmission have been investigated in both cellular and animal models. However, the impact of environmentally relevant Mn exposure on DA neurodevelopment is rather poorly understood. In the present study, the MMT dose of 100 μM (about 5 mg Mn/L) caused up-regulation of DA-related genes in association with cell body swelling and increase in the number of DA neurons of the ventral diencephalon subpopulation DC2. Furthermore, our analysis identified significant brain Mn bioaccumulation and enhancement of total dopamine levels in association with locomotor hyperactivity. Although DA levels were restored at adulthood, we observed a deficit in the acquisition and consolidation of memory. Collectively, these findings suggest that developmental exposure to low-level MMT-derived Mn is responsible for the selective alteration of diencephalic DA neurons and with long-lasting effects on fish explorative behaviour in adulthood.

Water temperature is an important environmental parameter influencing the distribution and the health of fishes and it plays a central role in ectothermic animals. The aim of this study is to determine the effects of environmental temperature on the brain proteome and the behavioural responses in zebrafish, a widely used animal model for environmental “omics” studies. Adult specimens of wild-type zebrafish were kept at 18 °C, 34 °C and 26 °C (control) for 21 days. Proteomic data revealed that several proteins involved in cytoskeletal organization, mitochondrial regulation and energy metabolism are differently regulated at the extreme temperatures. In particular, the expression of proteins associated to synapses and neurotransmitter release is down-regulated at 18 °C and 34 °C. In both thermal conditions, fish exhibited a reduced interest for the novel environment and an impairment of cognitive abilities during Y-Maze behavioural tests. The observed pathways of protein expression are possibly associated to functional alterations of the synaptic transmission that may result in cognitive functions impairment at central nervous system level as those revealed by behavioural tests. This study indicates that temperature variations can elicit biochemical changes that may affect fish health and behaviour. This combined approach provides insights into mechanisms supporting thermal acclimation and plasticity in fishes. Significance Environmental temperature variation may impact on all levels of biological life. Understanding the impact of thermal variation on the nervous system and animal behaviour is of primary importance since the results obtained can be applied from the ecological to the biomedical fields.

The synuclein (syn) family comprises three proteins: α-, β- and γ-syns. In humans, they are involved in neurodegenerative diseases such as Parkinson's disease and in tumors. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce and completely lacking in reptiles. In this study, the expression of genes coding for α-, β- and γ-syns was analyzed in the green lizard Anolis carolinensis by semiquantitative RT-PCR and Western blot. Results demonstrate good expression levels of the three syns in the lizard nervous system, similarly to human syns. This, together with the high identity between lizard and human syns, suggests that these proteins fulfill evolutionarily conserved functions. However, differences between lizard and humans in the expression of syn variants (two different variants of γ-syn were detected in A. carolinensis) and differences in some amino acids in key positions for the regulation of protein conformation and affinity for lipid and metal ions also suggest that these proteins may have acquired different functional specializations in the two lineages.

Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the β sheet-rich pathogenic isoform produced by the conformational conversion of the a-helix-rich physiological isoform PrPC. The peculiarity of PrPScis its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression "prion-like diseases" refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid β, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view.

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28 degrees C). After 21d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28 degrees C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28 degrees C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22 degrees C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28 degrees C in comparison with those maintained at 18 degrees C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28 degrees C than at 18 degrees C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.

The involvement of nitric oxide (NO) in the modulation of teleost osmoresponsive circuits is suggested by the facts that NO synthase enzymes are expressed in the neurosecretory systems and may be regulated by osmotic stimuli. The present paper is an overview on the research suggesting a role for NO in the central modulation of hormone release in the hypothalamo-neurohypophysial and the caudal neurosecretory systems of teleosts during the osmotic stress response. Active NOS enzymes are constitutively expressed by the magnocellular and parvocellular hypophysiotropic neurons and the caudal neurosecretory neurons of teleosts. Moreover, their expression may be regulated in response to the osmotic challenge. Available data suggests that the regulatory role of NO appeared early during vertebrate phylogeny and the neuroendocrine modulation by NO is conservative. Nonetheless, NO seems to have opposite effects in fish compared to mammals. Indeed, NO exerts excitatory effects on the electrical activity of the caudal neurosecretory neurons, influencing the amount of peptides released from the urophysis, while it inhibits hormone release from the magnocellular neurons in mammals.

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca2+/CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5′ and 3′ RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods. J. Comp. Neurol. 520:364–383, 2012. © 2011 Wiley Periodicals, Inc.

The structure of reptilian hard (beta)-keratins, their nucleotide and amino acid sequence, and the organization of their genes are presented. These 13-19 kDa proteins are basic, rich in glycine, proline and serine, and different from cytokeratins. Their mRNAs are expressed in beta-cells. The central part of beta-keratins (this region has been previously termed 'core-box' and is peculiar of all sauropsid proteins) is composed of two beta-folded regions and shows a high identity with avian beta-keratins. This central part present in all beta-keratins, including feather keratins, is the site of polymerization to build the framework of beta-keratin filaments. Beta-keratins appear cytokeratin-associated proteins. Their central region might have originated in an ancestral glycine-rich protein present in stem reptiles from which beta-keratins evolved and diversified into reptiles and birds. Stem reptiles of the Carboniferous period might have possessed glycine-rich proteins derived from exons/domains corresponding to the variable, glycine-rich region of cytokeratins. Beta-keratins might have derived from a gene coding for small glycine-rich keratin-associated proteins. The glycine-rich regions evolved differently in the lineage leading to modern reptiles and birds versus that leading to mammals. In the reptilian lineage some amino acid regions produced by point mutations and amino acid changes might have given rise to originate the central beta-pleated region. The latter allowed the formation of filamentous proteins (beta-keratins) associated with intermediate filament keratins and replaced them in beta-keratin cells. In the mammalian lineage no beta-pleated region was generated in their matrix proteins, the glycine-rich keratin-associated proteins. The latter evolved as glycine-tyrosine-rich, sulphur-rich, and ultra-sulphur-rich proteins that are used for building hairs, horns and nails.

Crocodilian keratinocytes accumulate keratin and form a corneous cell envelope of which the composition is poorly known. The present immunological study characterizes the molecular weight, isoelectric point (pI) and the protein pattern of alpha- and beta-keratins in the epidermis of crocodilians. Some acidic alpha-keratins of 47–68 kDa are present. Cross-reactive bands for loricrin (70, 66, 55 kDa), sciellin (66, 55–57 kDa), and filaggrin-AE2-positive keratins (67, 55 kDa) are detected while caveolin is absent. These proteins may participate in the formation of the cornified cell membranes, especially in hinge regions among scales. Beta-keratins of 17–20 kDa and of prevalent basic pI (7.0–8.4) are also present. Acidic beta-keratins of 10–16 kDa are scarce and may represent altered forms of the original basic proteins. Crocodilian beta-keratins are not recognized by a lizard beta-keratin antibody (A68B), and by a turtle beta-keratin antibody (A685). This result indicates that these antibodies recognize specific epitopes in different reptiles. Conversely, crocodilian beta-keratins cross-react with the Beta-universal antibody indicating they share a specific 20 amino acid epitope with avian beta-keratins. Although crocodilian beta-keratins are larger proteins than those present in birds our results indicate presence of shared epitopes between avian and crocodilian beta-keratins which give good indication for the future determination of the sequence of these proteins.

Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of "non verified" sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α-syn-like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates.

Little is known about proteins involved in the formation of the stratum corneum in the avian apteric epidermis. The present immunocytochemical, autoradiographic and electrophoretic study shows that antibodies against characteristic proteins of mammalian cornification (alpha-keratins, loricrin, sciellin, filaggrin, transglutaminase) recognize avian epidermal proteins. This suggests the presence of avian protein with epitopes common to related mammalian proteins. These proteins may also be involved in the formation of the cornified core and cell envelope of mature avian corneocytes. The immunoblotting study suggests that protein bands, cross-reactive for antibodies against loricrin (45, 52-57 kDa), sciellin (54, 84 kDa), filaggrin (32, 38, 45-48 kDa), and transglutaminase (40, 50, 58 kDa), are present in the avian epidermis. Immunocytochemistry shows that immunoreactivity for the above proteins is localized in the transitional and lowermost corneous layer of apteric epidermis. Their epitopes are rapidly masked/altered in cornifying cells and are no longer detectable in mature corneocytes. In scaled epidermis a thick layer made of beta-keratins of 14-18, 20-22, and 33 kDa is formed. Only in feathered epidermis (not in scale epidermis), an antifeather chicken beta-keratin antibody recognized a protein band at 8-12 kDa. This small beta-keratin is probably suitable for the formation of long, axial filaments in elongated barb, barbule and calamus cells. Conversely, the larger beta-keratins in scales are irregularly deposited forming flat plates. Tritiated histidine coupled to autoradiography show an absence of both keratohyalin and histidine-rich proteins in adult feathered and scaled epidermis. Most of the labeling appears in proteins within the range of beta- and alpha-keratins. These data on apteric epidermis support the hypothesis of an evolution of the apteric and interfollicular epidermis from the expansion of hinge regions of protoavian archosaurians.

Caveolae, the flask-shaped membrane invaginations abundant in endothelial cells, have acquired a prominent role in signal transduction. Evidence, that events occurring in caveolae participate in cell survival and angiogenesis, has been recently substantiated by the identification of two novel caveolar constituents: prostacyclin synthase (PGIS) and the cellular form of prion protein (PrPc). We have shown that PGIS, previously described as an endoplasmic reticulum component, is bound to caveolin-1 (cav-1) and localized in caveolae in human endothelial cells. By generating prostacyclin, PGIS is involved in angiogenesis. Previous observations regarding the localization of PrPc in caveolae-like membrane domains (CLDs) have been recently confirmed and extended. It has been demonstrated that PrPc is bound to cav-1 and, by recruiting Fyn kinase, can participate in signal transduction events connected to cell survival and differentiation. The new entries of PGIS and PrPc in caveolar components place caveolae and CLDs at the centre of a network, where cells decide whether to proliferate or differentiate and whether to survive or to suicide by apoptosis. © 2002 Elsevier Science Inc. All rights reserved.

In this paper, an experimental approach was used to test for the parallel effects of temperature (T) increase on the antipredator behaviour and the cholinergic expression in the juvenile European sea bass (Dicentrarchus labrax L.). The effects of three T treatments (18, 22 and 26°C) were tested on the main behavioural components of the antipredator response towards live aquatic predators and aerial simulated attacks, whereas brain cholinergic expression was evaluated by choline acetyltransferase (ChAT) immunoblotting (Western blot) at the extreme values of the thermal range (18 and 26°C). Antipredator responses towards a live fish were analysed over pre-exposure and exposure phases within a short temporal scale (20 s before and after the stimulus). The results suggest that T modulates several quantitative components of the antipredator behaviour. The mean shoaling index (shoal cohesiveness) was higher at 22°C than at 18 and 26°C during both the pre-stimulus and the exposure phase. Conversely, the mean distances from the predator and the tank bottom were, respectively, lower and higher at 26°C than in the other two treatments. In regard to the antipredator response on the aerial stimulus, comparisons across treatments revealed statistically significant differences between fish performing freezing or latency to recovery, suggesting that the fright reaction has a higher persistence at the coldest T (18°C) than at 22 and 26°C. Western blot analysis revealed a reduction in brain ChAT expression in fish acclimated to 26°C compared to those at 18°C. Results were discussed in the light of the relationships between behavioural traits, metabolism and their consequences on the population level, as a response to climate change in coastal habitats.

The present study analyzes the structure and the main proteins of reptilian claws. Mature claws are formed by two to four layers of keratinocytes, a transitional layer of spindle-shaped cells and a thick corneous layer. Transitional cells elongate and merge into a compact corneous layer that is immunoreactive for beta-keratins, now indicated as sauropsid keratin-associated proteins (sKAPs). Most proteins extracted from claws in representative reptiles have a molecular weight of 13–20 kDa, an acidic to basic isoelectric point, and are identified from the positive immunoreactivity to beta-keratin antibodies. The comparative analysis between lizard and avian claw beta-keratins shows the presence of an internal region of 20 amino acids with the highest identity, indicated as core-box, within an extended 32-amino acid region with a prevalent beta-sheet secondary conformation. This region is structurally equivalent to a 32-amino acid region present in scale beta-keratins of most reptiles. Both reptilian and avian keratins contain glycine-rich regions for stabilization of the beta-keratin polymer. The N- and C-regions contain most cysteine for disulphide-bonds formation. Claw proteins contain higher amount of cysteine and glycine than other scale proteins, suggesting that claw proteins are specialized cysteine–glycine-rich proteins suited to produce a very hard corneous material.

Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages probably derived from the localization of specialized areas of dermal–epidermal interaction in the integument. The horny scales and the other derivatives were formed from large areas of dermal–epidermal interaction. The evolution of these skin appendages was characterized by the production of specific coiled-coil keratins and associated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsids contain a double beta-folded sequence of about 20 amino acids, known as the core-box. The core-box shows 60%–95% sequence identity with known reptilian and avian proteins. The core-box determines the polymerization of these proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequences for these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolution in reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestral glycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrix proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral protein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently in the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high glycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, which evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In therapsids-mammals, mutations of the ancestral proteins formed the high glycine-tyrosine or the high cysteine proteins but no core-box was produced in the matrix proteins of the hard corneous material of mammalian derivatives.

Nucleotide and deduced amino acid sequences of three β-keratins of Nile crocodile scales are presented. Using 5′- and 3′-RACE analysis, two cDNA sequences of 1 kb (Cr-gptrp-1) and 1.5 kb (Cr-gptrp-2) were determined, corresponding to 17.4 and 19.3 kDa proteins, respectively, and a pI of 8.0. In genomic DNA amplifications, we determined that the 5′-UTR of Cr-gptrp-2 contains an intron of 621 nucleotides. In addition, we isolated a third gene (Cr-gptrp-3) in genomic DNA amplifications that exhibits seven amino acid differences with Cr-gptrp-2. Genomic organization of the sequenced crocodilian β-keratin genes is similar to avian β-keratin genes. Deduced proteins are rich in glycine, proline, serine, and tyrosine, and contain cysteines toward the N- and C-terminal regions, likely for the formation of disulfide bonds. Prediction of the secondary structure suggests that the central core box of 20 amino acids contains two β-strands and has 75–90% identity with chick β-keratins. Toward the C-terminus, numerous glycine–glycine–tyrosine and glycine–glycine–leucine repeats are present, which may contribute to making crocodile scales hard. In situ hybridization shows expression of β-keratin genes in differentiating β-cells of epidermal transitional layers. Phylogenetic analysis of the available archosaurian and lepidosaurian β-keratins suggests that feather keratins diversified early from nonfeather keratins, deep in archosaur evolution. However, only the complete knowledge of all crocodilian β-keratins will confirm whether feather keratins have an origin independent of those in bird scales, which preceded the split between birds and crocodiles. J Exp Zool (Mol. Dev. Evol.) 312B:42–57, 2009. © 2008 Wiley-Liss, Inc.

Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as "feather keratin". This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.

Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine–proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine–proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life.

Lizard scales are composed of alpha-(cyto-) keratins and beta-keratins. The characterization of the molecular weight and isoelectric point (p I ) of alpha- and beta-keratins of lizard epidermis ( Podarcis sicula ) has been done by using two-dimensional electrophoresis, immunoblotting, and immunocytochemistry. Antibodies against cytokeratins, against a chicken scale beta-keratin or against lizard beta-keratin bands of 15–16 kDa, have been used to recognize alpha- and beta-keratins. Acid and basic cytokeratins of 42–67 kDa show a p I from 5.0 to 8.9. This indicates the presence of specific keratins for the formation of the stratum corneum. Main protein spots of beta-keratin at 15–17 kDa, and p I at 8.5, 8.2, and 6.7, and one spot at 10 kDa and p I at 7.3 were recognized. Therefore, beta-keratins are mainly basic proteins, and are used for the formation of the hard corneous layer of the epidermis. Ultrastructural immunocytochemistry confirms that beta-keratin is packed into large and dense bundles of beta-keratin cells of lizard epidermis. The use of a probe against a lizard beta-keratin in situ-hybridization studies confirms that the mRNA for beta-keratins is present in beta-cells and is localized around or even associated with beta-keratin filaments.

Snake scales contain specialized hard keratins (beta-keratins) and alpha- or cyto-keratins in their epidermis. The number, isoelectric point, and the evolution of these proteins in snakes and their similarity with those of other vertebrates are not known. In the present study, alpha- and beta-keratins of snake molts and of the whole epidermis have been studied by using two-dimensional electrophoresis and immunocytochemistry. Specific keratins in snake epidermis have been identified by using antibodies that recognize acidic and basic cytokeratins and avian or lizard scale beta-keratin. Alpha keratins of 40–70 kDa and isoelectric point (pI) at 4.5–7.0 are present in molts. The study suggests that cytokeratins in snakes are acidic or neutral, in contrast to mammals and birds where basic keratins are also present. Beta keratins of 10–15 kDa and a pI of 6.5–8.5 are found in molts. Some beta-keratins appear as basic proteins (pI 8.2) comparable to those present in the epidermis of other reptiles. Some basic “beta-keratins” associate with cytokeratins as matrix proteins and replace cytokeratins forming the corneous material of the mature beta-layer of snake scales, as in other reptiles. The study also suggests that more forms of beta-keratins (more than three different types) are present in the epidermis of snakes.

The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.

The β-keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of β-keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3′- and 5′-rapid amplification of cDNA ends analyses five specific gecko β-keratin cDNA sequences. The cDNA coding sequences encoded putative glycine-proline-serine–rich proteins of 16.8–18 kDa containing 169–191 amino acids, especially 17.8–23% glycine, 8.4–14.8% proline, 14.2–18.1% serine. Glycine-rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (β-pleated fold) likely responsible for the formation of β-keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis show a higher β-keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and β-cells. Expression in adhesive setae of climbing lamellae was shown by RT-PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian β-keratins. Developmental Dynamics 236:374–388, 2007. © 2006 Wiley-Liss, Inc.

Beta-keratins form the hard corneous material of reptilian scales. In the present review, the distribution and molecular characteristics of beta-keratins in reptiles are presented. In lepidosaurians immunoreactive, protein bands at 12-18 kDa are generally present with less frequent proteins at higher molecular weight. In chelonians, bands at 13-18 and 22-24 kDa are detected. In crocodilians, bands at 14-20 kDa and weaker bands at 30-32 kDa are seen. Protein bands above 25 kDa are probably polymerized beta-keratins or aggregates. Two-dimensional gel electrophoresis shows that beta-keratins are mainly basic and that acidic-neutral keratins may derive from post-translational modifications. Beta-keratins comprise glycine-proline-rich and cystein-proline-rich proteins of 13-19 kDa. Beta-keratin genes may or may not contain introns and are present in multiple copies with a linear organization as in avian beta-keratin genes. Despite amino acid differences toward N- and C-terminals all beta-keratins share high homology in their central, beta-folded region of 20 amino acids, indicated as core-box. This region is implicated in the formation of beta-keratin filaments of scales, claws, and feathers. The homology of the core-box suggests that these proteins evolved from a progenitor sequence present in the stem of reptiles. Beta-keratins have diversified in their amino acid sequences producing secondary (and tertiary) conformations that suited them for their mechanical role in scales. In birds, a small beta-keratin has allowed the formation of feathers. It is suggested that beta-keratins represent the reptilian counterpart of keratin associated or matrix proteins present in mammalian hairs, claws, and horns.

The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.

β-keratins of reptilian scales have been recently cloned and characterized in some lizards. Here we report for the first time the sequence of some β-keratins from the snake Elaphe guttata. Five different cDNAs were obtained using 5′- and 3′-RACE analyses. Four sequences differ by only few nucleotides in the coding region, whereas the last cDNA shows, in this region, only 84% of identity. The gene corresponding to one of the cDNA sequences has a single intron present in the 5′-untranslated region. This genomic organization is similar to that of birds' β-keratins. Cloning and Southern blotting analysis suggest that snake β-keratins belong to a family of high-related genes as for geckos. PCR analysis suggests a head-to-tail orientation of genes in the same chromosome. In situ hybridization detected β-keratin transcripts almost exclusively in differentiating oberhautchen and β-cells of the snake epidermis in renewal phase. This is confirmed by Northern blotting that showed, in this phase, a high expression of two different transcripts whereas only the longer transcript is expressed at a much lower level in resting skin. The cDNA coding sequences encoded putative glycine-proline-serine rich proteins containing 137–139 amino acids, with apparent isoelectric point at 7.5 and 8.2. A central region, rich in proline, shows over 50% homology with avian scale, claw, and feather keratins. The prediction of secondary structure shows mainly a random coil conformation and few β-strand regions in the central region, likely involved in the formation of a fibrous framework of β-keratins. This region was possibly present in basic reptiles that originated reptiles and birds. Developmental Dynamics 236:1939–1953, 2007. © 2007 Wiley-Liss, Inc.

Beta-keratins are responsible for the mechanical resistance of scales in reptiles. In a scaleless crotalus snake (Crotalus atrox), large areas of the skin are completely devoid of scales, and the skin appears delicate and wrinkled. The epidermis of this snake has been assessed for the presence of beta-keratin by immunocytochemistry and immunoblotting using an antibody against chicken scale beta-keratin. This antibody recognizes beta-keratins in normal snake scales with molecular weights of 15-18 kDa and isoelectric points at 6.8, 7.5, 8.3 and 9.4. This indicates that beta-keratins of the stratum corneum are mainly basic proteins, so may interact with cytokeratins of the epidermis, most of which appear acidic (isoelectric points 4.5-5.5). A beta-layer and beta-keratin immunoreactivity are completely absent in moults of the scaleless mutant, and the corneous layer comprises a multilayered alpha-layer covered by a flat oberhautchen. In conclusion, the present study shows that a lack of beta-keratins is correlated with the loss of scales and mechanical protection in the skin of this mutant snake.

Scales of lizards contain beta-keratin of poorly known composition. In the present study, a rat polyclonal serum against a lizard beta-keratin of 14-15 kDa has been produced and the relative protein has been immunolocalized in the epidermis. The observations for the first time show that the isolated protein band derives from the extraction of a protein component of the beta-keratin filaments of lizard epidermis. In immunoblots and immunocytochemistry, the antiserum recognizes most lizard beta-keratins, but produces a variable cross-reactivity with snake beta-keratins, and weak or no reactivity with beta-keratins isolated from tuatara, turtles, alligator and birds. In bidimensional immunoblots of lizard epidermis, three main spots at 15-16 kDa with isoelectric point at 7.0, 7.6 and 8.0, and an unresolved large spot at 29-30 kDa and with pI at 7.5-8.0, are obtained, may be derived from the aggregation of smaller beta-keratin proteins. The ultrastructural immunolocalization with the antibody against lizard beta-keratin shows that only small and large beta-keratin filaments of beta-cells of lizard epidermis are labeled. Keratin bundles in oberhautchen cells are less immunolabeled. Beta-keratin is rapidly polymerized into beta-packets that merge into larger beta-keratin filaments. No labeling is present over other cell organelles or cell layers of lizard epidermis, and is absent in non-epidermal cells. The antiserum recognizes epitope(s) characteristics for lizard beta-keratins, partially recognized in snakes and absent in non-lepidosaurian species. This result indicates that beta-keratins among different reptilian groups posses different immunoreactive regions.