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Journal Cover Journal of Applied Ichthyology
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
     ISSN (Print) 0175-8659 - ISSN (Online) 1439-0426
     Published by John Wiley and Sons Homepage  [1602 journals]   [SJR: 0.427]   [H-I: 33]
  • Identification of cis‐regulatory elements in the upstream regions of
           zebrafish runx3 through in silico analysis: implications for function
    • Abstract: RUNX3 encodes a member of the runt domain family of transcription factors. In mammals this family includes three genes (RUNX1‐3) and their protein products function as context‐dependent transcription factors, either transcriptional activators or repressors, during developmental processes such as hematopoiesis, neurogenesis, and osteogenesis; all are proto‐oncogenes or tumour suppressors. All three genes were shown to be transcribed from two promoters, giving rise to protein products bearing either the P1 or the P2 N‐termini, translated respectively from transcripts originating from the distal (P1)‐ or the proximal (P2)‐promoters. Understanding their differential regulation and interaction may help explain how RUNX factors contribute to such different and often opposing biological processes. In this study, we have identified putative molecular players affecting zebrafish runx3 transcription by using a computational approach to search for cis‐regulatory transcription factor binding sites (TFBSs) in the runx3 promoter regions of zebrafish (Danio rerio) and fugu (Takifugu rubripes). From the data obtained it was possible to identify the sites most likely involved in regulating expression of runx3 in zebrafish. Our comparative approach reduced substantially the number of putative TFBSs in the runx3 promoter regions; reassuringly, published TFs identified as transcriptional regulators of Runx3 are confirmed by our in silico analysis. Our data now provides the basis for focused in vitro and/or in vivo experimental tests of the transcriptional regulatory activities of strong candidate regulators of zebrafish runx3. RUNX3 encodes a member of the runt domain family of transcription factors. In mammals this family includes three genes (RUNX1‐3) and their protein products function as context‐dependent transcription factors, either transcriptional activators or repressors, during developmental processes such as hematopoiesis, neurogenesis, and osteogenesis; all are proto‐oncogenes or tumour suppressors. All three genes were shown to be transcribed from two promoters, giving rise to protein products bearing either the P1 or the P2 N‐termini, translated respectively from transcripts originating from the distal (P1)‐ or the proximal (P2)‐promoters. Understanding their differential regulation and interaction may help explain how RUNX factors contribute to such different and often opposing biological processes. In this study, we have identified putative molecular players affecting zebrafish runx3 transcription by using a computational approach to search for cis‐regulatory transcription factor binding sites (TFBSs) in the runx3 promoter regions of zebrafish (Danio rerio) and fugu (Takifugu rubripes). From the data obtained it was possible to identify the sites most likely involved in regulating expression of runx3 in zebrafish. Our comparative approach reduced substantially the number of putative TFBSs in the runx3 promoter regions; reassuringly, published TFs identified as transcriptional regulators of Runx3 are confirmed by our in silico analysis. Our data now provides the basis for focused in vitro and/or in vivo experimental tests of the transcriptional regulatory activities of strong candidate regulators of zebrafish runx3.
  • Characterization of regulatory elements in the medaka osterix promoter
           required for osteoblast expression
    • Abstract: The zinc finger transcription factor Osterix/Sp7 is an essential regulator of osteoblastogenesis. In mammals, osterix expression is regulated by Runx2, Msx2 and Dlx5 but recent findings suggest that also retinoic acid plays an important role for osteoblast differentiation and function. Yet, how these and other factors act on the osterix promoter is largely unknown. Expression, knock‐down and promoter analyses have indicated that the function of Osterix in osteoblasts is conserved in teleosts and mammals. In the present study, we have used the teleost medaka to identify and characterize a region containing potential retinoic acid response elements in the osterix promoter. We analysed whether this region is important for activity in osteoblasts in vivo, using transgenic medaka lines with modified osterix promoter regions. Promoter activity in vivo and in vitro revealed a short nucleotide sequence in the promoter with crucial positive regulatory function. Mutations of this element lead to a complete inactivation of the osterix promoter in osteoblasts and made it insensitive to retinoic acid treatment. The comparison with the regulatory regions of osterix in other species suggests that the function of this element is highly conserved in vertebrates.
  • The effects of dietary arachidonic acid on bone in flatfish larvae: the
           last but not the least of the essential fatty acids
    • Abstract: Flatfish can provide a reliable model to study developmental disorders in bone tissues occurring during morphogenesis in response to nutritional imbalances. To date, most studies dealing with the effect of dietary essential fatty acids (EFA) on skeletogenesis in fish have focused their investigation on the role of docohexanoic (22:6n−3, DHA) and eicosapentaenoic (20:5n–3, EPA) acids, but only a few have focused on investigating the effects of arachidonic acid (20:4n–6, ARA) on bone during fish larval development. Bone development and composition at larval stage have been demonstrated to be highly sensitive to dietary levels of EFA, in particular the EPA and ARA acids, both precursors for highly bioactive eicosanoids presenting opposite effects on bone metabolism. Since fish are not able to synthesize EFA, they need to obtain them from the diet. However, dietary imbalances in EPA and ARA in flatfish larvae may disrupt bone formation and osteoblast differentiation in skeletal tissues, leading to the incidence of skeletal deformities, reduced mineralization and problems of bone remodelling in the cranial region associated with impaired eye migration. These anomalies in skeletal structures are one of the most important factors that affect flatfish larval quality and hamper their production. Thus, we have reviewed the current state of knowledge about the effects of dietary ARA contents on skeletogenesis in Senegalese sole (Solea senegalensis), one of the main flatfish species cultured in Europe. Their larval quality still suffers for a high incidence of skeletal anomalies induced by dietary imbalances during metamorphosis.
  • Old, new and new‐old concepts about the evolution of teeth
    • Abstract: The evolutionary origin of teeth from dermal denticles (odontodes) that developed in the mouth cavity, designated as outside‐in hypothesis, has long been undisputed. The outside‐in hypothesis is based on the conclusion that dermal denticles and teeth fulfil the criteria of homology in an exemplary manner. Over the past 15 years, this hypothesis has been challenged. Proponents of the alternative inside‐out hypothesis suggest that teeth did not evolve from dermal denticles, that they are of endodermal origin (forming in conjunction with neural crest‐derived mesenchyme) and that they evolved several times independently in different lineages of vertebrates. Key arguments for the inside‐out hypothesis are mineralized structures of conodonts that are accepted as teeth, the exclusive acceptance of placoderm pharyngeal denticles as teeth, together with the rejection of the presence of teeth in basal placoderms. We summarize the results of recent studies that have been triggered by the fruitful discussion between the two conflicting hypotheses. New findings support the traditional outside‐in hypothesis: the mineralized elements of conodonts are not teeth, and the oral cusps in basal placoderms are true teeth. Furthermore, new developmental and molecular data clarify homology between teeth and dermal denticles. Today a new synthesis is emerging about the evolutionary origin of teeth from dermal denticles and about the unity of the elements of the dermal skeleton.
  • Molecular investigation of mechanical strain‐induced phenotypic
           plasticity in the ecologically important pharyngeal jaws of cichlid fish
    • Abstract: Phenotypic plasticity in the form of alterations to teleost skeletons can result from a range of environmental factors, such as the hardness of the prey, particularly when exposure occurs early during development. Determining the molecular underpinnings of teleost skeletal plasticity is hampered by a limited understanding of the molecular basis of bone remodeling in derived teleost fish, whose bones are acellular, lacking the cell type known to orchestrate bone remodeling in mammals. We are using a fitting molecular model for phenotypic plasticity research: the East African cichlid Astatoreochromis alluaudi, with the aim to shed light on the molecular basis of phenotypic plasticity and on the remodeling of acellular bones. For this fish, sustained ingestion of a hard diet induces a ‘molariform’ lower pharyngeal jaw (LPJ), with molar‐like teeth set in an enlarged, relatively dense jaw, while a softer diet results in a smaller, finer ‘papilliform’ LPJ morphology, representing the ‘ground state’ for this species. Through comparing genome‐wide transcription in molariform and papilliform LPJs, our previous research has shed light on the molecular basis of phenotypic plasticity in the teleost skeleton and by extension, on acellular bone remodeling. In this manuscript we construct a model for the molecular basis of mechanically induced skeletal plasticity in teleosts, which involves iterative cycles of strain and compensatory cellular proliferation. Furthermore, we propose a framework for testing the potential influence of phenotypic plasticity and genetic assimilation on adaptive radiations.
  • Endoskeleton/Exo (dermal) skeleton — Mesoderm/Neural Crest: Two pair
           of problems and a shifting paradigm
    • Abstract: Vertebrates have both more than one skeleton and multiple skeletal systems reflecting origins from two germ layers (mesoderm and neural crest), locations of skeletal elements as external or internal (exo‐ and endoskeleton), and separate craniofacial, axial and appendicular skeleton systems. Current studies reinforce the exoskeleton as consisting of bone and dentine to the exclusion of cartilage. The endoskeleton is based in cartilage, which, depending on lineage may be replaced by bone. Current understanding of the nature of the exo‐ and endoskeletons, modes of ossification (intramembranous, endochondral, perichondral), and the contributions of mesodermal and neural crest cells to the skeleton(s) is summarized and then discussed in more depth using fin rays in paired and unpaired fins as examples. Recent studies demonstrating that fin rays and scales are mesodermal and not neural crest in origin are reviewed and whether trunk neural crest cells have skeletogenic potential is considered. These results are discussed in the context of whether scales and fin rays should continue to be regarded as components of the exoskeleton. The conclusion is that they should, thereby separating germ layer of origin from classification of skeletal elements as exo‐ or endoskeletal; germ layer of origin does not provide unambiguous evidence for classification of elements as parts of the exo‐ or endoskeletons. Recent studies indicating that turtle shells are mesodermal in origin provides further evidence of the contribution of mesoderm to skeletal elements traditionally regarded as part of the exoskeleton. Therefore, exoskeleton is not synonymous with neural crest origin. These studies on fish fin rays/scales and turtle shells herald a new paradigm in which germ layer of origin (mesoderm or neural crest) does not equate with classification of elements as parts of the endo‐ or exoskeleton.
  • Fish is Fish: the use of experimental model species to reveal causes of
           skeletal diversity in evolution and disease
    • Abstract: Fishes are wonderfully diverse. This variety is a result of the ability of ray‐finned fishes to adapt to a wide range of environments, and has made them more specious than the rest of vertebrates combined. With such diversity it is easy to dismiss comparisons between distantly related fishes in efforts to understand the biology of a particular fish species. However, shared ancestry and the conservation of developmental mechanisms, morphological features and physiology provide the ability to use comparative analyses between different organisms to understand mechanisms of development and physiology. The use of species that are amenable to experimental investigation provides tools to approach questions that would not be feasible in other ‘non‐model’ organisms. For example, the use of small teleost fishes such as zebrafish and medaka has been powerful for analysis of gene function and mechanisms of disease in humans, including skeletal diseases. However, use of these fish to aid in understanding variation and disease in other fishes has been largely unexplored. This is especially evident in aquaculture research. Here we highlight the utility of these small laboratory fishes to study genetic and developmental factors that underlie skeletal malformations that occur under farming conditions. We highlight several areas in which model species can serve as a resource for identifying the causes of variation in economically important fish species as well as to assess strategies to alleviate the expression of the variant phenotypes in farmed fish. We focus on genetic causes of skeletal deformities in the zebrafish and medaka that closely resemble phenotypes observed both in farmed as well as natural populations of fishes.
  • Issue Information
  • Interdisciplinary Approaches in Fish Skeletal Biology
  • Conference Group Photo
  • Table of Content
  • Fish skeletal biology and beyond
  • Skull developmental modularity: a view from a single bone – or two
    • Abstract: Reviewed are recent studies that connect development and evolution of skull bones in teleosts. Development uses genetic information to build a structured, modular phenotype, and since selection acts on the phenotype, developmental modularity may influence evolvability. Just how is a complex developing morphology spatially partitioned into modules' Here I briefly examine cellular, molecular genetic, and multivariate statistical approaches to the identification of developmental modules. Furthermore I review our evidence that developmental modularity provides evolutionarily labile regions within the skull and hence potentially biases evolutionary change in a positive manner. This view is rather different from early ones in the field of evolutionary developmental biology, in which developmental constraint due to patterns such as heterochronies were supposed to negatively impact evolution.
  • Preliminary study for phenotypic description of vertebral abnormalities in
           triploid trout subjected to prolonged deficiency in phosphorus
    • Abstract: In trout farmed in freshwater, the use of low phosphorus diet and other concurrent changes in food formulations could affect the normal growth of the skeleton and enhance the occurrence of vertebral abnormalities. Hence, it is important to refine diagnostic of malformations to quickly detect signs of P deficiency during production. Two practical diets consisting of P‐deficient (0.5% total P) and P‐sufficient (0.9% total P) were formulated. Experiments were conducted on all‐female juvenile triploid rainbow trout (initial mass ~60 g) until fish fed continuously with P‐sufficient diet reached
  • Responses of different body compartments to acute dietary phosphorus
           deficiency in juvenile triploid rainbow trout (Oncorhynchus mykiss,
    • Abstract: Rainbow trout (Oncorhynchus mykiss) is an important production species as well as one of the most studied fish models, particularly regarding nutritional physiology. Due to negative environmental impacts linked with rainbow trout farm effluents, significant restrictions have been established in numerous regions to reduce dietary phosphorus (P) outputs. However, questions have arisen regarding the link between abnormal skeletal development and mineralization and insufficient dietary P availability during rapid fish growth (juvenile fish). Despite significant work to understand the dynamics of P‐deficiency and the resulting impact on tissue mineralization, the extent of the early responses in rainbow trout fed low‐P diets is not well described. The aim of this study was to explore the early‐responses of scales, vertebrae and carcass P and ash in rainbow trout fed low‐P vs sufficient‐P controls. Two practical diets (sufficient: 0.45% available P and deficient: 0.29% available P) were fed over a 5‐week period to triploid rainbow trout (initial mass 60.8 ± 1.6 g). Ash and P contents were used to assess mineral status of the different tissues. The relative loss of mineral and coefficient of variation were also calculated to compare the relative response and the inter‐individual variability. After 4 weeks of P deprivation, no detectable effects were observed on growth performance, mortality or feed intake. However, as early as the second week onward, ash and P levels in scales and carcasses were significantly lower (p 
  • Effects of thyroid hormone treatment on the mineral density and mechanical
           properties of the African barb (Labeobarbus intermedius) skeleton
    • Abstract: Here we describe the results of a preliminary study to evaluate the response of the cellular skeleton of the large African barb, Labeobarbus intermedius, to exposure to high levels of the thyroid hormone T3 for 1 and 3 months. We examined the effects in terms of mineral density and mechanical properties of the operculum bone, as well as evaluated and compared the light microscopy features of this bone between the treatment groups and the untreated control group. We found a significant increase in bone mineral density in the treated groups compared to untreated controls, and a tendency towards a corresponding increase of bone material stiffness (Young's modulus). These findings suggest that thyroid hormone enrichment may contribute to improved skeletal properties in pond‐raised fish, and help moderate osteomalacia, a commonly seen problem in aquaculture.
  • Effects of thyroid hormone level alterations on the development of
           supraneural series in zebrafish, Danio rerio
    • Abstract: The effects of alterations in the level of thyroid hormones (THs) on the development of the supraneural series were studied in zebrafish, Danio rerio. Hyper‐ and hypothyroidism were revealed to influence the rate and timing of development of the supraneurals. The developmental changes induced by THs result in changes in the definitive number and morphology of supraneural bones. High TH‐level leads to an arrested development of the supraneural series resulting in the absence or drastic reduction of the number of elements. TH‐deficiency leads to a significant retardation of the development of the supraneural series resulting in the appearance of additional elements. These data may be an important piece of the puzzle that may help our understanding how supraneural number and form has changed in evolution.
  • Poly‐ and oligomerization of scales in the blue bream Ballerus
           ballerus (Cyprinidae) as a consequence of thyroid status regulation
    • Abstract: Poly‐ and oligomerization of serial elements are among the ways to enhance, weaken or even change organ's function and might contribute to adaptation of animals. Involvement of thyroid hormones in development and formation of adult phenotype of scale cover was found recently. Cyprinid fish blue bream Ballerus ballerus has relatively high number of scales in lateral line (62–77) and may serve as appropriate object to examine the role of thyroid regulation of scale number in fish. Individuals kept under different hormonal conditions manifest highly differential number of scales. Experimental variation of scale number (49–114) broadly exceeds the limits of natural variation known for species. Goitrogen‐treated fish have an increased scale number (polymerization), while triidothyronine‐treated fish have a reduced scale number (oligomerization). Additionally fish of most prolonged retardation of development (goitrogen‐treated) demonstrate phenotype of partially naked skin. This event of reduction of squamation and partial nakedness occurred due to underdevelopment. Also we suggest the regulation of the development by the thyroid hormones is thought to be a perspective approach to mark periods of determination of number of serial elements.
  • Length–weight relationships of seven freshwater fishes from the
           JiangSu province, China
    • Abstract: The length‐weight relationships (LWRs) are presented for seven freshwater fish species (Odontobutis potamophila, Protosalanx chinensis, Acheilognathus chankaensis, Megalobrama skolkovii, Pelteobagrus vachelli, Macropodus chinensis, Pseudobrama simoni) from JiangSu province in China. A total of 1340 specimens were sampled and measured between August 2012 and September 2013. No information regarding the LWRs of these species was available in FishBase.
  • Effect of stocking strategy on distribution and recapture rate of common
           carp Cyprinus carpio L., in a large and shallow temperate lake:
           implications for recreational put‐and‐take fisheries
    • Abstract: It is hypothesized that the stocking procedure influences survival, growth and distribution of introduced fishes; however, there is still limited information on the effect of various stocking strategies on recaptures in natural freshwaters. The present study aim was to investigate how the rate and distribution of anglers' catches of common carp (Cyprinus carpio) vary with the stocking season (spring, summer and autumn), lake area, method (shore and offshore releases), and fish size (≤500 and >500 g) in the large and shallow Lake Balaton, Hungary. In 2010, 4500 two‐summer‐old individually‐tagged common carp were stocked to test 36 release set‐ups (three seasons × three lake areas × two methods × two size groups). Anglers reported the date, location and fish size (standard length and weight) on 787 recaptures within 2 years after the release. Recapture rate was highest in summer and lowest in autumn stockings, but was not affected by the stocking area, method or fish size. Regarding space, the widest dispersals were in recaptures in autumn and in the centre of the lake, but fish movement was not influenced by the stocking method or fish size. In conclusion, in summer the stocking quotas should be evenly distributed along the entire shoreline; early spring stockings may be optimized for transport costs and concentrated by each lake basin. Late autumn stockings should be avoided, and the capacity of effective wintering ponds should be developed. This study also provides a good framework for testing fisheries management alternatives in other intensively fished habitats.
  • Skeletal malformations in Senegalese sole (Solea senegalensis Kaup, 1858):
           gross morphology and radiographic correlation
    • Abstract: Senegalese sole (Solea senegalensis) is a flatfish of increasingly interest in aquaculture in recent years. The establishment of the production requires the determination of the optimal conditions for development, growth and welfare of the sole in tanks. One important problem in cultured Senegalese sole is the presence of skeletal malformations that alter the morphology of the fish and cause the consumer's rejection. In contrast to the external study of the fish, the radiographic imaging allows to observe the existing types of deformities. The objective of this study was to define a straightforward evaluation method of skeletal alterations in Senegalese sole by means of computed radiography (CR). Fish between 255 and 352 days after hatching were randomly sampled, categorized according to their external appearance by the farm staff, and CR evaluated. Based on our previous research on skeletal malformations in Senegalese sole, we assessed the changes in the vertebrae and vertebral arches, processes and spines. Deformities varied from slight loss of structure of a single vertebra, to multiple vertebral deformities, fusions and alterations in intervertebral spaces that may result in lordosis/kyphosis or scoliosis. Anomalies in the abdominal region, caudal region and caudal complex were given a score: zero, absence of deformities; one, minor malformations; two, major malformations without altering body shape; and three, severe skeletal changes modifying gross morphology of the fish. An overall score for each fish was assumed as the highest number obtained in the three regions. The results showed that the caudal complex was the region with higher rate of deformities, usually minor; more severe malformations appeared in the caudal region. A high number of anomalies without effect on the external morphology were observed, and some gross changes in body shape did not correspond to bone alterations. CR allows the characterization of externally undetectable deformities in juvenile sole in a direct way, and is useful to discern the evolution and influence of the malformations on the gross morphology.
  • X‐ray‐based morphometrics: an approach to diagnose vertebral
           abnormalities in under‐mineralized vertebrae of juvenile triploid
           all‐female rainbow trout (Oncorhynchus mykiss) fed with a phosphorus
           deficient diet
    • Abstract: Extended phosphorus (P) deficiency has been linked to an increase of skeletal deformities in farmed salmonids. To date, X‐ray examination still is the most practical way to detect abnormalities that are not externally visible. The aim of this study was to develop a standardized method based on empirical measurements that provides an early radiograph‐based detection of deformities. Triploid all‐female rainbow trout (60.8 ± 1.6 g) were fed with phosphorus (P)‐deficient and P‐sufficient diets (total P = 0.5% and 0.92%, respectively). Experiments were carried out in 12 circular re‐circulating tanks (n = 140/tank; 8.5–52.7 kg m−3; 12 ± 0.3°C; 18L: 6D). All fish were x‐rayed at week 5, 15 and 24 to monitor vertebral abnormalities. Fish were assigned to a vertebral phenotype (normal, biconcave or compressed) when all vertebrae of the caudal region (V31‐44) showed the same type of abnormalities. At week 9, 18 and 27, ten fish per tank were randomly sampled according to their vertebral phenotype. Vertebrae V38–V39 were collected and a second X‐ray was performed to confirm the above‐mentioned phenotypes. Morphometrics (vertebral length and height; intervertebral distances and vertebral body angles) were measured directly on radiographs. For all parameters measured, comparisons were made between normal P‐sufficient fish and different scenarios of vertebral development (normal, restored, stable and aggravated) in P‐deficient fish. The two parameters showing the most sensitivity were the median intervertebral distance and dorsal/ventral vertebral endplate angles. When analysed together, these two parameters allow a quantitative distinction of the different vertebral phenotypes. Measuring dorsal/ventral vertebral endplate angles seems a promising approach to detect aberrant vertebral development at early stages as slight changes were observed in P‐deficient vertebrae that were assessed as normal by visual examination. Further studies including histological examinations and comparison with other radiological imaging techniques are required to confirm these results.
  • Development of the axial skeleton in the bay snook Petenia splendida
           Günther, 1862 (Perciformes: Cichlidae)
    • Abstract: We report the first description of the ontogenic development of the axial skeleton and unpaired fins of hatchery reared bay snook (Petenia splendida, Perciformes: Cichlidae) from hatching (5.3–5.5 mm in standard length, SL) to the juvenile stage (16.1–17.5 mm SL) by means of alcian blue‐alizarin red staining. The study of axial skeleton development was complemented by digital analysis of its level of ossification (number of red pixels) in order to identify major changes in vertebral column and caudal fin complex development. The main events of the skeletogenesis of the vertebral column and unpaired fins in P. splendida larvae were comprised between the onset of notochord segmentation and completion of the caudal fin complex at 6 (171 degree days post hatch, ddph; 6.4–6.5 mm SL) and 24 days post hatching (dph) (684 ddph, 8.9–9.4 mm SL), respectively; whereas the anal and dorsal fins completed morphogenesis at older ages (36 dph, 1026 ddph, 11.0–11.8 mm SL). In this sense, the development of a subcarangiform swimming mode in early juveniles was achieved with complete ossification of vertebral bodies and caudal lepidotrichia, as well as partial ossification of the caudal‐fin complex elements (hypurals, epurals, uroneural and parahypural). This information can be considered as a reference for other studies evaluating the quality of larvae and the influence of rearing biotic and abiotic factors in the skeletogenesis of this cichlid species and the occurrence of skeletal deformities.
  • A comparison of the larval and juvenile dentition in Polypterus senegalus
    • Abstract: Polypterus senegalus is a freshwater fish belonging to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its dentition has been well characterized in terms of distribution and shape of teeth in different life stages. Additionally, structural features of the first‐generation teeth have been briefly described. However, at present it is not known how the primary dentition is patterned and if this pattern is similar to that found in juveniles. In this preliminary study, to answer this question we investigate the tooth pattern of a larval P. senegalus based on serial sections and 3D reconstructions, and compare dental features in larvae and juveniles. Polypterus senegalus passive apterolarvae possess a well developed primary dentition. Dentigerous bones develop either prior to the teeth (dentary, retroarticular), or as an attachment site concomitant with the expanding dentition (coronoid 1). Most teeth are attached and erupted, and thus considered functional, and their pulp houses a blood vessel, two features that contrast with earlier findings. The primary teeth do not display a level of maturation that could reflect a particular order of development. New teeth are added anteriorly, and likely also posteriorly, to allow the tooth row to grow to keep pace with the growing jaw. Replacement teeth are absent at this stage. Thus, the pattern observed in juveniles, with alternate (odd and even) positions displaying a different level of tooth maturation, is not present in the larva. Most strikingly, in the larva, considerable symmetry exists between apparently random patterns on both dentaries, eliciting intriguing questions on left‐right control to be addressed in future studies.
  • Immune responses and intestinal morphology of striped catfish,
           Pangasianodon hypophthalmus (Sauvage, 1878), fed dietary nucleotides
    • Abstract: The present study was conducted to evaluate the effects of dietary nucleotides (NT) on growth performance, immune responses, and intestinal morphology of striped catfish, Pangasianodon hypophthalmus. Different levels of NT (0, 0.25. 0.5, 0.75, and 1% weight per weight, WW−1) were used in the basal diet and then randomly allocated to triplicate groups of fish with an average initial weight of 1.52 ± 0.11 g for 10 weeks. Nucleotide supplementation did not improve growth parameters significantly (P > 0.05). Feeding fish with a NT‐containing diet significantly improved ACH50 activity and IgM levels in comparison to the control group (P 
  • Growth and reproduction of the non‐native icefish Neosalanx
           taihuensis Chen, 1956 (Salangidae) in a plateau lake, southwestern China
    • Abstract: Growth, reproduction and abundance traits of the invasive icefish Neosalanx taihuensis Chen, 1956 were investigated monthly from July 2009 to May 2011 in Lake Erhai on the Yunnan‐Guizhou Plateau, south‐western China, in order to explore the changes in life‐history traits after translocation. The results indicated that the icefish exhibited obvious plasticity in growth and reproduction traits. Growth of the fish in Lake Erhai was faster than that in native waters and in other translocated reservoirs. By fitting the von Bertalanffy growth model to the data, it was estimated that icefish obtain an asymptotic size of 96.12 mm, a K of 1.61, and a t0 of ‐0.26; the calculated overall growth performance index φ′ was 4.17. The strategy of reproduction changed from multiple‐ to single‐spawning. The spawning period was from October to December with the absolute and relative fecundities of 1250 ± 169 eggs per ind and 2557 ± 245 eggs per g, respectively. Plasticity in icefish growth and reproduction in Lake Erhai greatly facilitated its population establishment, making it one of the most abundant fish species. The icefish invasion in the lake may be one of the reasons for the decrease or extinction of native fish species populations, and some measures for the control of this invasive fish are suggested.
  • Vertebra deformities in wild Atlantic salmon caught in the Figgjo River,
           southwest Norway
    • Abstract: In order to investigate the occurrence of vertebra deformities in wild Atlantic salmon, adult river migrating salmon were caught in the Figgjo River, Norway in 2010, 2011 and 2012 (n = 24; 21; 20). Based on a definition of a deformed fish having one or more deformed vertebrae, 28 out of 65 fish were classified as deformed. The majority were minor deviations of the morphology in vertebra no 2 and in vertebrae in the ural region (52–56). Vertebral fusions were observed, the majority of which were located in the post‐cranial and posterior‐truncal region of the vertebral column. Fusions typically resulted in a single large vertebra with a relatively normal amphicoelous structure, albeit with multiple neural and hemal arches. This indicates that wild salmon have a strong capability of ‘repairing’ vertebra pathologies, as this stage of a vertebra deformity should not be considered as pathological anymore. Occurrence of individuals with fusions varied over the sampling period (2010: 29%, 2011: 19%, 2012: 5%). Typical deformities observed in farmed salmon, e.g. vertebra compressions in the tail region, which often cause down‐grading, financial losses and reduced welfare, were not observed in any of the analysed fish and can therefore be considered as ‘farming specific’. This is the first study of vertebra deformities in adult wild Atlantic salmon, and demonstrates that these fish can develop deformities up to a level observed in harvest‐size farmed salmon. However, the severity of these malformations was found to be low compared to those commonly observed in farmed salmon. The results of this study may serve as a basis for future research on vertebra deformities in Atlantic salmon.
  • Architecture, mineralization and development of the axial skeleton in
           Acipenseriformes, and occurrences of axial anomalies in rearing
           conditions; can current knowledge in teleost fish help'
    • Abstract: We review current knowledge of axial skeleton biology in Acipenseriformes with the aim to know whether available data can help in understanding axial anomalies (anomalies of the vertebral column) in reared sturgeons or if further investigations are needed prior to undertake such analysis. In particular, accurate data on axial skeleton development, structure and mineralization are required to understand these pathologies. We show that such knowledge is fragmented and incomplete when compared to the information available in teleost fish. In Acipenseriformes, we only know that (i) the axial skeleton morphology is highly distinctive, as mostly cartilaginous, (ii) the vertebrae are composed of several elements organized around a persistent and unconstricted notochord, and (iii) mineralization only concerns a few of these elements and occurs late in ontogeny, but virtually no descriptions demonstrating the presence of bone (perichondral, membranous…) or mineralized cartilage is available in the literature. In addition, few studies concern axial anomalies in the wild or in rearing conditions, and the determinism of these pathologies is largely unknown. This is in contrast with the numerous studies dealing with the axial skeleton and its anomalies in many farmed teleost species, in which axial anomalies can result from numerous factors, such as unfavourable abiotic conditions, inappropriate nutrition and genetic factors. However, even if some parallels can be established between teleosts and sturgeons, it appears that current knowledge of the axial skeleton in teleosts may not help much in the study of axial anomalies in sturgeons. Knowledge of axial skeleton biology in sturgeon has to be therefore deeply improved prior to undertake investigations focusing on these pathologies.
  • Diet composition, feed preferences and mouth morphology of early stage
           silver therapon (Leiopotherapon plumbeus, Kner 1864) larvae reared in
           outdoor tanks
    • Abstract: This study examined the diet composition, feeding preferences, and mouth morphology of the silver therapon (Leiopotherapon plumbeus, Kner 1864) larvae under captive conditions. Larvae were reared in outdoor tanks (4 m3) with natural food grown 2 weeks prior to start of larval rearing. Food preference was measured by the Chesson's electivity index (αi). Gut content analysis of larvae sampled between 5 and 25 days after hatching (DAH) showed the dominance in the diet by zooplankton, mainly copepod nauplii, cladocerans and insect larvae. Small fish larvae (5–9 DAH; 3.32–6.29 mm standard length) preferred cladocerans, ciliates and copepod nauplii; whereas older larvae (12–25 DAH; 5.45–19.26 mm standard length) preferred insect larvae over cladocerans and adult insects. The mouth gape size at 5 DAH was 359 μm and increased to 3.75 mm at 40 DAH when body size grew at an average rate of 0.59 mm d−1. The standard length (SL) of L. plumbeus larvae was strongly associated with mouth size (r2 = 0.98, P 
  • Presence of prohibited fishes in the Brazilian aquarium trade:
           effectiveness of laws, management options and future prospects
  • Evaluation of Lactobacillus paracasei subsp. tolerans isolated from
           Jenyn's sprat (Ramnogaster arcuata) as probiotic for juvenile rainbow
           trout Oncorhynchus mykiss (Walbaum, 1792)
    • Abstract: A lactic acid bacterial strain, Lactobacillus paracasei subsp. tolerans F2, isolated from the intestine of Ramnogaster arcuata, was evaluated as a growth promoter in juvenile rainbow trout Oncorhynchus mykiss (Walbaum, 1972) farming. In addition, the safety of the strain was assessed according to the FAO recommendations. Strain F2 was susceptible to most antibiotics tested and no evidence of hemolytic activity was found. When the strain of Lactobacillus paracasei subsp. tolerans F2 was administered with food, no adverse effects on health were observed and fish biomass increased 12% more in the treatment group than in the control group. Significant differences were detected in the specific growth rate and feed conversion ratio. In the group receiving Lactobacillus paracasei subsp. tolerans F2‐supplemented feed, quantitative differences in the microbial composition of fish feces with respect to the control group were observed. An important decrease in fungi and enterobacteria was observed in feces from the treatment group, coincident with an increase in lactic acid bacteria. This result would indicate a change in the composition of the intestinal microbiota of fish treated with the putative probiotic. These results suggest that the strain of Lactobacillus paracasei subsp. tolerans F2 has the application potential to improve the performance in rainbow trout farming.
  • Length–weight relationships of fish species associated with the
           mangrove forest in the southwestern Terminos Lagoon, Campeche (Mexico)
    • Abstract: Length–weight relationships (LWR) were calculated for 18 species representing 11 families of fishes. A total of 958 fish specimens from the southwestern Terminos Lagoon, Campeche (Mexico), several of which had none or few LWRs available in FishBase, were sampled (except December) using a shrimp otter trawl net from September 2003 to August 2004. Sample sizes ranged from seven Mugil curema and Archosargus probatocephalus individuals to 250 Eugerres plumieri.
  • Occurrence of Apristurus species in the Galicia Bank Seamount (NE
    • Abstract: The aim of this study was to identify some of the Apristurus species by combining morphometric and genetic tools. Several specimens of the genus Apristurus were caught on the Galicia Bank Seamount (NE Atlantic), between 1460 and 1809 m depths, during a multidisciplinary survey carried out in 2011 within the framework of the INDEMARES Project. Morphometric and genetic analyses were conducted to aid the identification of the specimens collected. A total of 20 specimens were identified, of which 18 corresponded to Apristurus aphyodes (Nakaya and Stehmann, ), one to A. profundorum (Goode and Bean, ) and one to A. melanoasper Iglesias, Nakaya & Stehmann, . Genetic results based on the mtDNA COI sequences (682–690 bp fragment of the COI gene) support the identification of A. profundorum and A. melanoasper, with a bootstrap of 99 and 96%, respectively. The identification of A. aphyodes was also performed using a 499 bp fragment of the 16S mitochondrial gene. These are the first records of the Apristurus species from Galician waters, which extends their known area of distribution and provides more information on different biological and ecological aspects of this complex taxonomic group.
  • Growth pattern of an endemic and endangered West Balkan Peninsula trout
           species (Salmo lourosensis, Delling, 2010)
  • Changes in fatty acid profiles of three tissue types in channel catfish
           Ictalurus punctatus (Rafinesque, 1818) transferred from river to pond
    • Abstract: Little information is available regarding the time scale over which tissue fatty acid (FA) profiles change when fish transition between prey resources with different FA profiles, specifically when fish move from a main river channel to a floodplain habitat. The objective of this study was to evaluate changes in the FA composition of muscle, liver, and adipose fin tissues of channel catfish Ictalurus punctatus transferred from the Kaskaskia River, Illinois to earthen ponds at the Center for Fisheries, Aquaculture, and Aquatic Sciences Pond Facility, Makanda, Illinois, mimicking migration from river to floodplain habitat. Over time, n‐3 and C18 polyunsaturated FA (C18 PUFA) decreased in all tissues, whereas monounsaturated fatty acids (MUFA) increased with pond residence time. Liver profiles changed more rapidly than the muscle or adipose fin profiles (significant change in
  • Morphological parameters of abundant and threatened chondrichthyans of the
           northwestern Mediterranean Sea
    • Abstract: The diversity of chondrichthyans in the Mediterranean Sea is relatively high; however, available data indicate that this group is declining in abundance and several species are becoming rare. As a result, the collection of biological data is a priority for demographic models, stock assessments, and food web analysis. In the present study, we report morphological parameters and length–weight relationships of several chondrichthyan species, both abundant and threatened, from the western Mediterranean Sea. Samples were obtained with commercial and scientific bottom trawl vessels between 2001 and 2013. A total of 893 individuals belonging to 11 families and 20 species were weighed and total lengths measured. In addition, seven species of large demersal sharks were measured and length–length relationships obtained to study the relationships between different body length measurements. All species showed positive allometric or isometric growth, except for Centroscymnus coelolepis. The results of the length–weight relationships reveal differences between the western Mediterranean and nearby areas, depending on the species studied.
  • Spatiotemporal occurrence and feeding habits of tonguefish, Cynoglossus
           lighti Norman, 1925, larvae in Ariake Bay, Japan
    • Abstract: Spatiotemporal occurrence and feeding habits of tonguefish (Cynoglossus lighti Norman, 1925) larvae were investigated in an offshore area (>5 m in depth) of the inner part of Ariake Bay, Japan. All specimens were symmetric, free‐swimming larvae. Although their seasonal abundance and distribution in the study site varied from year to year, spawning started in June and the larval abundance was high in August and September with a wide distribution in the inner part of the bay. Both present and previous study results strongly suggest that larvae may settle primarily in the estuary and near‐shore areas of Ariake Bay after their wide distribution in the offshore area of the bay during the free‐swimming stage. Larvae showed a clear feeding rhythm in which they fed on prey mainly during the daytime. Larvae fed exclusively on copepods, and identified prey were mostly Paracalanidae (mainly Parvocalanus crassirostris), Microsetella norvegica, and Oithona davisae. Pre‐metamorphosis larvae fed primarily on Paracalanidae and O. davisae, whereas O. davisae formed a smaller proportion of the early‐metamorphosis diet. In early metamorphoses, larvae fed preferentially on Paracalanidae and M. norvegica.
  • Length–weight relationships for freshwater fish species from the
           Pantanal of the Negro River, Brazil
    • Abstract: The present study describes the length‐weight relationships (LWRs) of 28 species captured in Santa Virgínia Bay, in the Pantanal of the Negro River, Mato Grosso do Sul State, Brazil, over 3 years (1999 to 2001). The study provides the first references on LWRs for thirteen of these species.
  • Occurrence of the Kessler's gudgeon Romanogobio kesslerii (Dybowski, 1862)
           (Cyprinidae) in the Upper Vistula River (Poland)
  • Application of mesohabitat fish use information to identify guilds for
           lotic systems
  • Occurrence of the boarfish, Capros aper (Linneaus, 1758) in Edremit Bay,
           northern Aegean Sea (Osteichtyes: Caproidae)
  • Weight–length and length–length relationships for reef fish
           species from the Cape Verde Archipelago (tropical north‐eastern
    • Abstract: This study reports weight–length and length–length relationships for selected coastal reef fish species of the Cape Verde Archipelago (tropical north‐eastern Atlantic). Specimens were caught with different types of gear (long‐lines, hand‐lines, purse‐seines and traps) during commercial fishing activities and sampled during fish market operations. A total of 8328 individuals were sampled, representing 29 species from 14 Families. This study provides the first references on weight–length and length–length relationships for five and 23 fish species worldwide, for 10 and 24 species for the Eastern Atlantic and for 12 and 26 species for Cape Verde Archipelago, respectively. Additionally, it provides revised weight–length relationships for 11 species from Cape Verde waters.
  • First length–weight relationships of 11 fish species in the Aegean
    • Abstract: Weight‐length relationships were established for eleven marine fish species caught in the SE Aegean Sea, Turkey. Additionally, a bibliographic review of such relationships for these species was conducted. Based on the results, the values of b parameter varied between 2.477 and 3.496, with one species having isometric growth, five negative and six positive allometric growth. Furthermore, for Aulopus filamentosus there exist no information in the literature, whilst for Callanthias ruber and Gnathophis mystax, there are no such information available from the Mediterranean.
  • Phenotypic plasticity during early ontogeny in cultured turbot
           (Scophthalmus maximus): changes in dorsal and anal fin ray counts by water
    • Abstract: Plastic responses to environmental conditions are crucial among ectothermic organisms, and any traits induced early in ontogeny may have long‐term effects. Here, we studied how turbot larvae altered their morphology in three different thermal environments and assessed whether different thermal regimes affected the survival, growth, development and morphology of juvenile turbot at the end of metamorphosis. Therefore, newly hatched Turbot (Scopthalmus maximux L.) larvae were divided into three groups and reared at 14, 18 and 22°C until 80 days after hatching. Environmental temperature was seen to affect several turbot traits. Low temperature induced low dorsal and anal fin ray counts, but had no effect on gross body morphology. There were differences in growth and skeletal development rates, but no differences in survival. There were also no differences in the skeletal malformations occurring in response to environmental temperature treatment. Collectively, these results supported the idea of the existence of a thermal plastic response, as found, in other fish species.
  • Exploring the molecular link between swim‐training and caudal fin
           development in zebrafish (Danio rerio) larvae
    • Abstract: In vitro and in vivo studies have shown that mechanical forces play an important role during development. The molecular mechanisms via which mechanical forces regulate development have been extensively investigated by in vitro studies. However, knowledge about the molecular pathways that mediate the effect of mechanical forces during development in vivo is limited. Previously, we showed that swim‐training increased maximum normalized curvatures in the caudal fin (suggesting that the caudal fin experienced increased mechanical loads) and prioritized the development of skeletal structures in the caudal fin. Therefore, we used the zebrafish caudal fin to explore the molecular link between an increased swimming activity and development in vivo. Whole genome microarray analysis of caudal fins of zebrafish subjected to swim‐training and control fish identified 46 genes which were up‐regulated with a fold change of 1.5 or larger at 10 dpf. Fourteen genes were expressed specifically in the following tissues in the caudal fin: the neural tube, the tissue surrounding the hypurals, the finfold, or muscle fibers. Subsequently, we identified two muscle specific genes, aste1 (asteroid homolog 1) and zgc:65811, which showed an increased expression specifically in the caudal fin in response to swim‐training. This makes these genes interesting candidate genes for further research on the molecular link between mechanical forces and caudal fin development. Our study is the first to investigate the molecular link between swim‐training and caudal fin development and offers a system that can provide a deeper understanding of the link between mechanical and molecular signals during development in vivo.
  • Hybrid salmonids: ploidy effect on skeletal meristic characteristics and
           sea lice infection susceptibility
    • Abstract: This study examined how triploidization and species hybridization affects skeletal related meristic characteristics in salmonids with the goal of understanding the maternal and paternal contribution. The study also investigated the hybrid sea louse infection rate in hybrid salmonids. In order to do so, a number of vertebrae, scales along the lateral line, and dorsal fin rays were measured in diploid and triploid Atlantic salmon, Arctic char, and Atlantic salmon (female) X Arctic char (male) hybrids. The success of triploidization was 100%, and some spontaneous triploid individuals (~15%) were found among the hybrids. In general, hybrids displayed intermediate counts for all three characteristics with salmon displaying the highest number of dorsal fin rays, and char displaying the highest number of scales and vertebrae. The effect of triploidization was always strongest among the hybrids. However, the direction of the effect differed between structures in the hybrid; triploidization increased the number of vertebrae and reduced the number of fin rays towards the level observed in diploid char, and reduced the number of scales towards the level observed in diploid salmon. We conducted a second experiment whereby diploid Atlantic salmon and Atlantic salmon (female) X Arctic char (male) hybrids were exposed to an experimental challenge with the parasitic salmon louse (Lepeophtheirus salmonis). There was no difference in the sea‐lice infection level between Atlantic salmon and the hybrids. This study shows that the present model with diploid and triploid Atlantic salmon (female) X Arctic char (male) hybrids may be a useful tool for the study of which traits are maternally and paternally inherited in salmonids. The understanding of morphological development with regard to meristic traits could be advantageous for both hybrid and triploid fish domestication.
  • Exploring the effect of exercise on the transcriptome of zebrafish larvae
           (Danio rerio)
    • Abstract: In adult vertebrates, endurance training leads to physiological, metabolical and molecular adaptations which improve endurance performance. Only very few studies have focused on adaptive responses to endurance training during early vertebrate development, and molecular data is limited. Here, we explored the effect of swim‐training on the transcriptome of the zebrafish during early development on a quantitative and spatial gene expression level. We subjected larval zebrafish from 5 to 14 dpf (days post fertilization) to swim‐training and performed a whole genome microarray analysis of trained and control fish sampled at 10 dpf. In addition, we investigated if swim‐training affected the expression of genes involved in muscle growth and structure with quantitative real‐time PCR in trained and control fish sampled at 5 and 14 dpf. To obtain a general overview of the effects of swim‐training on the transcriptome, we selected 52 genes from the whole genome microarray analysis based on a number of criteria. In situ hybridization demonstrated that 15 genes were specifically expressed in the brain, muscle, kidneys, liver, pancreas or intestines. Thus, swim‐training led to molecular changes already after 6 days of swim‐training and in a variety of organ systems. In addition, the expression of slow fiber markers was increased after 10 days of swim‐training, indicating that muscle can already shift towards a slow aerobic phenotype during zebrafish larval development. Taken together, this study demonstrates that significant changes occur, even at early stages, as an adaptive response to endurance training during early vertebrate development.
  • Radioactive contamination causes only minor effect on fluctuating
           asymmetry of two fish species from the Chernobyl area
    • Abstract: The western part of the Bryansk region (Russia) was seriously contaminated with radioactive isotopes by the Chernobyl disaster in 1986. Contamination level of this area is similar to contaminated regions in Ukraine and Belarus. We studied the fluctuating asymmetry of two cyprinid species, roach Rutilus rutilus and bleak Alburnus alburnus, to assess the effect of radioactive contamination on developmental stability. Fluctuating asymmetry represents random deviations from perfect, usually bilateral symmetry, and is often used to assess developmental stability. Fish were collected in summer 1992 from the Desna and Iput rivers of the Bryansk region with similar hydrological characteristics but with differing levels of radioactive contamination. Contamination in the Desna River exceeded the background level of non‐contaminated areas, but was considerably lower than in the Iput River. Roach were 3–5 years old and bleak 1–2 years old. In total, 78 specimens were analyzed, with 13 roach and 26 bleak from each location. We examined 39 meristic and 18 morphometric characters in cranial bones of roach and 23 meristic and 13 morphometric characters in bleak. No significant differences in meristic or morphometric characters were found between roach from the contaminated and reference locations. In bleak, we found significantly higher fluctuating asymmetry in meristic (P test, P 
  • Atlantic salmon (Salmo salar L.) in the border of distribution range:
           patterns of osteological variation
    • Abstract: Osteological differentiation and developmental stability of Atlantic salmon juveniles were studied in eight rivers of the White, Barents and the Baltic seas basins. Mean values and fluctuating asymmetry (random deviations from perfect bilateral symmetry, measure of developmental instability, FA) were measured on skull bones using shape of the bones and number of teeth and sensory pores. Shape of skull bones allows correct identification of juveniles from different rivers with probability as high as 80%. Number of teeth and sensory pores allow only 46%. The most pronounced differences were found between three samples from the Pechora River basin, i.e. northeastern border of the species range, and five other samples. Pattern of osteological differentiation of samples does not coincide with genetic differentiation. Based on genetic markers, salmon from the Baltic Sea basin differ considerably from fish of the White and Barents seas basins. Morphological data show similarity between the Baltic and the White seas salmon and differences from Pechora River (the Barents Sea basin) fish. These results suggest that inter‐population osteological differences in Atlantic salmon are mostly determined by local conditions in rivers. Eastern populations also differ from all others by a higher FA, indicating their lower developmental stability. A significant negative correlation between FA and winter temperature suggest that lower winter temperatures, directly or indirectly, are stressful factor causing decrease in developmental stability of salmon near the eastern border of their distributional range.
  • Ets1 regulates the transcription of a cartilage‐specific S100
           protein in gilthead seabream
    • Abstract: A novel S100 calcium‐binding protein has been recently identified in teleost fish. Its expression is restricted in vivo to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization and its function has been associated in vitro with mechanisms of extracellular matrix mineralization. To get more insights into this mineralogenic role, the transcriptional regulation of S100 gene was investigated using luciferase reporter constructs. The occurrence of several silencers was revealed through the analysis of the basal activity of promoter constructs in HEK‐293 cells. Among those, a silencer located in the region −883/−768 had the capacity to shut down completely the activity of S100 gene promoter. The presence of several putative binding sites for Ets1, a transcription factor regulating the expression of several cartilage‐related proteins, was predicted through in silico analysis. Analysis of luciferase activity in cells expressing zebrafish Ets1a revealed that regions −636/−513 and −82/+62 contain active Ets1 binding sites and decrease luciferase activity upon mutation of specific sites confirmed the effectiveness of Ets1 binding at positions −552/−539 and −517/−501. In conclusion, this work provided novel evidence for the transcriptional regulation of fish cartilage‐specific S100 protein by Ets1.
  • Spatiotemporal expression and retinoic acid regulation of bone
           morphogenetic proteins 2, 4 and 16 in Senegalese sole
    • Abstract: Bone morphogenetic proteins (BMPs) are involved in various physiological processes from early life stages throughout adulthood. Specific characteristics of BMPs have been used to define different subfamilies and BMP2/4 subfamily (composed of BMP2 and BMP4) has been linked to osteogenesis and skeleton development. BMP16 was recently identified as a new member of the BMP2/4 subfamily and reported as a teleost fish‐specific form. In this work, we collected a comprehensive set of ray‐finned fish BMP2, BMP4 and BMP16 sequences and demonstrated, through its presence in Holostei, that BMP16 is not restricted to teleost fish genome. Comparative analysis of BMP2, BMP4 and BMP16 primary structures revealed that most of the residues required for protein stabilization, dimer formation, glycosylation and receptor binding are substantially conserved between the three proteins, suggesting that BMP16, BMP2 and BMP4 may share similar mechanisms of action. In contrast, comparative analysis of gene expression profiles during Senegalese sole development revealed differences in onset and extent of gene expression, indicating that BMP16, BMP2 and BMP4 may contribute to different developmental and physiological processes. High levels of transcripts in adult calcified tissues and the up‐regulation of gene expression by retinoic acid, a known regulator of skeletal development, suggests that BMP16 shares with BMP2 and BMP4 a role in bone metabolism and skeletal development. This study provides new insights into the taxonomic distribution and the spatiotemporal expression of BMP16 gene, and suggests that it may share structural and functional similarities with other members of the BMP2/4 protein subfamily.
  • Detection of nitric oxide by diaminofluorescein visualizes the skeleton in
           living zebrafish
    • Abstract: Several in vivo stainings, such as Calcein, Alizarin Red and Quercetin are commonly used to visualize ossification in living teleost specimen. These staining techniques represent important tools for bone research in fish, but do not visualize cartilage. In the present study, we show that nitric oxide (NO) labelling by DAF‐FM DA visualizes both bone and cartilage in vivo during zebrafish skeletogenesis. NO detection performed in Tg(osterix:mCherry) or in combination with Alizarin Red in wild‐type zebrafish reveals that intense staining through NO labelling colocalizes with the appearance of osteoblasts and characterizes ossified structures. Cartilage structures are clearly distinguished in the living larvae, although the labelling is less intensive when compared to ossified structures. This method is the first and easy to handle alternative to cartilage and bone double stainings on fixed samples. In contrast to most live skeletal stainings, which only stain the mineralized bone structures, this protocol in addition allows in vivo visualization of cartilage.
  • Can zebrafish be a valid model to study Paget's disease of bone'
    • Abstract: Paget's disease of bone (PDB) is the second most frequent metabolic bone disease after osteoporosis. Genetic factors play an important role in PDB, but to date the only PDB causal gene identified is the Sequestosome 1 (SQSTM1) gene. Because the zebrafish has been validated as a model for human genetic diseases, the objective was to investigate if the gene structure and chromosomal environment of the SQSTM1 were similar between zebrafish and humans, thus providing a basis for developing a mutant fish capable of modeling PDB. Through a comparative in silico analysis, we confirmed that zebrafish sqstm1 shares not only the same gene structure as its fish and mammalian orthologs, but they also present, within their promoter regions, similar putative binding sites for common transcriptional factors known to affect bone metabolism. The synteny of SQSTM1 was also determined and results indicate that the cluster of surrounding genes was conserved throughout evolution. The protein comparison revealed a high degree of conservation, particularly in the functional domains of the protein. The most common mutation in PDB patients is p.Pro392Leu and the residue Pro392 was found to be 100% conserved in all species analyzed, including zebrafish, confirming its known functional relevance. In conclusion, this study demonstrated that SQSTM1 is well conserved throughout evolution and therefore fish models such as the zebrafish could be an interesting tool to further investigate the biological role of SQSTM1 in PDB and in bone development.
  • Genomics of zebrafish hoxba and hoxbb loci
  • A statistical approach to mutation detection in zebrafish with
           next‐generation sequencing
    • Abstract: The zebrafish is an excellent model organism for forward‐genetics, with attributes such as small size, rapid development and straightforward imaging enabling mutagenesis screens for a wide variety of phenotypes. For the majority of these screens over the last few decades, the mutations were mapped using bulk segregant analysis (BSA) to establish approximate chromosomal locations, followed by fine mapping using microsatellite markers on hundreds (or thousands) of embryos. This process is very time consuming despite the large clutch sizes of the zebrafish. Next‐generation sequencing (NGS) technologies have drastically improved the speed of this process, but there is no consensus on the best method for performing the BSA and fine‐mapping analysis on NGS data. Here we describe a simple statistical approach to this problem using difference‐in‐homozygosity as a single variable with a normal distribution. This approach was used to accurately map and identify the causative mutation in a zebrafish line with a recessive mineralization disorder.
  • Zebrafish (Danio rerio) in calcium‐poor water mobilise calcium and
           phosphorus from scales
    • Abstract: Calcium is of vital importance in vertebrates and plasma levels are tightly regulated. Terrestrial vertebrates depend for their calcium uptake exclusively on the diet, while fish have an essentially infinite directly accessible source of calcium in the surrounding water. The need to store calcium seems thus less evident in fish. It was the goal of the present study to investigate the possible role of the scale compartment of zebrafish (Danio rerio) as an accessible pool for calcium and phosphorus. We restricted the calcium availability to zebrafish both in water and diet. Following 12 days restriction, we found that scalar contents of calcium and phosphorus had declined, while magnesium was unaffected, indicating resorption of calcium hydroxyapatite. Osteoclast activity on scales of calcium‐restricted fish increased, as we conclude from enlarged demineralized areas on scales, increased TRAcP activity staining, and higher ctsk gene expression. Osteoblasts respond to calcium limitation by increasing expression of col1a2 and alpl. In the presence of water and dietary calcium, removal of scales from one flank of the fish does not affect scalar mineral contents, scale demineralization and osteoclastic TRAcP activity; expression of sp7, alpl and ctsk are up‐regulated. Apparently, a high demand for calcium in zebrafish is preferentially complemented with exogenous calcium, while calcium is recruited from the scales when environmental calcium is limited. We conclude that the scales represent an important deposit of available calcium and phosphorus from which these minerals can be recruited in periods of high demand. This finding contributes to a niche for scales as model in bone research.
  • Development and characterization of eleven microsatellite markers for an
           endangered cavefish (Triplophysa rosa Chen and Yang, 2005) using 454
    • Abstract: Triplophysa rosa is an endangered cave‐dwelling and endemic fish species found only in Chongqing, southwestern China. The genetic data available for this species is very limited. Polymorphic microsatellites were identified in the genome of T. rosa using 454 sequencing. Of the 145 loci screened, 106 were amplified successfully and 11 showed polymorphic patterns. The number of alleles ranged from 2 to 6, and the observed and expected heterozygosity varied from 0.061 to 0.543 (mean = 0.349) and from 0.248 to 0.789 (mean = 0.551), respectively. The polymorphic information content (PIC) ranged from 0.215 to 0.744 (mean = 0.486), indicating moderate levels of polymorphism. In addition, cross‐species amplification was tested for the 106 loci in Triplophysa moquensis, which showed a high level of transferability (76.4%), implying that the microsatellite markers developed here could be used effectively for other closely related species.
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