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Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage., (© 2021 Houseright et al.)

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

The establishment of reproductive barriers between populations can fuel the evolution of new species. A genetic framework for this process posits that "incompatible" interactions between genes can evolve that result in reduced survival or reproduction in hybrids. However, progress has been slow in identifying individual genes that underlie hybrid incompatibilities. We used a combination of approaches to map the genes that drive the development of an incompatibility that causes melanoma in swordtail fish hybrids. One of the genes involved in this incompatibility also causes melanoma in hybrids between distantly related species. Moreover, this melanoma reduces survival in the wild, likely because of progressive degradation of the fin. This work identifies genes underlying a vertebrate hybrid incompatibility and provides a glimpse into the action of these genes in natural hybrid populations., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We evaluated the relationship of stocking density to survival, growth performance and fin condition of European perch Perca fluviatilis with hand feeding and self-feeders. Hand-fed perch (body weight 19.1 ± 5.1 g and total length 107 ± 9 mm) were reared at 0.5, 1.0, 1.5 and 2.0 fish/L. Self-feeding perch (body weight 25.4 ± 3.9 g and total length 128 ± 7 mm) were reared at stocking densities of 0.6, 1.0 and 1.4 fish/L. Pond-reared perch served as a comparison group for fin damage assessment. We found no differences in survival rate among stocking densities with either feeding method. Hand-fed fish displayed the highest weight gain and SGR at stocking density of 0.5 fish/L. The self-feeding fish showed a non-linear association of weight gain with stocking density with the highest growth at 1.0 fish/L. Fin length was noticeably greater in pond-reared fish compared with RAS-reared fish regardless of feeding method. In both experiments, fin length relative to standard length showed a negative relationship with stocking density, with pectoral fins showing the greatest effect. Fin condition deteriorated with increasing stocking density, and growth was highest at 0.5 and 1.0 fish/L in hand-fed and self-feeding fish, respectively., (© 2019 John Wiley & Sons Ltd.)

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease., Competing Interests: AR, KH, KH, AT, HI, CT, DS, JR, NK, AR, KH, CT, IS, WZ, AM, MW, DD, SR, LP No competing interests declared, CS is an employee of GlaxoSmithKline Research and Development Ltd. The author declares no other competing interests exist., (© 2019, Rahman et al.)

Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3a wue1/wue1 ) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3a wue1/wue1 mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish.

Reduced bone quality or mineral density predict susceptibility to fracture and also attenuate subsequent repair. Bone regrowth is also compromised by bacterial infection, which exacerbates fracture site inflammation. Because of the cellular complexity of fracture repair, as well as genetic and environmental influences, there is a need for models that permit visualisation of the fracture repair process under clinically relevant conditions. To characterise the process of fracture repair in zebrafish, we employed a crush fracture of fin rays, coupled with histological and transgenic labelling of cellular responses; the results demonstrate a strong similarity to the phased response in humans. We applied our analysis to a zebrafish model of osteogenesis imperfecta (OI), which shows reduced bone quality, spontaneous fractures and propensity for non-unions. We found deficiencies in the formation of a bone callus during fracture repair in our OI model and showed that clinically employed antiresorptive bisphosphonates can reduce spontaneous fractures in OI fish and also measurably reduce fracture callus remodelling in wild-type fish. The csf1ra mutant, which has reduced osteoclast numbers, also showed reduced callus remodelling. Exposure to excessive bisphosphonate, however, disrupted callus repair. Intriguingly, neutrophils initially colonised the fracture site, but were later completely excluded. However, when fractures were infected with Staphylococcus aureus , neutrophils were retained and compromised repair. This work elevates the zebrafish bone fracture model and indicates its utility in assessing conditions of relevance to an orthopaedic setting with medium throughput.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant genetic disorder in humans characterized by the gradual ossification of fibrous tissues, including skeletal muscle, tendons, and ligaments. In humans, mutations in the Type I BMP/TGFβ family member receptor gene, ACVR1, are associated with FOP. Zebrafish acvr1l, previously known as alk8, is the functional ortholog of human ACVR1. We previously created and characterized the first adult zebrafish model for FOP by generating animals harboring heat shock-inducible mCherry-tagged constitutively active Acvr1l (Q204D). Since injury is a known trigger for heterotopic ossification (HO) development in human FOP patients, in this study, we investigated several injury models in Acvr1l Q204D -expressing zebrafish and the subsequent formation of HO. We performed studies of Activin A injection, cardiotoxin (CTX) injection, and caudal fin clip injury. We found that none of these methods resulted in HO formation at the site of injury. However, some of the cardiotoxin-injected and caudal fin-clipped animals did exhibit HO at distant sites, including the body cavity and along the spine. We describe these results in the context of new and exciting reports on FOP, and discuss future studies to better understand the etiology and progression of this disease.

Pectoral fin healing in fin spines and rays were examined in juvenile Atlantic sturgeon Acipenser oxyrinchus oxyrinchus following three different sampling techniques (n = 8-9 fish per treatment): entire leading fin spine removed, a 1-2 cm portion removed near the point of articulation, or a 1-2 cm portion removed from a secondary fin ray. Also, to determine whether antibiotic treatment influences healing, an additional group of fish (n = 8) was not given an injection of an oxytetracycline (OTC)-based antibiotic following removal of the entire leading fin spine. Following fin sampling, fish from different treatments were mixed equally between three large (4,000 I) recirculating systems and fin-ray healing and mortality were monitored over a 12 month period. To assess healing, blood samples were collected at 4 months to measure immune system responses, radiographs were taken at 4, 8 and 12 months to assess the degree of calcification in regions of damaged fins and fins were analyzed histologically at 12 months. Fish grew from a mean weight of 1.8 to 3.2 kg during the experiment and survival was near 100% in all treatments, with only one fish dying of unknown causes. Leukocyte counts, an indication of health status and survival were similar among treatments and in groups with or without antibiotic injection. Radiographs revealed mineralization took longer in fish with the entire leading fin spine removed and was the slowest near the point of articulation, presumably due to the greater structural support for the pectoral fin at this location. Histological sampling indicated spines and rays had similar healing patterns. Following injury, an orderly matrix of collagen bundles and many evenly spaced scleroblasts were present, transitioning to Sharpey fibres, with concentric layers forming lamellar bone. Healing and mineralization were characterized as periosteal osteogenesis and included embedded osteocytes surrounded by an osteoid seam. Chondroid formation was apparent in a few fractures not associated with treatments. The duration of time for external wound healing and internal mineralization of spines and rays depended on the fin treatment, with the slowest healing observed in fish with the most tissue removed, the entire leading fin spine., (© 2018 The Fisheries Society of the British Isles.)

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

X-ray micro-computed tomography scans were used to examine the caudal-fin structure of an unusual double-tailed deformity in an adult brown surgeonfish Acanthurus nigrofuscus from the Great Barrier Reef. In both this case and in a similar double-tailed deformity in a juvenile Tomini surgeonfish Ctenochaetus tominiensis from the Philippines, the caudal fin was duplicated along the dorsoventral axis. Detailed examination of the A. nigrofuscus specimen revealed that the deformity was associated with duplication and reflection of the hypural plates and the posterior vertebrae, yet the fish survived to adulthood, indicating that the effects of duplication on survival may be limited., (© 2018 The Fisheries Society of the British Isles.)

A new species of myxozoan, Thelohanellus goldi n. sp. is described using morphological and molecular data, parasitizing the fin filaments from 18 of 25 host specimens (72.5%) of Carassius auratus collected from different ornamental fish farms of India. Mature spore of the new species were oval to spherical in frontal view having rounded posterior ends and tapering anterior end measures 8.7-10.26 (9.50) × 4.10-7.89 (5.84) μm. The single large polar capsule, round to oval in shape but slightly pointed at the anterior end measuring 4.91-7.63 (5.60) × 2.3-3.1 (2.96) μm and located just below the anterior end of the spore. Polar filament only at distal end with 5-6 loose coils. The most differentiating feature from closely related species was carried out by morpho-taxonomic affinities with previously described species which are tremendously supported by molecular taxonomy by partial sequencing of the 18S rDNA gene resulted in a total of 2124 bp fragment of newly obtained small subunit ribosomal RNA gene sequence of the new species which Exhibit 93-95% homogeneity with other closely related species available in GenBank. The BLAST search and high genetic diversity of distance matrix of Myxobolus sp. did not properly match with any available sequences in GenBank and make sister clade with Thelohanellus caudatus and Thelohanellus habibpuri in the Thelohanellus clade including most of Thelohanellus spp. The study of evolutionary history enables us to understand the evolution of modern species and supports some uncertain topologies which are being presented regarding the morphometric analysis. The severity of myxozoan infection has been assessed in this article by observing the histopathological changes of fins of the C. auratus along with the diversity, distribution and taxonomic description of the new Thelohanellus species with their new host and locality records., (Copyright © 2018 Elsevier B.V. All rights reserved.)

As one of three akt isoforms, akt2 plays a key role in the regulation of widely divergent cellular processes in mammals. However, its role and underlying mechanisms in zebrafish remain largely unknown. To elucidate the function of akt2 in zebrafish, we generated zebrafish lacking akt2 gene via CRISPR/Cas9 technology. Akt2-null zebrafish exhibit partial lethality and severe growth deficiency, which is different from those observed in akt2-null mice. Furthermore, akt2-null zebrafish display deficiency in fin ray development, but their cartilage is not affected. Similar to observations in akt2-null mice, akt2-null zebrafish display impaired glucose homeostasis. However, in contrast to that in akt2-null mice, insulin level is lower in akt2-null zebrafish, implicating the symptoms of type I diabetes exhibited in akt2-null zebrafish. In addition, transcriptome analysis reveals that the genes involved in metabolism and osteogenesis are disturbed in akt2-null zebrafish. Taken together, these data not only support an important role of akt2 in zebrafish survival, growth, bone development and glucose homeostasis, but also suggest that akt2 has divergent functions between mice and zebrafish, even though they are evolutionarily conserved., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid- and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.

Lufenuron is a benzoylurea insecticide that interfere in chitin synthesis in insects. Although lufenuron is widely used in agriculture and aquaculture, rare are studies described that relates to possible toxic effects in fish. This work aimed to evaluate acute and chronic toxic effects of benzoylurea pesticide (lufenuron) on biological parameters of Colossoma macropomum (Tambaqui). In the acute test, juveniles of Tambaqui were divided into control group and five experimental groups with exposure from 0.1 to 0.9 mg/L of lufenuron for 96 h. Animals were also submitted to chronic toxicity test for four months in concentrations of 0.1 and 0.3 mg/L of lufenuron, the concentration used in the treatment of ectoparasites in fish and 50% of LC50 96 h, respectively. The presence of hemorrhages was observed in eyes, fins and operculum of fish exposed to 0.7 and 0.9 mg/L of lufenuron. Histological analysis showed changes in the morphology of fish gills submitted to acute toxicity test, as lamellar aneurysm and blood congestion inside lamellae. Lufenuron promoted damage in fish retina as in ability to respond to stimuli in photoreceptors and in ON-bipolar cells in acute test. In chronic test, blood glucose analysis and morphometric parameters showed no significant differences (p > 0.05). In general, Tambaqui exhibited behaviors associated with stress when exposed to lufenuron. Thus, lufenuron showed several toxic effects in relation to biological parameters in Tambaqui. This concerns about the use and discard of lufenuron, and indicates the requirement of environmental actions to prevent potential contamination of aquatic biota., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Gyrodactylus salmonis is a common ectoparasite on the fins and body of North American salmonids in fresh water. In this study, the spatial distribution of G. salmonis on 60 captive hatchery-reared rainbow trout, Oncorhynchus mykiss , is reported. The highest parasite densities occurred on 5 × 5-mm(2) sections of the dorsal fin followed by the trunk, other fins, and the olfactory chamber, with the lowest densities on the head. The finding of infections within the olfactory chamber of 93% of the fish was unexpected. One possibility is that such infections represented spillover from high-density infrapopulations that occur on the skin and fins. However, this possibility is unlikely, because worm densities at various sites along the body surface of infected fish did not correlate with densities within the olfactory chamber. The parasite conceivably enters the chamber either via water incurrent or by crawling in from the head and subsequently remaining at this site to feed and reproduce. Results from scanning electron microscopy are consistent with physical modification to the olfactory epithelium associated with the attachment/reattachment of the opisthaptor and epithelial grazing.

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation.

This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX-induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0-5 hours) of wound-healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up-regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life., (© 2015 by the Wound Healing Society.)

A new species of the genus Thelohanellus Kudo, 1933 (Myxosporea, Bivalvulida) was isolated from the fins of goldfish Carassius auratus auratus (Linnaeus 1758). The fish had been imported from China by an Austrian retailer. Nodules from the margins of the fins contained pyriform myxospores with a singular polar capsule. In valvular view, the spores measured 12.2 µm in length and 6.4 µm in width. In sutural view, the thickness was 2.9 µm. The polar capsule measured 4.2 × 3.1 µm and contained a polar filament with 8 to 9 coils. Histological sections showed plasmodia of 0.2 to 4.0 mm diameter with the earlier developmental stages of the parasite in the periphery and the mature spores closer to the center. In the transmission electron microscope examination, the different developmental stages could be observed. Morphological data, host specificity, tissue tropism, and molecular analysis of the small subunit rDNA identify this parasite as a new species of Thelohanellus, which we have named Thelohanellus hoffmanni sp. nov.

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells., (© 2014. Published by The Company of Biologists Ltd.)

Interleukin-1 (IL-1), the 'gatekeeper' of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

From a forward genetic screen for epidermal defects in zebrafish, we identified a loss-of-function mutation in Kindlin-1, an essential regulator of integrin function. The mutation generates a premature stop codon, deleting the integrin-binding site. The mutant zebrafish develops cell-matrix and cell-cell adhesion defects in the basal epidermis leading to progressive fin rupturing, and was therefore designated rupturing-of-fins (rof). Similar defects were observed in the epidermis of Kindler syndrome patients, carrying a loss-of-function mutation in kindlin-1. Mutational analysis and rescue experiments in zebrafish revealed that residues K610, W612, and I647 in the F3 domain are essential for Kindlin-1 function in vivo, and that Kindlin-2 can functionally compensate for the loss of Kindlin-1. The fin phenotype of rof/kindlin-1 mutants resembles that of badfin mutants, carrying a mutation in integrin α3. We show here that this mutation impairs the biosynthesis of integrin α3β1 and causes cell-matrix and cell-cell defects in vivo. Whereas both Integrin-linked kinase (Ilk) and Kindlin-1 cooperate with Integrin α3β1 to resist trauma-induced epidermal defects, Kindlin-1 and Ilk, surprisingly, do not act synergistically but in parallel. Thus, the rof/kindlin-1 mutant zebrafish provides a unique model system to study epidermal adhesion mechanisms in vivo.

Nonlethal sampling is becoming a common method to diagnose fish diseases, especially with the availability of molecular testing. Viral hemorrhagic septicemia virus (VHSV) is a viral pathogen of finfish distributed worldwide. Although VHSV has been known to occur in some parts of the world for decades, a new genotype, IVb, recently emerged in the Laurentian Great Lakes of northeastern North America. Golden shiners (Notemigonus crysoleucas; Mitchill, 1814) and fathead minnows (Pimephales promelas; Rafinesque, 1820) were exposed to VHSV-IVb doses between 10(2) and 10(6) plaque forming units per fish by intraperitoneal injection at 10°C. Both species experienced significant mortality after exposure, ranging from 38% to 52% in golden shiners and from 35% to 95% in fathead minnows. In golden shiners, a fin or gill sample was somewhat less sensitive at detecting VHSV-IVb by quantitative reverse transcription polymerase chain reaction (qRT-PCR) than a pooled organ sample (consisting of liver, anterior and posterior kidney, spleen, and heart), however the relative sensitivity increased when a fin and gill sample were tested in parallel. In fathead minnows, a fin or gill sample tested alone or in parallel was relatively more sensitive than a pooled organ sample by qRT-PCR. Specificity was 100% for all sample types in both species. The results suggest that fin and gill biopsies are useful tools to test for VHSV in live fish.

A 10-year-old female false killer whale (Pseudorca crassidens) developed skin lesions in the left breast fin. Histopathologically, the lesions consisted of multiple granulomas spread diffusely into the deep dermis and bone; characteristically, each granuloma had septate, branching fungal hyphae and chlamydospores surrounded by eosinophilic Splendore-Hoeppli materials. Macrophages, epithelioid cells and multinucleated giant cells in the granulomas reacted mainly to anti-SRA-E5 antibody against human macrophage scavenger receptor type I. Fusarium solani was isolated and its gene was detected from the skin samples. Mycotic skin lesions by Fusarium spp. reported so far in marine mammals were regarded as superficial dermatitis; therefore, the present case is very uncommon in that the lesions spread deeper into the skin.

Philasterides dicentrarchi is a histophagous scuticociliate infecting Korean olive flounder farms in Jeju Island, South Korea, where it causes significant economic losses. However, the route of entry of these parasites in olive flounder is currently unknown. In the present study, we attempted experimental infection with different doses (concentrations) of P. dicentrarchi by intraperitoneal (IP) injection; maximum cumulative mortality of 95% was recorded on the administration of 2.1×10(8) ciliates/ml. In small size group (3 cm length, 24±2 gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5 cm length, 47±3 gwt), the ciliates were predominantly occurred with associated symptoms of liquefaction in the central nervous system. The major clinico-pathological manifestations were loss of scales, appearance of bleached spots that coalesced to form brownish patches, hemorrhagic, and severe dermal necrotic lesion. In addition there was degeneration of muscle fibers, necrosis of the hepatic parenchyma, and severe edema of the intestinal walls, extensive fouling, necrotic degeneration and hyperplasia in the gill bronchial epithelium. In this study, many ciliates were found in the blood vessel, brain, dorsal fins, muscle, kidney, and vertebra of infected flounder. The histopathological investigations are of significant importance in view of possible routes of entry into the host and doses of ciliates that trigger fast infection for potential utility of drugs as a strategy for the control of P. dicentrarchi in farmed olive flounder., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study focused on several wetlands in Laguna del Tigre National Park (Guatemala) as part of Conservation International's Rapid Assessment Program. Sediment and water samples were collected from a laguna near Xan field, Guatemala's largest oil facility, and three other sites for determination of levels of polycyclic aromatic hydrocarbons (PAHs). Cichlid fish (Thorichthys meeki and Vieja synspila) were collected for determination of DNA strand breakage (by gel electrophoresis), chromosomal breakage (flow cytometry), and fin erosion. For T. meeki from Xan field, chromosomal breakage and strand breakage was greater than in at least two of the three reference sites. For V. synspila, chromosomal breakage and strand breakage were greater in Xan than one of the two reference sites. Fin erosion was observed only at the Xan laguna. Genetic biomarker effects and fin erosion, along with patterns of aqueous PAH concentrations, indicate that fish are affected by anthropogenic contaminants. PAHs were elevated at some reference sites, but environmental forensic analysis suggested a pyrogenic or diagenic origin. It is possible that oil field brines injected into the ground water caused fin erosion and genotoxicity in fish at Xan field, and it is also possible that pyrogenic PAHs influence levels of DNA damage in reference sites. These analyses represent one of the first efforts to examine genotoxicity in native Mesoamerican cichlids.

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The study reported in this paper tested the hypothesis that the developmental and reproductive health of mosquitofish (Gambusia affinis) exposed to pulp and paper effluent in the Dengcun River would differ from that of mosquitofish living in a reference site. We also studied whether morphological characteristics such as the anal fin and hemal spines of mosquitofish could serve as indicators for evaluating the androgenic effect and mosquitofish population security in the Dengcun River. Male and female mosquitofish were captured at three sites contaminated by pulp and paper effluent in the Dengcun River in Sihui, South China, and at a nearby uncontaminated reference site. Samples were collected from the sampling sites on the same day in August 2009. We compared the populations by total length, wet body and liver mass, gonad mass, and population composition. We also compared the populations according to number of anal fin segments, oocyte and embryo count, anal fin and hemal spine morphology among females, and by sperm count and viability among males, and observed the gonadal and liver histology of both males and females. Female mosquitofish exposed to pulp and paper effluent in the Dengcun River were generally smaller in length and mass, had a greater number of anal fin segments and more embryos, but had significantly fewer oocytes in comparison with those living at the reference site. The higher number of anal fin ray 3 segments and the increased ray 4:6 length ratio observed among fish taken from the Dengcun River sites indicated that they might be subject to the androgenic effect. Furthermore, the significantly different hemal spine morphology of the effluent-affected females also indicated the pulp and paper mills effluents in Dengcun River might contain androgenic substance(s). Male mosquitofish at the sites exposed to effluent had a higher number of anal fin segments and greater testis mass in comparison with those living at the reference site. No evidence of intersex was found in either males or females, although histopathological tests on females revealed histologic abnormalities in the liver and gonads. It can be concluded that pulp and paper effluent contamination in the Dengcun River has affected a number of developmental parameters and reproductive characteristics in mosquitofish, with possible adverse effects on reproduction in this population., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A key feature of inflammatory cells is the ability to migrate to a site of injury or infection quickly and efficiently. Infectious agents can then be taken up by these inflammatory cells, preventing established infection. Inflammatory cell migration is driven by a complex interaction between inflammatory cells and their environment. In order to maintain health, inflammation needs to resolve, allowing the surrounding tissues to recover and heal. These processes are not fully understood and have been difficult to study in cell culture due to the complex interactions between cell types. We therefore use a range of techniques in near-transparent zebrafish (Danio rerio) larvae to study these migration events in a whole-organism, in vivo model. Using a transgenic zebrafish line that specifically marks neutrophils with green fluorescent protein, Tg(mpx:GFP)i114, we are able to follow neutrophil behaviour at a single cell level. Using these methods, the cellular processes involved in all phases of inflammation can be studied and better understood.

Basement membranes (BMs) are a complex, sheet-like network of specialized extracellular matrix that underlies epithelial cells and surrounds muscle cells. They provide adherence between neighboring tissues, permit some flexibility of these adherent structures, and can act as a store for growth factors and as a guide for cell migration. The BM is not just a static structure; its deposition and remodeling are important for many processes including embryonic development, immune response, and wound healing. To date, dysfunction in BM deposition or remodeling has been linked to many human congenital disorders and diseases, affecting many different tissues in the body, including malformations, dystrophies, and cancer. However, many questions remain to be answered on the role BM proteins, and their mutations, play in the pathogenesis of human disease. In recent years, the zebrafish (Danio rerio) has emerged as a powerful animal model for human development and disease. In the first part of this chapter, we provide an overview of described defects caused by BM dysfunction in zebrafish, including development and function of notochord, muscle, central nervous system, skin, cardiovascular system, and kidney. In the second part, we will describe details of methods used to visualize and assess the structure of the BM in zebrafish, and to functionally analyze its different components., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pathological findings associated with scuticociliatosis in farmed Japanese flounder in Japan are described. Ten moribund fishes, farmed in Tottori Prefectural Fisheries Experimental Station, showed cutaneous ulcers, darkened skin, fin and tail rot, exophthalmia and alterations in swimming behaviour. Histopathologically, severe epidermal degeneration and necrosis, hyperplasia of branchial epithelium, myositis, myelitis, encephalitis associated with heavy accumulation of scuticociliates in the periorbital cavity and optic nerve fiber were observed. Moreover, masses of ciliates were found to feed on the host tissues such as skeletal muscles, gills and brain, causing severe degenerative changes associated with abundant neutrophilic and lymphocytic infiltration. These findings suggest that the present scuticociliate, Miamiensis avidus, is a highly invasive and destructive pathogen infecting Japanese flounder and capable of developing systemic fatal infection.

We report here the first documented case of a cetacean fatality from entanglement in recreational Spectra fishing twine. Spectra twine is a relatively new microfilament braided twine that is marketed to replace nylon monofilament twine in rod and reel fisheries. Following the case of this entangled bottlenose dolphin (Tursiops truncatus), we conducted tests with Spectra and comparable monofilament twines on Tursiops tissue from stranded animals to compare the abrasion properties of the twines. We found that Spectra twine was significantly more abrasive on bottlenose dolphin fluke tissue than a similar strength and diameter monofilament. With the same forces applied, the Spectra twine cut deeper than the monofilament, exhibiting a linear relationship with force applied where the monofilament appeared to reach a maximum depth of penetration of approximately 2 mm. These tests may explain why this bottlenose dolphin was so severely debilitated from carrying a relatively light load of twine over a short period of time (20 days). Future public and corporate outreach will be essential to minimize the effect that this increasingly popular fishing twine will have on non-target species., (Copyright 2010 Elsevier Ltd. All rights reserved.)