Incorporación de Tecnología Tridimensional mediante Tomografía Microcomputarizada en el Estudio de la Anatomía de Salmónidos: Una Evaluación de Utilidad / Integration of Three-Dimensional Technology through Microcomputed Tomography in the Study of Salmonid Anatomy: A Utility Evaluation

Los salmónidos, durante su desarrollo, pueden experimentar ocasionalmente deformaciones esqueléticas. Para su diagnóstico se emplean diversas metodologías, entre las que se incluyen radiografías, técnicas histológicas, diafanización con tinciones de alizarina y azul de alcián, así como el uso del microscopio electrónico de barrido (SEM), cada una con sus inherentes ventajas y desventajas. Este estudio tuvo como finalidad evaluar y comparar la eficacia de la tomografía microcomputarizada (Micro-CT) para el análisis anatómico, reconstruyendo tridimensionalmente las imágenes y contrastándolas con los resultados obtenidos mediante la técnica de diafanización. Se analizaron las aletas caudales de cinco ejemplares de salmón Oncorhynchus kisutch: dos sujetos a diafanización y tres procesados para análisis mediante Micro-CT utilizando el equipo BRUKER SkyScan 1272. La técnica de Micro-CT demostró superioridad en la resolución de las estructuras óseas, facilitando una exploración detallada de las variaciones morfológicas y la distribución de la densidad mineral. Este enfoque permitió identificar anomalías en la morfología y crecimiento de las últimas vértebras y lepidotriquias dorsales, así como una densidad incrementada en lepidotriquias dorsales malformadas. La mayor resolución proporcionada por la Micro-CT no solo potencia nuestra comprensión de la ontogenia piscícola y su adaptación a ambientes diversos, sino que además inaugura perspectivas innovadoras para el estudio de la evolución de las estrategias locomotoras y las respuestas adaptativas frente a cambios ambientales a través del tiempo.

SUMMARY: During their development, some species of salmonids may occasionally experience skeletal deformations. Several methodologies are currently being used for the diagnosis of such malformations, among which X-rays, histological techniques, diaphanization coupled either with Alizarin Red or Alcian Blue stains, as well as Scanning Electron Microscopy (SEM) can be mentioned. Each one of those methods presents inherent advantages and disadvantages. The purpose of this study was twofold: Firstly, to evaluate and compare the effectiveness of microcomputed tomography (Micro-CT) technology for anatomical analysis, three-dimensionally reconstructing the obtained images; and secondly, to contrast those images with the results obtained through the diaphanization technique. The caudal fins of five specimens of the Oncorhynchus kisutch salmon were analyzed: Two specimens were subjected to diaphanization and three were processed for Micro-CT analysis, using the BRUKER SkyScan 1272 equipment. The Micro-CT technology demonstrated superiority in the resolution of bone structures, facilitating a detailed exploration of morphological variations, as well as the distribution of mineral density. This experimental approach allowed us to identify anomalies in the morphology and growth of the last vertebrae and dorsal lepidotrichiae, as well as an increased mineral density in the malformed dorsal lepidotrichiae. The higher resolution provided by Micro-CT not only enhances our understanding of the fish ontogeny and its adaptation to diverse environments, but also opens innovative perspectives for the study of the evolution of locomotor strategies and adaptive responses to environmental changes.

Draft genomes and reference transcriptomes extend the coding potential of the fish pathogen Piscirickettsia salmonis

Background: Draft and complete genome sequences from bacteria are key tools to understand genetic determinants involved in pathogenesis in several disease models. Piscirickettsia salmonis is a Gram-negative bacterium responsible for the Salmon Rickettsial Syndrome (SRS), a bacterial disease that threatens the sustainability of the Chilean salmon industry. In previous reports, complete and draft genome sequences have been generated and annotated. However, the lack of transcriptome data underestimates the genetic potential, does not provide information about transcriptional units and contributes to disseminate annotation errors. Results: Here we present the draft genome and transcriptome sequences of four P. salmonis strains. We have identified the transcriptional architecture of previously characterized virulence factors and trait-specific genes associated to cation uptake, metal efflux, antibiotic resistance, secretion systems and other virulence factors. Conclusions: This data has provided a refined genome annotation and also new insights on the transcriptional structures and coding potential of this fish pathogen.

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

Novel antimicrobial activity of a dichloromethane extract obtained from red seaweed Ceramium rubrum (Hudson) (Rhodophyta: Florideophyceae) against Yersinia ruckeri and Saprolegnia parasitica, agents that cause diseases in salmonids

Background Enteric red mouth disease and Saprolegniasis, which are caused by the bacteria Yersinia ruckeri and the oomycete Saprolegnia parasitica, respectively, are important illnesses that affect salmonid farming. Sanitary problems in farms are addressed by the prevention of disease outbreaks or by the treatment of diseases with chemicals. Environmental and governmental restrictions, toxicity and high treatment costs limit the use of drugs. Marine organisms, such as algae, sponges and corals, have developed an antimicrobial defense strategy based on the production of bioactive metabolites. Among these organisms, seaweeds offer a particularly rich source of potential new drugs. Hence, many pharmacologically active substances have been isolated from seaweeds. In the Ceramium genus, Ceramium rubrum has been emphasized by several authors for its antimicrobial properties. Based on this background, the present study focused on the antimicrobial activity of a lipophilic extract of C. rubrum on Y. ruckeri and S. parasitica. Results The alga, collected from the Pacific coast of Chile, underwent an ethanol extraction, and the concentrated extract was partitioned between water and dichloromethane. From the dichloromethane extract, fatty acids, fatty acid esters, one hydrocarbon and phytol were identified by Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antimicrobial study showed that the whole extract was more active than the individual components, which suggests a strong synergistic effect among the components. Conclusions These results may constitute a basis for promising future applied research that could investigate the use of C. rubrum seaweed as a source of antimicrobial compounds against fish pathogens.

Fish ladders: safe fish passage or hotspot for predation?

Fish ladders are a strategy for conserving biodiversity, as they can provide connectivity between fragmented habitats and reduce predation on shoals that accumulate immediately below dams. Although the impact of predation downstream of reservoirs has been investigated, especially in juvenile salmonids during their downstream movements, nothing is known about predation on Neotropical fish in the attraction and containment areas commonly found in translocation facilities. This study analysed predation in a fish passage system at the Lajeado Dam on the Tocantins River in Brazil. The abundance, distribution, and the permanence (time spent) of large predatory fish along the ladder, the injuries imposed by piranhas during passage and the presence of other vertebrate predators were investigated. From December 2002 to October 2003, sampling was conducted in four regions (downstream, along the ladder, in the forebay, and upstream of the reservoir) using gillnets, cast nets and counts or visual observations. The captured fish were tagged with thread and beads, and any mutilations were registered. Fish, birds and dolphins were the main predator groups observed, with a predominance of the first two groups. The entrance to the ladder, in the downstream region, was the area with the highest number of large predators and was the only region with relevant non-fish vertebrates. The main predatory fish species were Rhaphiodon vulpinus, Hydrolycus armatus, and Serrasalmus rhombeus. Tagged individuals were detected predating along the ladder for up to 90 days. Mutilations caused by Serrasalmus attacks were noted in 36% of species and 4% of individuals at the top of the ladder. Our results suggested that the high density of fish in the restricted ladder environment, which is associated with injuries suffered along the ladder course and the presence of multiple predator groups with different predation strategies, transformed the fish corridor into a hotspot for predation.

Passagens para peixes têm sido consideradas como estratégias para conservação da biodiversidade, visando além de assegurar os deslocamentos entre habitats fragmentados, atenuar a mortalidade por predação dos estratos populacionais que se concentram nos trechos imediatamente abaixo de barragens. Embora a mortalidade por predação no trecho a jusante de reservatórios tenha sido bem investigada, especialmente sobre os juvenis de salmonídeos em movimentos descendentes, nada se sabe acerca da predação de peixes neotropicais em áreas de atração e confinamento de peixes, eventos comuns a essas facilidades de transposição. Nesse estudo são analisados aspectos da predação em um sistema de transposição de peixes (barragem de Lajeado, rio Tocantins, Brasil), buscando avaliar a abundância, a distribuição e o tempo de permanência de grandes peixes predadores na escada, as injúrias impostas por piranhas durante a passagem e a ocorrência de outros vertebrados predadores. Para isso foram realizadas amostragens a jusante, ao longo da escada, no trecho imediatamente acima da barragem e a montante do reservatório, utilizando redes de espera, tarrafas e contagens ou registros visuais durante o período de um ano (Novembro de 2002 a Outubro de 2003). Marcações com linha e missanga e registro de mutilações foram também realizados em concomitância. Peixes, aves, quelônios e cetáceos foram os principais grupos de predadores observados, com predomínio dos dois primeiros. A área nas imediações da entrada da escada, a jusante, foi a região com maior número de grandes predadores, sendo a única de relevância para vertebrados não peixes. Indivíduos marcados permaneceram na escada exercendo a piscivoria por até 90 dias. Rhaphiodon vulpinus, Hydrolycus armatus e Serrasalmus rhombeus foram os principais peixes predadores. Mutilações por ataques de Serrasalmus atingiram 36% das espécies e alcançaram 4% dos indivíduos no topo da escada. Os resultados sugerem que a alta densidade ...

Anti-peptide antibodies: a tool for detecting IL-8 in salmonids

Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar, presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95 percent and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish.

Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids

Background: Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-alpha has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-alpha detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-alpha antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-alpha of salmonid fish. TNF-alpha epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-alpha, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-alpha positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-alpha function.

Infectious pancreatic necrosis virus (IPNV) enumeration through epifluorescence microscopy: technical aspects

A method for counting Infectious pancreatic necrosis virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.

Swimming performance of the migratory Neotropical fish Leporinus reinhardti (Characiformes: Anostomidae)

The fishways built in Latin America were mainly based on those used in the northern hemisphere for passing salmonids upstream. However, the swimming capacity in terms of speed and endurance is a major factor concerning the design of a fishway, and this information is not available for migratory neotropical fish species. Therefore, studies on swimming performance of neotropical species is fundamental. The aim of this study was to estimate the critical, sustained and prolonged speeds for the piau-três-pintas Leporinus reinhardti, and to evaluate the influence of water temperature on the swimming performance of the species. Variable (increasing) and fixed velocity tests were performed in a respirometer. When compared to other species whose critical speed data are available, the piau showed higher speeds, for a given total length. Fatigue times per prolonged speed class were estimated as well as prolonged speed per fatigue time class. The changing point from sustained to prolonged swimming was estimated to occur approximately at a speed of seven body-lengths per second (1.12 m/s for an individual with 16 cm total length: the first maturation length). In spite of the great swimming performance of piau, the adoption of efficient directives in fishway projects to allow the passage of neotropical ichthyofauna, will depend on a more detailed analysis of the swimming capacity of different species

As passagens para peixes construídas na América Latina são principalmente baseadas nos dispositivos existentes no hemisfério norte que, por sua vez, foram dimensionados para salmonídeos. Entretanto, a capacidade natatória das espécies migradoras, em termos de resistência e velocidade, é o principal fator a ser considerado no dimensionamento de uma passagem para peixes e estas informações ainda não são conhecidas para as espécies migradoras neotropicais. Dessa forma, o estudo da capacidade natatória das espécies neotropicais tornou-se uma necessidade. Este estudo tem como objetivo estimar as velocidades crítica, sustentável e prolongada do piau-três-pintas, Leporinus reinhardti, bem como avaliar a influência da temperatura da água na capacidade natatória. Testes de velocidade variável e fixa foram desenvolvidos em um aparato experimental do tipo respirômetro. Quando comparado a outras espécies, cujos dados de velocidade crítica são disponíveis, observa-se que o piau apresenta velocidades superiores para um mesmo comprimento total. Tempo de fadiga por classe de velocidade prolongada foram obtidos bem como a velocidade prolongada por classes de tempo de fadiga. O ponto de mudança do modo sustentável para o modo prolongado de natação foi estimado ocorrer em, aproximadamente, sete comprimentos por segundo (1.12 m/s para indivíduos com 16 cm de comprimento total: comprimento de primeira maturação). Apesar da grande capacidade natatória do piau, a adoção de critérios eficientes para o projeto de mecanismos de transposição para peixes neotropicais dependerá de análise detalhada da capacidade natatória de diferentes espécies

Difilobotriasis humana en la Patagonia, Argentina / Human diphyllobothriasis in Patagonia, Argentina

El objetivo del trabajo fué detectar hospedadores humanos en la zona andino patagónica argentina, teniendo en cuenta las prácticas de pesca desportiva y la importancia de los salmónidos dentro de la zona. entre 1986 y 1995 se implementaron Campañas de Información en los laboratorios de Análisis Clínicos de la región andinopatogónica argentina, destinadas a lograr una detección más eficiente de la difilobotriasis, a través de análisis coproparasitológicos. Adicionalmente, se confeccionaron planillas destinadas a recoger información sobre las características de la infección, del tratamiento y del paciente. Durante este período se detectaron 13 nuevos casos humanos, por identificación directa del parásito o por la presencia de huevos en materia fecal. Las características de las infecciones responden a las descriptas para el género Diphyllobothrium. En la región, los salmónidos son los peces predilectos en la pesca deportiva. Estos peces, frecuentemente parasitados con larvas, constituyen la principal fuente de contagio para el hombre al ser consumidos insuficientemente cocidos o ahumados en frío

Registro de nuevos casos de difilobotriasis humana en Chile (1981-1992), incluído un caso de infección múltiple por diphyllobothrium latum / Record of new cases of human diphyllobothriasis in Chile (1981-1992), including a case of multiple diphyllobotrium latum infection

New records of human diphyllobothriasis in Chile (1981-1992), with a case of multiple diphyllobotrium latum infection

Primer registro de camallanus corderoi Torres, Teuber y Miranda, 1990 (nematoda: camallanidae) en salmónidos cultivados en el lago Puyehue, Chile / First report of camallanus corderoi Torres, Teuber and Miranda, 1990 (nematoda: camallanidae) in cultured salmonids from Puyehue lake, Chile

First report of camallanus corderoi Torres, Teuber and Miranda, 1990 (Nematoda: Camallanidae) in cultured salmonids from Puyehue lake, Chile

Ecological aspects of nematode parasites of introduced salmonids from Valdivia river basin, Chile

Between 1986 and 1987 fishes distributed among the following species introduced in Chile, and from different sectors of the Valdivia river basin (39graus 30' - 40 graus00', 73 graus 30' - 71 graus 45'W), were examined: 348 Salmo trutta, 242 salmo gairdneri, 24 Cyprinus carpio and 52 Gambusia affinis holbrooki. The presence of Camallanus corderoi and Contracaecum sp. in S. gairdneri and of C. corderoi in S. trutta is recorded in Chile for the first time. Cyprinus carpio and G. a. holbrooki did not present infections by nematodes. The prevalence and mean intensity of the infections by nematodes presented significant differences among some sectors of the Valdivia river basin. In general, the prevalence and intensity of the infections by C. corderoi were greater than those by Contracaecum sp. The infections in S. gairdneri were higher than in S. trutta. The sex of the hosts had no influence on the prevalence and intensity of the infections by both nematodes. The length of the hosts did have an influence, except in the case of the infections by Contracaecum sp. in S, gairdneri. The infrapopulations of both nematode species showed over dispersion in most cases. The diet of the examined salmonids suggests that they would become infected principally throught the consuption of autochthonous fishes