Differential Gene Expression between Fall- and Spring-Run Chinook Salmon Assessed by Long Serial Analysis of Gene Expression

Of all Pacific salmonids, Chinook salmon Oncorhynchus tshawytscha display the greatest variability in return times to freshwater. The molecular mechanisms of these differential return times have not been well described. Current methods, such as long serial analysis of gene expression (LongSAGE) and microarrays, allow gene expression to be analyzed for thousands of genes simultaneously. To investigate whether differential gene expression is observed between fall- and spring-run Chinook salmon from California's Central Valley, LongSAGE libraries were constructed. Three libraries containing between 25,512 and 29,372 sequenced tags (21 base pairs/tag) were generated using messenger RNA from the brains of adult Chinook salmon returning in fall and spring and from one ocean-caught Chinook salmon. Tags were annotated to genes using complementary DNA libraries from Atlantic salmon Salmo salar and rainbow trout O. mykiss. Differentially expressed genes, as estimated by differences in the number of sequence tags, were found in all pairwise comparisons of libraries (freshwater versus saltwater = 40 genes; fall versus spring = 11 genes: and spawning versus nonspawning = 51 genes). The gene for ependymin, an extracellular glycoprotein involved in behavioral plasticity in fish, exhibited the most differential expression among the three groupings. Reverse transcription polymerase chain reaction analysis verified the differential expression of ependymin between the fall- and spring-run samples. These LongSAGE libraries, the first reported for Chinook salmon, provide a window of the transcriptional changes during Chinook salmon return migration to freshwater and spawning and increase the amount of expressed sequence data.

From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Avian Piscivores as Vectors for Myxobolus cerebralis in the Greater Yellowstone Ecosystem

Myxobolus cerebralis, the cause of whirling disease in salmonids, has dispersed to waters in 25 states within the USA, often by an unknown vector. Its incidence in Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri within the highly protected environment of Yellowstone Lake, Yellowstone National Park, is a prime example. Given the local abundances of piscivorous birds, we sought to clarify their potential role in the dissemination of M. cerebralis. Six individuals from each of three bird species (American white pelican Pelecanus erythrorhynchos, double-crested cormorant Phalacrocorax auritus, and great blue heron Ardea herodias) were fed known-infected or uninfected rainbow trout O. mykiss. Fecal material produced during 10-d periods before and after feeding was collected to determine whether M. cerebralis could be detected and, if so, whether it remained viable after passage through the gastrointestinal tract of these birds. For all (100%) of the nine birds fed known-infected fish, fecal samples collected during days 1–4 after feeding tested positive for M. cerebralis by polymerase chain reaction. In addition, tubificid worms Tubifex tubifex that were fed fecal material from known-infected great blue herons produced triactinomyxons in laboratory cultures, confirming the persistent viability of the parasite. No triactinomyxons were produced from T. tubifex fed fecal material from known-infected American white pelicans or double-crested cormorants, indicating a potential loss of parasite viability in these species. Great blue herons have the ability to concentrate and release viable myxospores into shallow-water habitats that are highly suitable for T. tubifex, thereby supporting a positive feedback loop in which the proliferation of M. cerebralis is enhanced. The presence of avian piscivores as an important component of aquatic ecosystems should continue to be supported. However, given the distances traveled by great blue herons between rookeries and foraging areas in just days, any practices that unnaturally attract them may heighten the probability of M. cerebralis dispersal and proliferation within the Greater Yellowstone Ecosystem.

Appearance of red mark syndrome / cold water strawberry disease in Switzerland and Austria

Red mark syndrome (RMS) or cold water strawberry disease (CWSD) is a non-lethal skin disease of rainbow trout Oncorhynchus mykiss that is of high economic importance in the UK. The disease is temperature-dependent, with up to 60% morbidity at water temperatures below 15 degrees C. Although CWSD is horizontally transmissible, the aetiology is still unknown. Here we describe the first cases of RMS on the European mainland in the alpine regions of Switzerland and Austria. In Switzerland, morbidity remained around 1% after the first outbreak, whereas in Austria no further cases were diagnosed.

Molecular epidemiology of Flavobacterium psychrophilum from Swiss fish farms

Bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum are 2 of the major diseases causing high fish mortality in salmonid fish farms. The molecular epidemiology of F. psychrophilum is still largely unknown. Multilocus sequence typing (MLST) has been previously used for this pathogen and underscored a correlation between clonal complexes and host fish species. Here we used MLST to study the relationships among 112 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta fario and S. t. lacustris in Swiss fish farms between 1993 and 2012. The isolates belonged to 27 different sequence types (STs). Most of the Swiss outbreaks were associated with strains belonging to clonal complexes CC-ST2 and CC-ST90, found in both rainbow trout and brown trout and represented by several STs. Eight ST singletons could not be connected to any known clonal complex. Already reported from other parts of Europe and North America, CC-ST2 was the most frequent clonal complex observed, and it caused the majority of outbreaks in Switzerland, with CC-ST90 being the second most important type. In the tightly interconnected Swiss fish farms, no association between clonal complex and host fish was detected, but a temporal evolution of the frequency of some STs was observed. The occurrence of sporadic STs suggests high F. psychrophilum diversity and may reflect the presence of different sequence types in the environment.

Benzo(a)pyrene Metabolism and EROD and GST Biotransformation Activity in the Liver of Red- and White-Blooded Antarctic Fish.

Climate change and anthropogenic pollution are of increasing concern in remote areas such as Antarctica. The evolutionary adaptation of Antarctic notothenioid fish to the cold and stable Southern Ocean led to a low plasticity of their physiological functions, what may limit their capacity to deal with altered temperature regimes and pollution in the Antarctic environment. Using a biochemical approach, we aimed to assess the hepatic biotransformation capacities of Antarctic fish species by determining (i) the activities of ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), and (ii) the metabolic clearance of benzo(a)pyrene by hepatic S9 supernatants. In addition, we determined the thermal sensitivity of the xenobiotic biotransformation enzymes. We investigated the xenobiotic metabolism of the red-blooded Gobionotothen gibberifrons and Notothenia rossii, the hemoglobin-less Chaenocephalus aceratus and Champsocephalus gunnari, and the rainbow trout Oncorhynchus mykiss as a reference. Our results revealed similar metabolic enzyme activities and metabolic clearance rates between red- and white-blooded Antarctic fish, but significantly lower rates in comparison to rainbow trout. Therefore, bioaccumulation factors for metabolizable lipophilic contaminants may be higher in Antarctic than in temperate fish. Likewise, the thermal adaptive capacities and flexibilities of the EROD and GST activities in Antarctic fish were significantly lower than in rainbow trout. As a consequence, increasing water temperatures in the Southern Ocean will additionally compromise the already low detoxification capacities of Antarctic fish.

Field-Based Evaluations of Horizontal Flat-Plate Fish Screens

Diversions from streams are often screened to prevent the loss of or injury to fish. Hydraulic criteria meant to protect fish that encounter screens have been developed, but primarily for screens that are vertical to the water flow rather than horizontal. For this reason, we measured selected hydraulic variables and released wild rainbow trout Oncorhynchus mykiss over two types of horizontal flat-plate fish screens in the field. Our goal was to assess the efficacy of these screens under a variety of conditions in the field and provide information that could be used to develop criteria for safe fish passage. We evaluated three different inverted-weir screens over a range of strewn (0.24-1.77 m(3)/s) and diversion flows (0.10-0.31 m(3)/s). Approach velocities (AVs) ranged from 3 to 8 cm/s and sweeping velocities (SVs) from 69 to 143 cm/s. We also evaluated a simple backwatered screen over stream flows of 0.23-0.79 m(3)/s and diversion flows of 0.08-0.32 m(3)/s. The mean SVs for this screen ranged from 15 to 66 cm/s and the mean AVs from 1 to 5 cm/s. The survival rates of fish held for 24 h after passage over these screens exceeded 98%. Overall, the number of fish-screen contacts was low and the injuries related to passage were infrequent and consisted primarily of minor fin injuries. Our results indicate that screens of this type have great potential as safe and effective fish screens for small diversions. Care must be taken, however, to avoid operating conditions that produce shallow or no water over the screen surface, situations of high AVs and low SVs at backwatered screens, and situations producing a localized high AV with spiraling flow.

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Patogenia de piscirickettsiosis: estudio sobre la infectividad de Piscirickettsia salmonis y el efecto de sus productos extracelulares

La piscirickettsiosis es una enfermedad contagiosa sistémica de los peces teleósteos cuyo agente causal es Piscirickettsia salmonis, una bacteria gram negativa intracelular facultativa. Esta enfermedad se ha descrito esporádicamente en distintas áreas geográficas y especies de peces en el mundo, pero es endémica y particularmente severa en salmónidos criados en agua de mar en Chile. En esta tesis se investigaron algunos aspectos de la patogénesis de esta enfermedad, estudiándose la infectividad de P. salmonis, tanto in vitro como in vivo, y buscándose además evidencias de la capacidad de secretar exotoxinas por parte de esta bacteria. Los ensayos de infectividad en células CHSE-214, procedentes de embrión de salmón chinook (Oncorhynchus tshawytscha), mostraron que existe una rápida adherencia de la bacteria a la superficie de la membrana plasmática (≤ 5 min posinoculación) seguida de su incorporación al citoplasma de estas células, proceso que ocurre entre las 3 y las 6 h posinoculación. Por su parte, el estudio de infectividad in vivo, que se realizó en trucha arcoiris (O. mykiss), reveló que este proceso comprende tres etapas principales: (i) una fase de rápida adhesión a células epiteliales principalmente de piel y branquias, pero también del canal alimentario; (ii) una invasión progresiva desde los sitios de entrada hacia tejidos más profundos hasta alcanzar el torrente sanguíneo y; (iii) una rápida diseminación vía hematógena para alcanzar virtualmente todos los tejidos corporales. Finalmente, se demostró que P. salmonis puede secretar exotoxinas termolábiles que tienen un efecto citotóxico selectivo según la célula blanco expuesta y que, probablemente, son parte de los factores de virulencia involucrados en la patogénesis de la piscirickettsiosis.

Towards a more sustainable aquaculture: diets effects and environmental impact of aquaculture from a microbiome perspective

There is a lot of interest to optimize aquaculture production due to its overexploitation of marine resources, ocean pollution and habitat destruction. Since feed production is one of the greatest issues in aquaculture, feeding strategy optimization is important. The study of several different feed additives or supplementation is important to secure optimal growth, gut health, and function in farmed fish. Feed additives are typically supplied to ensure good health and to help the animal ward off pathogens during both normal and challenging conditions, which could stress animals and promote insurgence of pathologies or pathogens invasions. In this context has an increasing interest the study of host associated microbiome to understand the influence of novel functional feed on the health and physiology of animals. To achieve a more sustainable aquaculture sector, show a great importance the understanding of the environmental impact of this human activity in terms of habitat destruction, ocean pollution and reduction marine environments biodiversity. Marine microbiomes, either free-living or associated with multicellular hosts, is acquiring an increasing interest because their role in supporting the functioning and biodiversity of marine ecosystems, providing essential ecological services. Becoming extremely important to understand how these activities can affect marine microbiomes by altering their function and diversity. In this thesis work, we were able to present a comprehensive evaluation of different functional feeds assessing their effects in terms of growth and gut health of three fish species, Rainbow Trout (Oncorhynchus mykiss), Gilthead seabream (Sparus aurata) and Zebrafish (Danio rerio). We also explored the impact of Aquaculture on the surrounding marine microbiomes, using Patella caerulea as a model holobionts. Finally, we provided a synoptical study on the microbiomes of the water column and surface sediments in North-Western Adriatic Sea (Italy), providing the finest-scale mapping of marine microbiomes in the Mediterranean Sea.

Adaptation of rainbow fish to lake and stream habitats

Fish occupy a range of hydrological habitats that exert different demands on locomotor performance. We examined replicate natural populations of the rainbow fishes Melanotaenia eachamensis and M. duboulayi to determine if colonization of low-velocity (lake) habitats by fish from high-velocity (stream) habitats resulted in adaptation of locomotor morphology and performance. Relative to stream conspecifics, lake fish had more posteriorly positioned first dorsal and pelvic fins, and shorter second dorsal fin bases. Habitat dimorphism observed between wild-caught fish was determined to be heritable as it was retained in M. eachamensis offspring raised in a common garden. Repeated evolution of the same heritable phenotype in independently derived populations indicated body shape divergence was a consequence of natural selection. Morphological divergence between hydrological habitats did not support a priori expectations of deeper bodies and caudal peduncles in lake fish. However, observed divergence in fin positioning was consistent with a family-wide association between habitat and morphology, and with empirical studies on other fish species. As predicted, decreased demand for sustained swimming in takes resulted in a reduction in caudal red muscle area of lake fish relative to their stream counterparts. Melanotaenia duboulayi lake fish also had slower sustained swimming speeds (U-crit) than stream conspecifics. In M. eachamensis, habitat affected U-crit of males and females differently. Specifically, females exhibited the pattern observed in M. duboulayi (lake fish had faster U-crit than stream fish), but the opposite association was observed in males (stream males had slower Ucrit than lake males). Stream M. eachamensis also exhibited a reversed pattern of sexual dimorphism in U-crit (males slower than females) relative to all other groups (males faster than females). We suggest that M. eachamensis males from streams responded to factors other than water velocity. Although replication of muscle and U,,it phenotypes across same habitat populations within and/or among species was suggestive of adaptation, the common garden experiment did not confirm a genetic basis to these associations. Kinematic studies should consider the effect of the position and base length of dorsal fins.

Dietary Levamisole Influenced Hematological Parameters of Juvenile Pacu, Piaractus mesopotamicus (Holmberg 1887)

Diseases outbreaks are a major concern in intensive fish farming because fish are exposed to stressors which may negatively affect their physiology. This study set out to determine effects of dietary levamisole (Levamisole HCl; SIGMA (R)) on performance and hematology of pacu, Piaractus mesopotamicus, juveniles. Fish (55.94 g) were stocked into 24 plastic aquaria (500 L; 15 fish per aquarium) and fed for 30 d with a commercial diet with 0, 50, 100, 200, 400, and 800 mg/kg levamisole, and for an extra 15 d, with a control diet in a totally randomized design trial (n = 4). Biometrical and hematological data were collected. No significant differences in growth parameters were recorded for either control or supplemented diets. Hematological parameters, such as hemoglobin, plasma glucose, white blood count (WBC), and differential leukocyte count were influenced (P < 0.05) levamisole. WBC, lymphocytes, neutrophils, monocytes, eosinophils, and special granulocytic cell numbers decreased significantly after 15 d. Dietary levamisole at 100 mg/kg diet for 15 d increased leukocyte production in juvenile pacu. However, levamisole administration for more than 15 d presented toxicity to lymphopoietic tissues. Information about long-period administration, mode of action in weight gain, effects on hematology of levamisole in freshwater fish nutrition are scarce and necessary for its safe use in aquaculture.

Dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (Holmberg, 1887)

Lysine is the reference essential amino acid in fish feeds and usually the most limiting amino acid in feedstuffs. The dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (4.3 g) was determined using five isonitrogenous (32% CP) test diets containing graded levels of lysine (0.9, 1.17, 1.44, 1.69 and 1.96% of dry diet) fed three times a day to four groups of 18 fish for 74 days. Growth, body composition, nutrient retention and hematological parameters of pacu were analyzed. Analysis of variance showed that all growth performance parameters were significantly affected by dietary treatments. The lysine requirements estimated using regression analysis for maximum weight gain and feed efficiency were 1.45 and 1.51% of dry diet, respectively. Nitrogen retention efficiency increased with increasing levels of dietary lysine up to 1.43% (p<0.05). Whole-body protein increased (p<0.05) and whole-body lipid decreased (p<0.05) with increasing dietary lysine level. Thus, the lysine requirement of juvenile pacu was estimated as being 1.4-1.5% of dry diet or 4.4-4.7% of dietary protein. (c) 2009 Elsevier B.V. All rights reserved.

Estimating Amino Acid Requirement of Brazilian Freshwater Fish from Muscle Amino Acid Profile

Information on nutritional requirement of some Brazilian farmed fish species, especially essential amino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based on amino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, and curimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. The objective of the present study was to estimate amino acid requirements of these species and analyze similarities among amino acid composition of different fish species by cluster analysis. To estimate amino acid requirement, the following formula was used: amino acid requirement = [(amount of an individual amino acid in fish muscle tissue) x (average totalEAA requirement among channel catfish, Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(average fish muscle totalEAA). Most values found lie within the range of requirements determined for other omnivorous fish species, in exception of leucine requirement estimated for both species, and arginine requirement estimated for matrinxa alone. Rather than writing off the need for regular dose-response assays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa, results set solid base for the study of tropical species dietary amino acids requirements.