Benefits of the microalgae Spirulina and Schizochytrium in fish nutrition: a meta-analysis.

Use of microalgae in fish nutrition can relieve pressure on wild fish stocks, but there is no systematic quantitative evaluation of microalgae benefits. We conducted a metanalysis on the nutritional benefits of Spirulina and Schizochytrium as replacements of fishmeal and fish or plant oil, respectively. We reviewed 50 peer-reviewed studies involving 26 finfish species and 144 control vs microalgae replacement comparisons. Inclusion of Spirulina in the fish diet significantly improved growth compared to controls (SMD = 1.21; 95% CI 0.71-1.70), while inclusion of Schizochytrium maintained the content of omega-3 PUFA of the fish fillet compared to fish fed on fish or plant oils (SMD = 0.62; 95% CI - 0.51-1.76). Benefits were apparent at replacement levels as low as 0.025% in the case of Spirulina and 10% in the case of Schizochytrium oil. Dose-dependent effects were found for Spirulina replacement on growth, but not for Schizochytrium on omega-3 fillet content. Subgroup analysis and meta-regression revealed that ~ 24-27% of variation in effect sizes can be accounted by variation between fish families, the rest likely reflecting variation in experimental conditions. Overall, the evidence indicates that Spirulina and Schizochytrium replacement in aquafeeds can be used to improve fish growth and maintain fillet quality, respectively, but considerable uncertainty exists on the predicted responses. To reduce uncertainty and facilitate the transition towards more sustainable aquafeeds, we recommend that feeding trials using microalgae are conducted under commercially relevant conditions and that greater care is taken to report full results to account for sources of heterogeneity.

Density-dependent changes in neophobia and stress-coping styles in the world's oldest farmed fish.

Farmed fish are typically reared at densities much higher than those observed in the wild, but to what extent crowding results in abnormal behaviours that can impact welfare and stress coping styles is subject to debate. Neophobia (i.e. fear of the 'new') is thought to be adaptive under natural conditions by limiting risks, but it is potentially maladapted in captivity, where there are no predators or novel foods. We reared juvenile Nile tilapia (Oreochromis niloticus) for six weeks at either high (50 g l-1) or low density (14 g l-1), assessed the extent of skin and eye darkening (two proxies of chronic stress), and exposed them to a novel object in an open test arena, with and without cover, to assess the effects of density on neophobia and stress coping styles. Fish reared at high density were darker, more neophobic, less aggressive, less mobile and less likely to take risks than those reared at low density, and these effects were exacerbated when no cover was available. Thus, the reactive coping style shown by fish at high density was very different from the proactive coping style shown by fish at low density. Our findings provide novel insights into the plasticity of fish behaviour and the effects of aquaculture intensification on one of the world's oldest farmed and most invasive fish, and highlight the importance of considering context. Crowding could have a positive effect on the welfare of tilapia by reducing aggressive behaviour, but it can also make fish chronically stressed and more fearful, which could make them less invasive.

Eco-immunology of fish invasions: the role of MHC variation.

The relationship between invaders and the pathogens encountered in their new environment can have a large effect on invasion success. Invaders can become free from their natural pathogens and reallocate costly immune resources to growth and reproduction, thereby increasing invasion success. Release from enemies and relaxation of selective pressures could render newly founded populations more variable at immune-related genes, such as the major histocompatibility complex (MHC), particularly when they have different origins. Using rainbow and brown trout, two of the world's most successful fish invaders, we tested the general hypothesis that invaders should display high intrapopulation immunogenetic diversity and interpopulation divergence, due to the interplay between genetic drift and successive waves of genetically divergent introductions. We analysed genetic diversity and signatures of selection at the MHC class II ß immune-related locus. In both species, MHC diversity (allelic richness and heterozygosity) for southern hemisphere populations was similar to values reported for populations at their native range. However, MHC functional diversity was limited, and population immunogenetic structuring weaker than that observed using neutral markers. Depleted MHC functional diversity could reflect a decrease in immune response, immune-related assortative mating or selection for resistance to newly encountered parasites. Given that the role of MHC diversity in the survival of these populations remains unclear, depleted functional diversity of invasive salmonids could compromise their long-term persistence. A better understanding of the eco-immunology of invaders may help in managing and preventing the impact of biological invasions, a major cause of loss of biodiversity worldwide.

Using molecular markers for pedigree reconstruction of the greater amberjack (Seriola dumerili) in the absence of parental information.

Ensuring appropriate levels of genetic diversity in captive populations is essential to avoid inbreeding and loss of rare alleles by genetic drift. Pedigree reconstruction and parentage analysis in the absence of parental genotypes can be a challenging task that relies in the assignment of sibship relationships among the offspring. Here, we used eight highly variable microsatellite markers and three different assignment methods to reconstruct the most likely genotypes of a parental group of wild Seriola dumerili fish based on the genotypes of six cohorts of their offspring, to assess their relative contributions to the offspring. We found that a combination of the four most variable microsatellites was enough to identify the number of parents and their contribution to the offspring, suggesting that the variability of the markers can be more critical than the number of markers. Estimated effective population sizes were lower than the number of breeders and variable among years. The results suggest unequal parental contribution that should be accounted for breeding programs in the future.

Maintaining functional major histocompatibility complex diversity under inbreeding: the case of a selfing vertebrate.

Major histocompatibility complex (MHC) genes encode proteins that present pathogen-derived antigens to T-cells, initiating the adaptive immune response in vertebrates. Although populations with low MHC diversity tend to be more susceptible to pathogens, some bottlenecked populations persist and even increase in numbers despite low MHC diversity. Thus, the relative importance of MHC diversity versus genome-wide variability for the long-term viability of populations after bottlenecks and/or under high inbreeding is controversial. We tested the hypothesis that genome-wide inbreeding (estimated using microsatellites) should be more critical than MHC diversity alone in determining pathogen resistance in the self-fertilizing fish Kryptolebias marmoratus by analysing MHC diversity and parasite loads in natural and laboratory populations with different degrees of inbreeding. Both MHC and neutral diversities were lost after several generations of selfing, but we also found evidence of parasite selection acting on MHC diversity and of non-random loss of alleles, suggesting a possible selective advantage of those individuals with functionally divergent MHC, in accordance with the hypothesis of divergent allele advantage. Moreover, we found that parasite loads were better explained by including MHC diversity in the model than by genome-wide (microsatellites) heterozygosity alone. Our results suggest that immune-related overdominance could be the key in maintaining variables rates of selfing and outcrossing in K. marmoratus and other mixed-mating species.

Microsatellite standardization and evaluation of genotyping error in a large multi-partner research programme for conservation of Atlantic salmon (Salmo salar L.).

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.

Contrasting responses to selection in class I and class IIα major histocompatibility-linked markers in salmon.

Comparison of levels and patterns of genetic variation in natural populations either across loci or against neutral expectation can yield insight into locus-specific differences in the strength and direction of evolutionary forces. We used both approaches to test the hypotheses on patterns of selection on major histocompatibility (MH)-linked markers. We performed temporal analyses of class I and class IIα MH-linked markers and eight microsatellite loci in two Atlantic salmon populations in Ireland on two temporal scales: over six decades and 9 years in the rivers Burrishoole and Delphi, respectively. We also compared contemporary Burrishoole and Delphi samples with nearby populations for the same loci. On comparing patterns of temporal and spatial differentiation among classes of loci, the class IIα MH-linked marker was consistently identified as an outlier compared with patterns at the other microsatellite loci or neutral expectation. We found higher levels of temporal and spatial heterogeneity in heterozygosity (but not in allelic richness) for the class IIα MH-linked marker compared with microsatellites. Tests on both within- and among-population differentiation are consistent with directional selection acting on the class IIα-linked marker in both temporal and spatial comparisons, but only in temporal comparisons for the class I-linked marker. Our results indicate a complex pattern of selection on MH-linked markers in natural populations of Atlantic salmon. These findings highlight the importance of considering selection on MH-linked markers when using these markers for management and conservation purposes.

Evaluación de la calidad microbiológica del agua envasada en bolsas producida en Sincelejo - Colombia / Assessment of the microbiological quality of water packed in bags manufactured in Sincelejo - Colombia

Objetivo. Evaluar la calidad microbiológica y fisico-quimica del agua envasada en bolsas producidad en la ciudad de Sincelejo-Colombia con destino al consumo humano. Materiales y métodos. Para la estimación de organismos coliformes totales y fecales, Pseudomona aeruginosa y mesófilos en el agua envasada de 13 marcas, se utilizó el método de filtración por membrana (FxM). Resultados. El 92 % de las marcas de agua envasada en bolsa que se produce en la ciudad de Sincelejo presentaron bacterias mesófilas en su producto, mientras que en el 33% de ellas se encontraron coliformes totales. Cabe destacar que una marca presentó coliformes fecales, otra Pseudomonas aeruginosa y el reporte microbiano fue mayoren las envasadoras que poseían registro INVIMA. Conclusiones. Gran parte del agua envasada en bolsas de la ciudad de Sincelejo genera un riesgo a la salud de los consumidores, debido a la presencia de microorganismos patógenos, lo que está relacionado con inadecuados procesos de producción y a la intermitencia del suministro del agua utilizada como materia prima.

A critical review of adaptive genetic variation in Atlantic salmon: implications for conservation.

Here we critically review the scale and extent of adaptive genetic variation in Atlantic salmon (Salmo salar L.), an important model system in evolutionary and conservation biology that provides fundamental insights into population persistence, adaptive response and the effects of anthropogenic change. We consider the process of adaptation as the end product of natural selection, one that can best be viewed as the degree of matching between phenotype and environment. We recognise three potential sources of adaptive variation: heritable variation in phenotypic traits related to fitness, variation at the molecular level in genes influenced by selection, and variation in the way genes interact with the environment to produce phenotypes of varying plasticity. Of all phenotypic traits examined, variation in body size (or in correlated characters such as growth rates, age of seaward migration or age at sexual maturity) generally shows the highest heritability, as well as a strong effect on fitness. Thus, body size in Atlantic salmon tends to be positively correlated with freshwater and marine survival, as well as with fecundity, egg size, reproductive success, and offspring survival. By contrast, the fitness implications of variation in behavioural traits such as aggression, sheltering behaviour, or timing of migration are largely unknown. The adaptive significance of molecular variation in salmonids is also scant and largely circumstantial, despite extensive molecular screening on these species. Adaptive variation can result in local adaptations (LA) when, among other necessary conditions, populations live in patchy environments, exchange few or no migrants, and are subjected to differential selective pressures. Evidence for LA in Atlantic salmon is indirect and comes mostly from ecological correlates in fitness-related traits, the failure of many translocations, the poor performance of domesticated stocks, results of a few common-garden experiments (where different populations were raised in a common environment in an attempt to dissociate heritable from environmentally induced phenotypic variation), and the pattern of inherited resistance to some parasites and diseases. Genotype x environment interactions occurr for many fitness traits, suggesting that LA might be important. However, the scale and extent of adaptive variation remains poorly understood and probably varies, depending on habitat heterogeneity, environmental stability and the relative roles of selection and drift. As maladaptation often results from phenotype-environment mismatch, we argue that acting as if populations are not locally adapted carries a much greater risk of mismanagement than acting under the assumption for local adaptations when there are none. As such, an evolutionary approach to salmon conservation is required, aimed at maintaining the conditions necessary for natural selection to operate most efficiently and unhindered. This may require minimising alterations to native genotypes and habitats to which populations have likely become adapted, but also allowing for population size to reach or extend beyond carrying capacity to encourage competition and other sources of natural mortality.

Rapid evolution of the MH class I locus results in different allelic compositions in recently diverged populations of Atlantic salmon.

We compared major histocompatibility class I allelic diversity in two currently reproductively isolated Atlantic salmon (Salmo salar) populations (Irish and Norwegian) with a common postglacial origin in order to test for among-population differences in allelic composition and patterns of recombination and point mutation. We also examined the evidence for adaptive molecular divergence at this locus by analyzing the rate of amino acid replacement in relation to a neutral expectation. Contrary to our prediction, and in contrast to the situation for other genetic markers, the two populations have almost nonoverlapping sets of major histocompatibility class I alleles. Although there is a strong signal of point mutation that predates population divergence, recent recombination, acting in similar, but not identical, ways in both populations appears to be a significant force in creating new alleles. Moreover, selection acting on peptide-binding residues seems to favor new recombinant alleles and is likely to be responsible for the rapid divergence between populations.

Patterns of variability at the major histocompatibility class II alpha locus in Atlantic salmon contrast with those at the class I locus.

In order to investigate the mechanisms creating and maintaining variability at the major histocompatibility (MH) class II alpha (DAA) locus we examined patterns of polymorphism in two isolated Atlantic salmon populations which share a common post-glacial origin. As expected from their common origin, but contrary to the observation at the MH class I locus, these populations shared the majority of DAA alleles: out of 17 sequences observed, 11 were common to both populations. Recombination seems to play a more important role in the origin of new alleles at the class II alpha locus than at the class I locus. A greater than expected proportion of sites inferred to be positively selected (potentially peptide binding residues, PBRs) were found to be involved in recombination events, suggesting a mechanism for increasing MH variability through an interaction between recombination and natural selection. Thus it appears that although selection and recombination are important mechanisms for the evolution of both class II alpha and class I loci in the Atlantic salmon, the pattern of variability differs markedly between these classes of MH loci.

Mitochondrial DNA variation in Pleistocene and modern Atlantic salmon from the Iberian glacial refugium.

Current understanding of the postglacial colonization of Nearctic and Palearctic species relies heavily on inferences drawn from the phylogeographic analysis of contemporary generic variants. Modern postglacial populations are supposed to be representative of their Pleistocene ancestors, and their current distribution is assumed to reflect the different colonization success and dispersal patterns of refugial lineages. Yet, testing of phylogeographic models against ancestral genomes from glacial refugia has rarely been possible. Here we compare ND1 mitochondrial DNA variation in late Pleistocene (16,000-40,000 years before present), historical and contemporary Atlantic salmon (Salmo salar) populations from northern Spain and other regions of western Europe. Our study demonstrates the presence of Atlantic salmon in the Iberian glacial refugium during the last 40,000 years and points to the Iberian Peninsula as the likely source of the most common haplotype within the Atlantic lineage in Europe. However, our findings also suggest that there may have been significant changes in the genetic structure of the Iberian refugial stock since the last ice age, and question whether modern populations in refugial areas are representative of ice age populations. A common haplotype that persisted in the Iberian Peninsula during the Pleistocene last glacial maximum is now extremely rare or absent from European rivers, highlighting the need for caution when making phylogeographic inferences about the origin and distribution of modern genetic types.

Is the white-ivory assay of Drosophila melanogaster a useful tool in genetic toxicology?

The white-ivory assay of Drosophila is based on the detection of reversions to wild-type phenotype of ommatidia with the white-ivory mutation. A tandem quadruplication of this gene is used in order to increase the reversion probability. Although the exact mechanism implicated in reversion is not known, revertant spots are believed to arise as a consequence of intrachromosmal recombination or related phenomena. Since the white-ivory assay has not been broadly used, the number of chemicals tested until now is still limited. In this work, we have assayed 25 chemicals belonging to several chemical groups, i.e., crosslinking agents, DNA-topoisomerase inhibitors, antimetabolites/nucleotide pool inhibitors, cyclic-adduct inducers, halogenated hydrocarbons, bulky-adduct inducers, intercalating agents, oxidative damage inducers, and a multiple damage inducer, to validate this test. Cross-linking agents, halogenated hydrocarbons, and the multiple damage inducer, dounomycin, were positive. On the contrary, the three antimetabolites/nucleotide pool inhibitors tested were negative. The other chemical groups showed disparate results, since some chemicals were positive, whereas others were negative in each group. A comparison with the results obtained in the w/ w+ and mwh/flr3 assays shows that the wi assay detects a more restricted spectrum of damages than those, although, with respect to carcinogenicity, its sensitivity (0.76, with the 62 chemicals tested until now) is similar to that estimated for the mentioned somatic assays. The conclusion of this work, then, is that the wi assay is not recommended as a general screening test, because the background reversion frequencies show a high variability among solvents, the range of lesion-recognition is lower than in the w/ w+ and mwh/flr3 SMARTs, and the mechanism implicated in the white-ivory reversion is poorly understood.

Somatic recombination, gene amplification and cancer.

The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the background genotype for the outcome of the test. Up to a 60-fold variation was found between the different genotypes in the response to procarcinogens, evidently dependent on differences in the metabolic activation of procarcinogens. In 1989 Schiestl presented results on intrachromosomal recombination in the strain RS112 of Saccharomyces, which indicated a capability to detect a range of chemical carcinogens, which gave negative results in Ames Salmonella assay. Such a test system, which could identify a larger range of potential carcinogens than conventional short-term tests evidently would be of great value and it therefore seemed of importance to follow up the observations by Schiestl. However, studies within this programme on the same strain of Saccharomyces, as well as the strains D7 (measuring intragenic recombination, intergenic recombination, and point mutation) and JD1 (measuring intragenic recombination at two loci) could not support the observations and interpretation by Schiestl (1989). The Drosophila white-ivory system, which presumably responds primarily by intrachromosomal recombination, did not give positive results with these Salmonella-negative agents either. One system to measure mitotic recombination in mammalian cell cultures was developed in the present programme. It was based on heterozygous mutations in both alleles of the adenosine deaminase gene (ADA). The system selects for proficient recombinants generated by the deficient cells. So far only pilot experiments, indicating that this experimental system operates as planned, have been performed. 6.2 Mechanisms of mitotic recombination The induction of mosaic spots in the wing spot and the white/white+ assays is predominantly dependent on interchromosomal recombination. This is evident from the fact that heterozygous inversions reduce the frequency of spots. A relationship between the length of inversions and the reduction of spots was demonstrated in the white/white+ assay--the long inversion ln(l)sc4L

'Non-genotoxic' carcinogens evaluated using the white-ivory assay of Drosophila melanogaster.

Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster. These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination. Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative. These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D. melanogaster.