Freezing response of white bass (Morone chrysops) sperm cells

The water transport response during freezing of sperm cells of Morone chrysops (white bass, WB) was obtained using a shape-independent differential scanning calorimeter (DSC) technique. Sperm cell suspensions were frozen at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), or (2) with 5% (v/v) dimethyl sulfoxide (Me2SO). For calculations, the sperm cell was modeled as a cylinder of length 24.8 microm and diameter of 0.305 microm, while the osmotically inactive cell volume (Vb) was assumed to be 0.6 Vo, where Vo was the isotonic or the initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined, and ranged from Lpg = 0.51-1.7 x 10(-15) m3/Ns (0.003-0.01 microm/min-atm), and ELp = 83.6-131.3 kJ/mol (20.0-31.4 kcal/mol). The parameters obtained in this study suggest that the optimal rate of cooling for M. chrysops sperm cells is approximately 22 degrees C/min, a value that compares closely with experimentally determined optimal rates of cooling (approximately 16 degrees C/min).     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Characterization of a white bass (Morone chrysops) embryonic cell line with epithelial features

The genus Morone is an important one for U.S. aquaculture, but there has been no available cell line from this genus. We report here a cell line (the WBE line) derived from white bass embryos that has been grown for more than 80 passages over 21 mo in Dulbecco modified Eagle medium supplemented with fetal bovine serum. The WBE line showed epithelial features with positive immunohistochemical staining for cytokeratin and intercellular junctions when observed by electron microscopy. The doubling time, transformation characteristics, response to cryopreservation, and karyotype were determined. The WBE line was also tested by polymerase chain reaction to verify the species of origin and to screen for mycoplasma infection. This epithelial cell line may prove useful for the study of host-pathogen interactions that occur at the epithelial surface in this commercially important fish species.     

Temperature affects physiological stress responses to acute confinement in sunshine bass (Morone chrysops x Morone saxatilis)

Sunshine bass (Morone chrysopsxMorone saxatilis) were subjected to a 15-min low-water confinement stressor at temperatures ranging from 5 to 30 degrees C. Physiological responses were evaluated by measuring hematocrit, and plasma chloride, glucose and cortisol. Fish acclimated to 30 degrees C had initial glucose concentrations of 3.13 mM (564 mg/L) which were significantly lower than in fish acclimated to 5 and 10 degrees C (4.32 and 4.82 mM or 779 and 868 mg/l, respectively). Fish survived the conditions imposed at every temperature except 30 degrees C, where 15 out of 42 fish died during the stress and recovery protocol. The general pattern was an initial increase in hematocrit, followed by a delayed decrease in hematocrit and chloride, and an increase in plasma glucose and cortisol. In general, fish stressed at temperatures below 20 degrees C had lower and more delayed changes in plasma glucose and cortisol than fish tested at 20, 25 and 30 degrees C. Initial cortisol concentrations were 65 ng/ml and increased to above 200 ng/ml in fish held at 20 degrees C and above. At the higher temperatures, glucose concentrations were twice the initial concentration after stress and cortisol changes were four to five times the initial concentration after the stress. Quantitative responses for glucose and cortisol were moderate and recovery rapid in fish stressed at 10 and 15 degrees C; therefore, this range of water temperature is recommended when handling sunshine bass.     

High‐density SNP assay development for genetic analysis in maritime pine (Pinus pinaster)

Maritime pine provides essential ecosystem services in the south‐western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three‐generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene‐based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.     

Microsatellite DNA markers for parental assignment in hybrid striped bass (Morone saxatilis × Morone chrysops)

Development of nine polymorphic microsatellites from a genomic library of hybrid striped bass (female Morone chrysops × male Morone saxatilus) DNA is described. Breeding of hybrid striped bass for aquaculture is based largely on breeding wild fish. Molecular markers such as microsatellites will be useful tools for developing broodstock, estimating heritability for production traits, and selective breeding via marker‐assisted selection. The nine polymorphic microsatellites include six dinucleotide and three complex repeat motifs. The number of alleles detected among a sample of 10 individuals of each species was relatively low. All polymerase chain reaction primer pairs also amplified products in the sea bass Dicentrarchus labrax.     

Tissue essential fatty acid composition and competitive response to dietary manipulations in white bass (Morone chrysops), striped bass (M. saxatilis) and hybrid striped bass (M. chrysopsxM. saxatilis)

The effects of wide changes in dietary levels of docosahexaenoic (DHA) or arachidonic (ArA) acids on growth, survival and fatty acid composition in body tissues of Morone larvae were examined. White bass (WB, Morone chrysops), striped bass (SB, Morone saxatilis) and sunshine hybrid bass (HSB, M. chrysopsxM. saxatilis) larvae (day 24-46) were fed Artemia nauplii enriched with algal sources of varying proportions of DHA and ArA (from 0 to over 20% of total fatty acids). WB larvae fed DHA-deficient Artemia diet retarded over 50% of their potential growth, however, increasing dietary DHA/ArA ratios were associated with a significant growth improvement. The highest proportion of polyunsaturated fatty acids was found in WB neural tissue (approx. 50% of total fatty acids), while HSB neural tissue contained the highest proportion of saturated fatty acids (approx. 35% of total fatty acids). Within the neural tissues of all Morone larvae, both DHA and ArA were generally the most dominant as well as the most responding fatty acids to dietary manipulations (except in WB fed DHA or ArA deficient diets). HSB neural tissue was particularly efficient in retaining a significant amount of DHA in the face of dietary deficiency. However, WB neural tissue was the most responsive to dietary increase in DHA, accumulating a significantly higher amount of DHA (P<0.05) than SB or HSB. Results demonstrate significant differences in fatty acid composition and growth responsiveness to dietary manipulations between Morone larvae species and within specific tissues. WB weight gain and neural tissue composition was affected most by dietary changes in both DHA and ArA whereas SB and HSB tissue compositions were generally less affected by dietary manipulations.     

Genetic structure and differentiation of wild and domesticated populations of Capsicum annuum (Solanaceae) from Mexico

 Genetic variation and structure of ten wild, three domesticated and one wild-cultivated populations of pepper (Capsicum annuum) from northwestern Mexico were studied in order to find out if the domestication process has reduced the genetic variation of the modern cultivars of this species. The analysis was based on 12 polymorphic loci from nine isozymes. Wild populations were sampled in different habitats along a latitudinal gradient of ca. 500 km. All populations had high genetic variation (i.e. wild: A = 2.72, P = 90.8%, He = 0.445; wild-cultivated: A = 2.50, P = 92.3%, He = 0.461; domesticated: A = 2.60, P = 84.6%, He = 0.408), indicating little genetic erosion in modern cultivars of pepper. Genetic diversity estimated by Nei's method showed that most genetic variation is found within, rather than among populations. However, genetic differentiation is greater among cultivated (G _ST=0.167) than among wild (G _ST=0.056) populations. Wild populations had an average genetic identity (I) of 0.952, indicating little differentiation and high gene flow (Nm=4.21) among these populations. Average genetic identity between wild and domesticated populations was of I=0.818, revealing that the domestication process has modified the genetic composition of commercial varieties of pepper. Changes in genetic composition among commercial varieties seem to have occurred in different directions, as indicated by the average value of I = 0.817 among these populations. The high level of diversity found in wild populations of C. annuum suggests that the wild relatives of cultivated peppers are a valuable genetic resource which must be conserved. Received May 5, 1999 Accepted October 30, 2000     

The European sea bass: a key marine fish model in the wild and in aquaculture

The European sea bass (Dicentrarchus labrax L.) is a marine fish of key economic and cultural importance in Europe. It is now more an aquaculture than a fisheries species (>96% of the production in 2016), although modern rearing techniques date back only from the late 1980s. It also has high interest for evolutionary studies, as it is composed of two semispecies (Atlantic and Mediterranean lineages) that have come into secondary contact following the last glaciation. Based on quantitative genetics studies of most traits of interest over the past 10–15 years, selective breeding programs are now applied to this species, which is at the beginning of its domestication process. The availability of a good quality reference genome has accelerated the development of new genomic resources, including SNP arrays that will enable genomic selection to improve genetic gain. There is a need to improve feed efficiency, both for economic and environmental reasons, but this will require novel phenotyping approaches. Further developments will likely focus on the understanding of genotype‐by‐environment interactions, which will be important both for efficient breeding of farmed stocks and for improving knowledge of the evolution of natural populations. At the interface between both, the domestication process must be better understood to improve production and also to fully evaluate the possible impact of aquaculture escapees on wild populations. The latter is an important question for all large‐scale aquaculture productions.     

Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass (Morone chrysops x Morone saxatilis) to Streptococcus iniae infection

Three feeding trials were conducted to evaluate potential immunomodulatory effects of nucleotides in the diet of hybrid striped bass. A basal diet was formulated from menhaden fish meal to contain 40% crude protein and 10% lipid. An oligonucleotide product (Ascogen P) from brewer's yeast was added to the basal formulation at the manufacturer's recommended rate of 0.5% to produce the experimental diet. Each diet was fed to four replicate groups of juvenile hybrid striped bass for seven or eight weeks in two separate trials. After Trials 1 and 2, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. No significant difference in growth performance was observed between fish fed the basal and experimental diets. Body composition of whole fish, hematocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by dietary nucleotides. Neutrophil oxidative radical production of fish fed the nucleotide-supplemented diet was significantly (P=0.011) higher than in fish fed the basal diet. Significantly (P<0.05) enhanced survival after exposure to S. iniae also was generally observed in fish fed the nucleotide-supplemented diet. In addition, fish fed the nucleotide-supplemented diet tended to have a higher antibody response based on microtitration agglutination; however, the difference was not statistically significant because of high variation between individual fish. Long-term (16 weeks) administration of oligonucleotides in Trial 3 failed to show enhancement of immune responses between treatments. It is concluded that dietary oligonucleotides positively influenced immune responses and resistance of juvenile hybrid striped bass to S. iniae infection.     

Observations on the inflammatory response within the dermis of a white bass, Morone chrysops (Rafinesque), infected with Lernaea cruciata (Copepoda: Caligidea)

Infection of a serranid fish, Morone chrysops , with the adult female 'anchor worm' Lernaea cruciata was accompanied by an inflammatory response characterized by an extensive proliferation of fibrous connective tissue elements Both the dermis and musculature of the host were involved. No mineralization of tissues was observed. This constitutes the first report of L. cruciata from M. chrysops .     

The effect of storing hybrid bass (Morone saxatilis x M. chrysops) sera at freezing temperatures on antibody titers

The effect of storing hybrid bass sera at freezing temperatures on their enzyme-linked immunosorbent assay (ELISA) titers was determined, and the precipitate formed during storage was characterized. Frozen bass sera from fish vaccinated with bacterial sonicate showed no titer in either of two groups-one immunized with bacterial sonicate and the other with sonicate emulsified in adjuvant. Fresh sera exhibited average titers of 1:32 in both groups and 1:20 in convalescent serum.Hybrid bass antisera to bovine serum albumin showed maximum ELISA OD when sera were used after a short storage at 4 degrees C. Storage at freezing temperatures (-20 and -73 degrees C) reduced the OD value substantially. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the cryoprecipitate formed after freezing hybrid bass sera showed protein bands at 85 and 38 kDa, most probably the heavy and light chains, respectively, of bass IgM. Bands at 55, 52, 28, and 26 kDa may represent fibrinogen subunits. It is possible that the cryoprecipitate is composed of a fibrinogen net, which traps the tetrameric IgM molecules and precipitates them during frozen storage.     

Landscape models for nuclear genetic diversity and genetic structure in white-footed mice (Peromyscus leucopus)

Dramatic changes in the North American landscape over the last 12 000 years have shaped the genomes of the small mammals, such as the white-footed mouse (Peromyscus leucopus), which currently inhabit the region. However, very recent interactions of populations with each other and the environment are expected to leave the most pronounced signature on rapidly evolving nuclear microsatellite loci. We analyzed landscape characteristics and microsatellite markers of P. leucopus populations along a transect from southern Ohio to northern Michigan, in order to evaluate hypotheses about the spatial distribution of genetic heterogeneity. Genetic diversity increased to the north and was best approximated by a single-variable model based on habitat availability within a 0.5-km radius of trapping sites. Interpopulation differentiation measured by clustering analysis was highly variable and not significantly related to latitude or habitat availability. Interpopulation differentiation measured as F_ST values and chord distance was correlated with the proportion of habitat intervening, but was best explained by agricultural distance and by latitude. The observed gradients in diversity and interpopulation differentiation were consistent with recent habitat availability being the major constraint on effective population size in this system, and contradicted the predictions of both the postglacial expansion and core-periphery hypotheses.     

Single nucleotide polymorphism (SNP) discovery in mammals: a targeted-gene approach

Single nucleotide polymorphisms (SNPs) have rarely been exploited in nonhuman and nonmodel organism genetic studies. This is due partly to difficulties in finding SNPs in species where little DNA sequence data exist, as well as to a lack of robust and inexpensive genotyping methods. We have explored one SNP discovery method for molecular ecology, evolution, and conservation studies to evaluate the method and its limitations for population genetics in mammals. We made use of 'CATS' (or 'EPIC') primers to screen for novel SNPs in mammals. Most of these primer sets were designed from primates and/or rodents, for amplifying intron regions from conserved genes. We have screened 202 loci in 16 representatives of the major mammalian clades. Polymerase chain reaction (PCR) success correlated with phylogenetic distance from the human and mouse sequences used to design most primers; for example, specific PCR products from primates and the mouse amplified the most consistently and the marsupial and armadillo amplifications were least successful. Approximately 24% (opossum) to 65% (chimpanzee) of primers produced usable PCR product(s) in the mammals tested. Products produced generally high but variable levels of readable sequence and similarity to the expected genes. In a preliminary screen of chimpanzee DNA, 12 SNPs were identified from six (of 11) sequenced regions, yielding a SNP on average every 400 base pairs (bp). Given the progress in genome sequencing, and the large numbers of CATS-like primers published to date, this approach may yield sufficient SNPs per species for population and conservation genetic studies in nonmodel mammals and other organisms.     

Influence of genetic drift on patterns of genetic variation: The footprint of aquaculture practices in Sparus aurata (Teleostei: Sparidae)

Aquaculture finfish production based on floating cage technology has raised increasing concerns regarding the genetic integrity of natural populations. Accidental mass escapes can induce the loss of genetic diversity in wild populations by increasing genetic drift and inbreeding. Farm escapes probably represent an important issue in the gilthead sea bream (Sparus aurata), which accounted for 76.4% of total escapees recorded in Europe during a 3‐year survey. Here, we investigated patterns of genetic variation in farmed and wild populations of gilthead sea bream from the Western Mediterranean, a region of long gilthead sea bream farming. We focused on the role that genetic drift may play in shaping these patterns. Results based on microsatellite markers matched those observed in previous studies. Farmed populations showed lower levels of genetic diversity than wild populations and were genetically divergent from their wild counterparts. Overall, farmed populations showed the smallest effective population size and increased levels of relatedness compared to wild populations. The small broodstock size coupled with breeding practices that may favour the variance in individual reproductive success probably boosted genetic drift. This factor appeared to be a major driver of the genetic patterns observed in the gilthead sea bream populations analysed in the present study. These results further stress the importance of recommendations aimed at maintaining broodstock sizes as large as possible and equal sex‐ratios among breeders, as well as avoiding unequal contributions among parents.     

In vitro manipulations of vitamin C and vitamin E concentrations alter intracellular O2- production of hybrid striped bass (Morone chrysops x Morone saxatilis) head-kidney cells

To examine the mechanism by which vitamins C and E alter phagocyte function, a series of in vitro manipulations were conducted with cells isolated from the head-kidney of hybrid striped bass (average weight 680 g) fed a diet supplemented with minimum requirement levels of vitamins C and E for 2 weeks. Head-kidney phagocytes were cultured in media containing physiologically deficient (23 microM, adequate (45 microM) or excessive (182 microM) concentrations of vitamin C, and physiologically deficient (5 microM), adequate (9 microM) or excessive (32 microM) concentrations of vitamin E for 18 h. Following culture and stimulation, levels of reactive oxygen intermediates and hydrogen peroxide were determined. There were no effects of vitamin C or vitamin E concentrations on hydrogen peroxide or extracellular O2- generation. Intracellular O2- production, however, was significantly (P < or = 0.05) affected. When vitamin C was supplied at deficient levels to the medium, vitamin E elevated O2- production to levels not different from those of cells incubated with requirement levels of both vitamins. Similarly, when vitamin E was deficient in the media, vitamin C supplementation at requirement levels normalised intracellular O2- production. This data provides support for the presence of a vitamin C and vitamin E sparing mechanism in phagocytic head-kidney cells of hybrid striped bass and yield some insight into the mechanisms by which vitamin C and vitamin E function in immunomodulation.     

Comparison of population genetic estimates amongst wild,F1 and F2 cultured abalone (Haliotis midae)

Haliotis midae is South Africa's most important aquaculture species. The reproduction cycle is currently not closed as many farms rely on wild‐caught broodstock for seed production. However, there is an increasing interest in genetic improvement in commercial stocks, with a growing number of producers implementing selective breeding strategies. High throughput commercial production and mass spawning make it difficult to maintain breeding records; therefore, mostly mass selection is practised. The high fecundity and unequal parental contributions also often lead to increased levels of inbreeding. This study therefore aimed to assess the genetic effects of such breeding practices on commercial populations of H. midae. Using microsatellite loci, the genetic properties of a wild, an F1 and an F2 population were estimated and compared. Although there was no significant loss of genetic diversity amongst the cultured populations in comparison with the wild progenitor population, there was low‐to‐moderate genetic differentiation between populations. Relatedness amongst the F2 population was significant, and the rate of inbreeding was high. The effective population size for the F2 (±50) was also comparatively small with respect to the wild (∞) and F1 (±470) populations. These results suggest that farms need to give caution to breeding practices beyond the first (F1) generation and aim to increase effective population sizes and minimise inbreeding to ensure long‐term genetic gain and productivity. This study also confirms the usefulness of population genetic analyses for commercial breeding and stock management in the absence of extensive pedigree records.