Identification of young-of-the-year great hammerhead shark Sphyrna mokarran in northern Florida and South Carolina.

Two sharks, visually identified in the field as young-of-the-year (YOY) scalloped hammerhead Sphyrna lewini, were identified as great hammerhead Sphyrna mokarran based on nuclear-encoded single nucleotide polymorphisms (SNP) and sequences of mtDNA. Individuals were captured and released in Bulls Bay, SC, and Saint Joseph Bay, FL, in 2013 and 2014, respectively. These findings indicate S. mokarran may be pupping in or around these areas and highlight new regions that may be a productive focus for future research on early life history of S. mokarran.

A method for detecting recent changes in contemporary effective population size from linkage disequilibrium at linked and unlinked loci.

Estimation of contemporary effective population size (Ne) from linkage disequilibrium (LD) between unlinked pairs of genetic markers has become an important tool in the field of population and conservation genetics. If data pertaining to physical linkage or genomic position are available for genetic markers, estimates of recombination rate between loci can be combined with LD data to estimate contemporary Ne at various times in the past. We extend the well-known, LD-based method of estimating contemporary Ne to include linkage information and show via simulation that even relatively small, recent changes in Ne can be detected reliably with a modest number of single-nucleotide polymorphism (SNP) loci. We explore several issues important to interpretation of the results and quantify the bias in estimates of contemporary Ne associated with the assumption that all loci in a large SNP data set are unlinked. The approach is applied to an empirical data set of SNP genotypes from a population of a marine fish where a recent, temporary decline in Ne is known to have occurred.

Genetic variation of Prochilodus lineatus (Valenciennes, 1836) from Paraná, Baía, Miranda, and Corumbá rivers, Brazil.

The curimbatá (Prochilodus lineatus) is one of the migratory species in the Paraná River Basin impacted by the construction of dams. Mitochondrial DNA sequences and random amplified polymorphic DNA (RAPD) fragments were used to investigate genetic variability and geographic structure of five populations of curimbatá from the Paraná River Basin. A total of 1815 bp from seven polymerase chain reaction-amplified fragments representing five protein-coding mitochondrial genes were sequenced from 12 individuals. Estimates of nucleotide sequence divergence ranged from 0.00 to 0.95%. A total of 86 RAPD markers from 58 individuals were detected. Results from the Fisher exact test indicated that P. lineatus is not genetically subdivided, although significant differences in the frequencies of a few RAPD fragments were observed. This study provides useful information for stocking and management programs for resource planning of P. lineatus.

Selection and sex-biased dispersal in a coastal shark: the influence of philopatry on adaptive variation.

Sex-biased dispersal is expected to homogenize nuclear genetic variation relative to variation in genetic material inherited through the philopatric sex. When site fidelity occurs across a heterogeneous environment, local selective regimes may alter this pattern. We assessed spatial patterns of variation in nuclear-encoded, single nucleotide polymorphisms (SNPs) and sequences of the mitochondrial control region in bonnethead sharks (Sphyrna tiburo), a species thought to exhibit female philopatry, collected from summer habitats used for gestation. Geographic patterns of mtDNA haplotypes and putatively neutral SNPs confirmed female philopatry and male-mediated gene flow along the northeastern coast of the Gulf of Mexico. A total of 30 outlier SNP loci were identified; alleles at over half of these loci exhibited signatures of latitude-associated selection. Our results indicate that in species with sex-biased dispersal, philopatry can facilitate sorting of locally adaptive variation, with the dispersing sex facilitating movement of potentially adaptive variation among locations and environments.

Contemporary population structure and post-glacial genetic demography in a migratory marine species, the blacknose shark, Carcharhinus acronotus.

Patterns of population structure and historical genetic demography of blacknose sharks in the western North Atlantic Ocean were assessed using variation in nuclear-encoded microsatellites and sequences of mitochondrial (mt)DNA. Significant heterogeneity and/or inferred barriers to gene flow, based on microsatellites and/or mtDNA, revealed the occurrence of five genetic populations localized to five geographic regions: the southeastern U.S Atlantic coast, the eastern Gulf of Mexico, the western Gulf of Mexico, Bay of Campeche in the southern Gulf of Mexico and the Bahamas. Pairwise estimates of genetic divergence between sharks in the Bahamas and those in all other localities were more than an order of magnitude higher than between pairwise comparisons involving the other localities. Demographic modelling indicated that sharks in all five regions diverged after the last glacial maximum and, except for the Bahamas, experienced post-glacial, population expansion. The patterns of genetic variation also suggest that the southern Gulf of Mexico may have served as a glacial refuge and source for the expansion. Results of the study demonstrate that barriers to gene flow and historical genetic demography contributed to contemporary patterns of population structure in a coastal migratory species living in an otherwise continuous marine habitat. The results also indicate that for many marine species, failure to properly characterize barriers in terms of levels of contemporary gene flow could in part be due to inferences based solely on equilibrium assumptions. This could lead to erroneous conclusions regarding levels of connectivity in species of conservation concern.

Parthenogenesis in a whitetip reef shark Triaenodon obesus involves a reduction in ploidy.

Genetic analysis of a female whitetip reef shark Triaenodon obesus and her stillborn pup, assumed to be of parthenogenetic origin, revealed that the pup was homozygous at all 24 nuclear-encoded microsatellites assayed, consistent with the idea that diploidy in the pup had been restored via terminal fusion. Flow cytometric analysis, however, indicated that the genome size of the pup was no more than half that of the mother, and microscopy revealed that nuclear volume was c. 1.73 times larger in the mother than in the pup. Together these data suggest that the pup was genetically haploid, developing directly from an unfertilized egg; as far as is known, this is the first observation of a spontaneously produced haploid vertebrate.

Does mating behaviour affect connectivity in marine fishes? Comparative population genetics of two protogynous groupers (Family Serranidae).

Pelagic larval duration (PLD) has been hypothesized to be the primary predictor of connectivity in marine fishes; however, few studies have examined the effects that adult reproductive behaviour may have on realized dispersal. We assessed gene flow (connectivity) by documenting variation in microsatellites and mitochondrial DNA sequences in two protogynous species of groupers, the aggregate spawning red hind, Epinephelus guttatus, and the single-male, harem-spawning coney, Cephalopholis fulva, to ask whether reproductive strategy affects connectivity. Samples of both species were obtained from waters off three islands (Puerto Rico, St. Thomas and St. Croix) in the Caribbean Sea. Despite the notion that aggregate spawning of red hind may facilitate larval retention, stronger signals of population structure were detected in the harem-spawning coney. Heterogeneity and/or inferred barriers, based on microsatellites, involved St. Croix (red hind and coney) and the west coast of Puerto Rico (coney). Heterogeneity and/or inferred barriers, based on mitochondrial DNA, involved St. Croix (coney only). Genetic divergence in both species was stronger for microsatellites than for mitochondrial DNA, suggesting sex-biased dispersal in both species. Long-term migration rates, based on microsatellites, indicated asymmetric gene flow for both species in the same direction as mean surface currents in the region. Red hind had higher levels of variation in microsatellites and lower levels of variation in mitochondrial DNA. Long-term effective size and effective number of breeders were greater for red hind; estimates of θ(f) , a proxy for long-term effective female size, were the same in both species. Patterns of gene flow in both species appear to stem in part from shared aspects of larval and adult biology, local bathymetry and surface current patterns. Differences in connectivity and levels of genetic variation between the species, however, likely stem from differences in behaviour related to reproductive strategy.

Population structure in Chilean hake Merluccius gayi as revealed by mitochondrial DNA sequences.

Genetic variation and divergence among samples of Chilean hake Merluccius gayi, from three localities off the coast of Chile and one locality off the coast of northern Peru, were assessed using sequences from the control region of mitochondrial DNA. Homogeneity tests revealed occurrence of at least three distinct genetic stocks of M. gayi within the region sampled. Factors potentially contributing to genetic divergence among M. gayi probably include hydrodynamics and behaviour.

A genetic linkage map of red drum, Sciaenops ocellatus.

Second-generation, sex-specific genetic linkage maps were generated for the economically important estuarine-dependent marine fish Sciaenops ocellatus (red drum). The maps were based on F(1) progeny from each of two single-pair mating families. A total of 237 nuclear-encoded microsatellite markers were mapped to 25 linkage groups. The female map contained 226 markers, with a total length of 1270.9 centiMorgans (cM) and an average inter-marker interval of 6.53 cM; the male map contained 201 markers, with a total length of 1122.9 cM and an average inter-marker interval of 6.03 cM. The overall recombination rate was approximately equal in the two sexes (♀:♂=1.03:1). Recombination rates in a number of linkage intervals, however, differed significantly between the same sex in both families and between sexes within families. The former occurred in 2.4% of mapped intervals, while the latter occurred in 51.2% of mapped intervals. Sex-specific recombination rates varied within chromosomes, with regions of both female-biased and male-biased recombination. Original clones from which the microsatellite markers were generated were compared with genome sequence data for the spotted green puffer, Tetraodon nigroviridis; a total of 43 matches were located in 17 of 21 chromosomes of T. nigroviridis, while seven matches were in unknown portions of the T. nigroviridis genome. The map for red drum provides a new, useful tool for aquaculture, population genetics, and comparative genomics of this economically important marine species.

Spawning frequency of brood dams and sires in a marine fish stock-enhancement hatchery.

Parentage analysis, employing five hypervariable microsatellite markers, was used to follow spawning patterns of red drum Sciaenops ocellatus broodfish in two spawning tanks through most of a calendar year in a marine fish hatchery dedicated to stock enhancement. Five of six dams and all four sires spawned at least once during the year. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire translated into reduced genetic effective size (N e) per spawn by 40·6% in one tank and 50·8% in the other.

PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol's iodine solution.

A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol's iodine preservative. Destaining of Lugol's-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.

Plasma adrenocorticotropin, cortisol, and adrenocorticotropin/cortisol ratios in septic and normal-term foals.

BACKGROUND: Little information exists on the hypothalamic-pituitary-adrenal axis in septic foals. HYPOTHESIS: The plasma concentrations of adrenocorticotropin (ACTH) and cortisol are expected to be higher in septic foals as compared to normal foals. The concentrations of hormones in septic foals also are expected to differ further depending upon survival. ANIMALS: Twenty-eight control foals and 46 septic foals <14 days of age were included in this study. METHODS: Blood was collected in EDTA once from 28 normal foals born in the University of Georgia or Cornell University equine research herds and from 46 septic foals within 12 hours after admission to 1 of the 3 tertiary care referral centers involved in the study. Septic foal selection was based on a sepsis score of >11 or a positive blood culture. The control foals were age matched to the septic foals in the study. ACTH and cortisol concentrations were measured by a chemiluminescent immunoassay system. RESULTS: Cortisol concentrations in control foals did not vary with age. Septic foals had significantly higher mean ACTH, cortisol, and ACTH/cortisol ratios than did normal foals. Within the septic foal group, 28 foals survived to discharge, and 18 were euthanized or died. The mean age was not significantly different between the septic surviving and nonsurviving foals. The mean ACTH/cortisol ratio was significantly higher in the septic nonsurviving foals as compared to the septic surviving foals. CONCLUSIONS AND CLINICAL IMPORTANCE: Septic foals had higher hormone concentrations as compared to normal foals, which is an expected endocrine response to critical illness. The increased ACTH/cortisol ratio in nonsurviving septic foals in comparison to surviving septic foals could indicate hypothalamic-pituitary-adrenal axis dysfunction at the level of the adrenal gland in critically ill septic foals.

Plasma, subcutaneous tissue and bone concentrations of ceftiofur sodium after regional limb perfusion in horses.

REASONS FOR PERFORMING STUDY: Regional limb perfusion (RLP) is an effective treatment option for injuries and infections of the distal limb in horses. Using ceftiofur sodium in RLP has been studied due to its superior spectrum of Gram-positive organisms compared to aminoglycosides, but it is not known if this antimicrobial drug adequately penetrates subcutaneous tissue and bone. OBJECTIVE: To determine the concentration of ceftiofur in plasma, subcutaneous tissue and bone in horses after RLP. STUDY DESIGN: Experimental prospective study. METHODS: Six healthy horses were used in this study. Under standing sedation, an Esmarch tourniquet was applied to both proximal metacarpi and RLP was performed in each forelimb by injecting either ceftiofur sodium (experimental limb) or saline (control limb) i.v. in the lateral palmar digital vein. The experimental limb was injected with 2 g ceftiofur diluted to 60 mL with sterile saline. The control limb was injected with 60 mL of sterile saline. The tourniquet was left in place for 30 min post injection. Plasma, subcutaneous tissue and cortical bone samples were collected immediately after tourniquet removal (0.5 h), 12 and 24 h post injection. Ceftiofur concentrations and its active metabolite desfuroylceftiofur were analysed using high performance liquid chromatography-tandem mass spectrometry and the results were compared between control and experimental limbs using a Wilcoxon signed rank test. RESULTS: The median plasma concentrations were greater than the minimum inhibitory concentration (MIC) for common pathogens (1 µg/mL) at 0 and 12 h post RLP. The median subcutaneous tissue concentrations were greater than MIC (1 µg/g) at all 3 time points in the experimental limb. The median bone concentration was above MIC (1 µg/g) at time 0 in the experimental limb but was below MIC at 12 and 24 h in the experimental limb. CONCLUSIONS: Ceftiofur administration via RLP maintained plasma concentrations above MIC for 12 h. Subcutaneous tissue concentrations above MIC were maintained for 24 h. Bone concentrations were only above MIC immediately after tourniquet removal. Further research is needed to evaluate ceftiofur administration via RLP and its implications in disease states.

Application of hypervariable genetic markers to forensic identification of 'wild' from hatchery-raised red drum, Sciaenops ocellatus.

Forensic identification of 'wild' versus hatchery-produced (cultured) red drum (Sciaenops ocellatus), an economically important marine fish in the southern United States, was assessed using hypervariable nuclear-encoded microsatellites and sequences of mitochondrial DNA. Both genotype exclusion and likelihood-ratio tests successfully identified 'wild' and 'cultured' individuals within requisite error bounds and within the context of complete parental sampling. Of the two, genotype exclusion was more effective, producing satisfactory results with fewer microsatellites and larger allowable error rates. Assignment tests proved ineffective, most likely because of the low level of genetic divergence between the sampled populations. An optimal, minimum set of ten markers that will reduce potential genotyping costs is identified. Results of the study should allay concerns regarding identification of 'wild'-caught fish sold illegally.

Microsatellite variation among red snapper (Lutjanus campechanus) from the Gulf of Mexico.

Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (+/- SE) direct count heterozygosity values ranged from 0.148 +/- 0.025 to 0.902 +/- 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico.

Medical management of sand enteropathy in 62 horses.

REASONS FOR PERFORMING STUDY: Medical management of sand enteropathy is common in equine practice, but the clinical features and outcomes associated with medically managed sand enteropathy are not well described. OBJECTIVES: To review clinical features, therapeutic approaches and outcomes associated with primary medical management of sand enteropathy in the mature horse. METHODS: Medical record databases at 3 equine referral hospitals from January 2000 to April 2010 were reviewed for cases of sand enteropathy diagnosed via abdominal radiographs in mature horses that were initially managed medically. Data were collected and descriptive analyses compiled. Uni- and multivariate logistic regression was used to evaluate factors potentially associated with treatment failure. RESULTS: The medical records of 62 horses were analysed; 90% of horses survived to discharge and 50% of horses that had repeat abdominal radiographs taken demonstrated improvement in the degree of sand accumulation after treatment. Nine horses underwent exploratory laparotomy during hospitalisation, and colonic sand impaction was found in all 9, with a concurrent gastrointestinal lesion identified in 7. Four horses were subjected to euthanasia during or after surgery because of disease severity or complications. Need for exploratory laparotomy was the factor most strongly associated with nonsurvival. CONCLUSIONS: These data suggest that medical management can result in clinical and radiographic resolution of uncomplicated sand enteropathy in mature horses, and is associated with a good prognosis. Horses with sand enteropathy that exhibit persistent colic signs despite medical management are likely to have a concurrent gastrointestinal lesion, so prompt exploratory laparotomy should be considered in such cases. POTENTIAL RELEVANCE: Uncomplicated sand enteropathy can be managed medically in mature horses, and serial abdominal radiography can be used to monitor sand clearance. Surgery to evaluate for and correct concurrent gastrointestinal lesions should be recommended without delay in horses showing persistent colic signs.

Association of adrenocorticotrophin and cortisol concentrations with peripheral blood leukocyte cytokine gene expression in septic and nonseptic neonatal foals.

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) is influenced by the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in critically ill humans. Information about the association of cytokines with the HPA axis in neonatal foals is lacking. HYPOTHESIS/OBJECTIVES: The objectives were to describe for hospitalized septic and nonseptic foals (1) temporal changes in blood concentrations of ACTH, and cortisol, and leukocyte cytokine gene expression, and (2) coassociation of these HPA axis hormones with blood leukocyte cytokine gene expression. ANIMALS: Hospitalized septic foals (N = 15) and hospitalized nonseptic foals (N = 11). METHODS: Blood samples, obtained from study foals at admission (T = 0), and 24 (T = 1), 48 (T = 2), 72 (T = 3), and 96 (T = 4) hours after admission, were processed to isolate RNA from leukocytes and to harvest plasma and serum for hormone assays. Plasma ACTH and serum cortisol concentrations were determined by radioimmunoassay. Leukocyte mRNA expression of IL-1ß IL-6, IL-8, IL-10, and TNF-α was determined using RT-PCR. RESULTS: Cortisol concentrations were greater (P < .05) in foals at admission than at other time points. The expressions of IL-8 and IL-10 mRNA were lower (P < .05) at each time point in septic than in nonseptic foals. Among septic foals, ACTH was positively associated (P = .0026) with IL-6 mRNA expression. CONCLUSIONS: Sepsis influences secretion of the HPA axis hormones and expression of cytokines in foals. A positive association with the HPA axis and IL-6 expression was detected. The clinical importance of these findings requires additional study.

Silver staining for nucleolar organizing regions of vertebrate chromosomes.

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.

Genome size variation among north american minnows (Cyprinidae). I. Distribution of the variation in five species.

Genome sizes (nuclear DNA contents) were examined spectrophotometrically from ten individuals of each of five species of North American cyprinid fishes (minnows). The distributions of DNA values both within and between the five species were essentially continuous and normal. Differences between individuals within populations were significant and contributed to approximately 16 per cent of the total variation. Variation between individuals within species ranged from 4.7-13.5 per cent and averaged ca. 7.4 per cent. Variation between species ranged from 0-9.5 per cent and the average difference between any species pair was ca. 4.6 per cent. Statistical analyses showed that the methodology used was sufficient to detect significant differences in genome size as small as 2-3 per cent. Consideration of these data lead to the following tentative conclusions: (i) changes in genome size in cyprinids appear small in amount, frequent in occurrence, to involve both gains and losses of DNA, and to be cumulative and independent in effect; (ii) differences within and between cyprinid taxa are likely the result of accumulations of small changes in DNA quantity; and (iii) the primary focus of quantitative DNA variation in cyprinids is between individuals within populations. The extent of DNA quantity variation which occurs within species would appear to preclude any direct relationship between genome size variation and many of the organismal parameters (including speciation) which differentiate the five species. In short, the data suggest that a significant fraction of the cyprinid genome, perhaps more than 10 per cent, is free to vary quantitatively without phenotypic constraint or biological consequence. This fraction is considerably larger than that theoretically needed for the structural gene component.