Christopher C. Green

Reproductive Output of Gulf Killifish at Different Stocking Densities in Static Outdoor Tanks

Abstract Broodstock of Gulf killifish Fundulus grandis (initial mean [±SD] weight = 8.2 ± 1.6 g) were held in static outdoor tanks at a 2:1 (female:male) sex ratio and at densities of 15, 30, or 45 fish/m3. Eggs were collected twice per week from spawning mats suspended 8 cm below the water surface. Collected eggs were measured volumetrically and assessed for total number, egg size, and embryo viability. Significantly more eggs were collected from tanks with manufactured (Spawntex) spawning mats than from tanks with cured Spanish moss spawning mats. Significant differences in the mean number of eggs collected were present among density treatments in April, June, and July. Stocking density was also associated with mortality; the greatest mortality was observed in tanks at the highest density (45 fish/m3). There was a clear pattern in egg production in relation to lunar phase, with egg production peaks occurring between full and new moon phases for all three stocking densities. Mean gonadosomatic index valu...

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.

Influence of Substrate and Salinity on Air-Incubated Gulf Killifish Embryos

Abstract Gulf killifish Fundulus grandis have evolved the ability to utilize air incubation in marsh grass to protect embryos from aquatic predators and allow for synchronous hatching during the following diurnal peak tide. This incubation strategy provides a humid, oxygen-rich environment for the embryos to develop. The current project investigated practical materials that could be used in an aquaculture setting to mimic the air incubation strategy at different salinities, and determined the effect of substrate (moistened with 7.5-g/L salinity water) on embryo size after an extended incubation period (19 d) at 22°C. Air-incubated embryos were placed on synthetic foam, bamboo batting, or burlap cloth substrates. Substrates were moistened with 3.5-, 7.5-, 14-, 20-, or 27-g/L-salinity water, and hatching was induced by submersion in water after 19 d of incubation. Embryos incubated in water (3.5- and 7.5-g/L salinity) at the same temperature began hatching at 8 d and continued intermittently until 14 d. The...

Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments.

The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤ 2 mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1 mg/l DO), moderate hypoxia (2 mg/l DO), or normoxic conditions (control: DO>7.5 mg/l) for 12, 24, and 48 h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.

Characterization of Isolates of Streptococcus agalactiae from Diseased Farmed and Wild Marine Fish from the U.S. Gulf Coast, Latin America, and Thailand

We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybrid Striped Bass (Striped Bass Morone saxatilis × White Bass M. chrysops) and wild and cultured Gulf Killifish Fundulus grandis from coastal waters of the U.S. Gulf of Mexico (Gulf coast) and compared those isolates to strains from tilapias Oreochromis spp. reared in Mississippi, Thailand, Ecuador, and Honduras and to the original Gulf coast strain identified by Plumb et al. ( 1974 ). The isolates were subjected to phylogenetic, biochemical, and antibiotic susceptibility analyses. Genetic analysis was performed using partial sequence comparison of (1) the 16S ribosomal RNA (rRNA) gene; (2) the sipA gene, which encodes a surface immunogenic protein; (3) the cspA gene, which encodes a cell surface-associated protein; and (4) the secY gene, which encodes components of a general protein secretion pathway. Phylogenies inferred from sipA, secY, and cspA gene sequence comparisons were more discriminating than that inferred from the 16S rRNA gene sequence comparison. The U.S. Gulf coast strains showed a high degree of similarity to strains from South America and Central America and belonged to a unique group that can be distinguished from other group B streptococci. In agreement with the molecular findings, biochemical and antimicrobial resistance analyses demonstrated that the isolates recovered from the U.S. Gulf coast and Latin America were more similar to each other than to isolates from Thailand. Three laboratory challenge methods for inducing streptococcosis in Gulf Killifish were evaluated-intraperitoneal (IP) injection, immersion (IMM), and immersion plus abrasion (IMMA)-using serial dilutions of S. agalactiae isolate LADL 97-151, a representative U.S. Gulf coast strain. The dose that was lethal to 50% of test fish by 14 d postchallenge was approximately 2 CFU/fish via IP injection. In contrast, the fish that were challenged via IMM or IMMA presented cumulative mortality less than 40% by 14 d postchallenge.

Effects of Shading on the Reproductive Output and Embryo Viability of Gulf Killifish

Abstract Gulf killifish Fundulus grandis broodstock (7.3 ± 0.2 g [mean ± SE]) were held in static outdoor tanks at a 4:1 (female : male) sex ratio and a density of 20 fish/m3. Shade cloth was used to cover 0, 40, or 80% of the surface area of 12 randomly selected outdoor tanks. Eggs were collected from all tanks twice per week by using spawning mats suspended 15 cm below the water surface. Collected eggs were measured volumetrically and assessed for total number and embryo viability. The use of shade cloth had a significant effect on water temperature, the number of eggs collected, and embryo viability. Specifically, shading affected mean weekly water temperature, and the 80% treatment had significantly lower temperatures than both the 0% and 40% treatments during the months of June through August and than the 0% treatment in September. There was a clear cyclical reproductive pattern across all treatments, in which peaks in egg production occurred between the full and new moon phases for the majority of c...

Diet-induced fatty acid variation in critical tissues of a spawning estuarine fish and consequences for larval fitness.

Freshwater and marine fishes exhibit a dichotomy in biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) from shorter-chain precursors. Marine species generally lack this ability due to deficiencies in requisite desaturase or elongase enzymes. Gulf killifish Fundulus grandis is a euryhaline cyprinodont whose fatty acid (FA) dynamics have not been previously examined. We utilized experimental variations in exogenous FAs available to spawning F. grandis in order to construct a multitissue evaluation of FA allocation and quantify effects on reproductive output and offspring fitness. No significant decrease in fecundity occurred in animals consuming low levels of LC-PUFAs, although embryo viability rates were affected. Maternal dietary FA variation did produce differences in starvation tolerance, hypoosmoregulatory ability, and acute thermal stress tolerance for larvae. These variations occurred at elevated salinities and temperatures seldom if ever encountered by F. grandis in culture or natural environments and are unlikely to affect larval survival in these settings. FA composition of eggs and tissues from spawning females suggested potential biosynthesis of LC-PUFAs from shorter-chain precursors in F. grandis. Results of this study suggest that F. grandis possesses physiological mechanisms allowing maintenance of reproductive function when subjected to dietary deficiencies in FAs generally considered essential for marine fishes.

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K(+)) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K(+) supplementation (1.31±0.04mmol/L and 2.06±0.04mmol/L K(+); mean±SEM), one was maintained with crystal salt and no K(+) supplementation (0.33±0.05mmol/L K(+)), the fourth system was maintained using a standard marine mix salt (2.96±0.04mmol/L K(+)), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na(+)/K(+)-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K(+) supplementation resulted in 100% mortality within 24h. Mortality and dry mass were significantly influenced by K(+) concentration (P≤0.05). No differences were observed among treatment groups for NKA activity. At 1dph NKA mRNA expression was higher in the 0.3mmol [K(+)] group than in other treatment groups and at 7dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.

Variability of Egg Characteristics among Female White Bass and the Relationship between Egg Volume and Length at Hatch of Sunshine Bass

Abstract Domesticated white bass Morone chrysops can be used in selective breeding programs to produce sunshine bass (female white bass × male striped bass M. saxatilis). In addition to fast growth or favorable feed conversion, selection could be based on favorable egg or larval characteristics. Eggs from 12 white bass were fertilized with striped bass sperm to produce sunshine bass larvae and were individually photographed and incubated. The yolk sac larvae hatched from these eggs were also photographed within 3 h of hatching. Percent hatch ranged from 49% to 96%. Egg and larval characteristics were determined from the digital images. Total lipid and fatty acid composition were determined for eggs from each female. Average egg volume ranged from 0.32 to 0.42 mm3. Standard lengths of yolk sac larvae ranged from 2.51 to 3.62 mm. The relation between individual egg volumes and individual standard lengths explained only 1% of the variability in length at hatch. Average egg characteristics were generally unre...

Experimental inoculation of Louisiana red swamp crayfish Procambarus clarkii with white spot syndrome virus (WSSV)

The red swamp crayfish Procambarus clarkii represents an important aquaculture species responsible for over half of all commercial aquaculture profits in Louisiana, USA. White spot syndrome virus (WSSV) is highly pathogenic in crustacean species and induces mass mortality in aquaculture operations worldwide. Natural outbreaks of WSSV occur yearly in cultured populations of crayfish in Louisiana. The goal of this study was to better understand the infectivity of WSSV in P. clarkii, by determining the minimum lethal dose necessary to initiate infection and to measure the resulting cumulative mortality following infection with different doses. A real time quantitative PCR (qPCR) method was used to detect WSSV in DNA extracted from gill tissue to ensure P. clarkii study populations were WSSV-free before the start of trials. Viable viral particles were isolated from naturally infected P. clarkii gill tissue and quantified using a novel digital PCR approach. Three infectivity trials were performed, and WSSV inocula were created by serial dilution, generating 5 treatments per trial. Five crayfish (weighing ~25 g) per dilution per trial received viral inoculations. Mortality was monitored daily for the duration of the trial in order to construct a median lethal dose (LD50) curve, and probit regression analysis was used to determine LD50 concentrations of viral particles. Knowledge of the infectivity of WSSV in native crayfish populations is of critical importance to the management of the commercial crayfish aquaculture industry in Louisiana. This is the first study to investigate the infectivity and to determine the LD50 of the Louisiana strain of WSSV in native crayfish.