Salinity tolerance of laboratory-reared larvae of the California killifish, Fundulus parvipinnis Girard

Newly hatched larvae of the California killifish ( Fundulus parvipinnis ) reared in the laboratory, were tolerant of salinities from fresh water to 70‰. Their salinity tolerance was influenced by incubation salinity; larvae hatched in lower incubation salinities exhibited greater freshwater tolerance than those hatched in higher salinities. In gradual acclimation tests, the upper median lethal salinity for the larvae was 130‰. Freshwater tolerance of the larvae decreased with age; yolk sac larvae were completely tolerant of fresh water while larvae more than 15 days old were least resistant.     

Sulfide-hemoglobin interactions in the sulfide-tolerant salt marsh resident,the California killifish Fundulus parvipinnis

Summary Sulfide can potentially damage hemoglobin or be detoxified by hemoglobin. In the sulfide-tolerant California killifish neither seems to be the case at environmentally realistic (micromolar) and physiologically relevant (millimolar) sulfide concentrations. An 8-h exposure of killifish to 5 and 8 mmol sulfide · 1^-1 results in 50–100% mortality, but not due to sulfhemoglobin (where sulfide covalently binds to the porphyrin) nor ferric hemoglobin (Hb⁺), both dysfunctional hemoglobin derivatives. Killifish hemoglobin converts to sulfhemoglobin in vitro only in the presence of 1–5 mmol sulfide · 1^-1. The amount of sulfhemoglobin formed increases with time and heme concentration but decreases with pH. Hb⁺ binds sulfide as ferric hemoglobin sulfide (Hb⁺S, an unstable complex where sulfide ligates to the iron), and also as sulfhemoglobin. Killifish blood does not catalyze the oxidation of 10–500 mol sulfide · 1^-1 to any appreciable extent. Radiolabeled sulfide incubated with oxyhemoglobin or whole blood disappears at rates greater than in buffers, but only minimal amounts of thiosulfate and no sulfate nor sulfite are formed (elemental sulfur and bound sulfide not quantified). Sulfide disappearance rates increase linearly with initial sulfide concentration. Hb⁺ does catalyze the oxidation of sulfide to thiosulfate in vitro. Similar experiments on another sulfide-tolerant species, the long-jawed mudsucker Gillichthys mirabilis, produced similar results.Abbreviations ANOVA analysis of variance - BV benzyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high-pressure liquid chromatography - RBC red blood cells - SHb sulfhemoglobin     

Sulphide tolerance and adaptation in the California killifish, Fundulus parvipinnis, a salt marsh resident

Hydrogen sulphide is a toxicant naturally produced in hypoxic marine sediments, hydrocarbon and brine seeps and hydrothermal vents. The California killifish, a salt marsh resident, is remarkably tolerant of sulphide. The 50% lethal concentration is 700 μM total sulphide in 96 h, and 5 mM in 8 h (determined in flow-through, oxygenated sea water). Killifish exposed to sulphide produce thiosulphate which accumulates in the blood. The cytochrome c oxidase (a major site of toxicity) of the killifish is 50% inhibited by <1 μM sulphide. Killifish liver mitochondria are poisoned by 50–75 μM sulphide but can oxidize 10–20 μM sulphide to thiosulphate. Sulphide causes sulphhaemoglobin formation (and impairment of oxygen transport) at 1–5 mM in vitro and to a small extent at 2 mM in vivo . Killifish blood neither catalyses sulphide oxidation significantly nor binds sulphide at environmental (low) sulphide concentrations. Exposure to 200 μM and 700 μM sulphide over several days causes significant increases in lactate concentrations, indicating shift to anaerobic glycolysis. However, individuals with the most lactate die. In terms of diffusible H₂S, the killifish can withstand concentrations two to three orders of magnitude greater than would poison cytochrome c oxidase. The high sulphide tolerance of the killifish, particularly of concentrations typical of salt marshes, can be explained chiefly by mitochondrial sulphide oxidation. Sulphide tolerance and mitochondrial sulphide oxidation in the killifish have a constitutive basis, i.e. do not diminish in fish held in the laboratory in sulphide-free water for 1–2 months, and are improved by prior acclimation.     

Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis

California killifish (Fundulus parvipinnis) infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours rendering them more susceptible to predation by avian final hosts. Heavily infected killifish grow and reproduce normally, despite having thousands of cysts inside their braincases. This suggests that E. californiensis affects only specific locomotory behaviours. We hypothesised that changes in the serotonin and dopamine metabolism, essential for controlling locomotion and arousal may underlie this behaviour modification. We employed micropunch dissection and HPLC to analyse monoamine and monoamine metabolite concentrations in the brain regions of uninfected and experimentally infected fish. The parasites exerted density-dependent changes in monoaminergic activity distinct from those exhibited by fish subjected to stress. Specifically, E. californiensis inhibited a normally occurring, stress-induced elevation of serotonergic metabolism in the raphae nuclei. This effect was particularly evident in the experimentally infected fish, whose low-density infections were concentrated on the brainstem. Furthermore, high E. californiensis density was associated with increased dopaminergic activity in the hypothalamus and decreased serotonergic activity in the hippocampus. In conclusion, the altered monoaminergic metabolism may explain behavioural differences leading to increased predation of the infected killifish by their final host predators.     

Oxidative detoxification of sulfide by mitochondria of the California killifish Fundulus parvipinnis and the speckled sanddab Citharichthys sitgmaeus

Summary Earlier whole-animal experiments have shown that the California killifish (Fundulus parvipinnis) from tidal marshes is highly tolerant to sulfide while the speckled sanddab (Citharichtys stigmaeus) from the open coast is intolerant to sulfide. In the present paper, we demonstrate that the liver mitochondria of the California killifish detoxify sulfide by oxidizing it to thiosulfate and produce ATP in the process. Sulfide oxidation is obligately and stoichiometrically linked to mitochondrial electron transport to oxygen. Concentrations up to 20 M sulfide stimulate mitochondrial respiration while 50 M sulfide causes half-inhibition. Sulfide oxidation by mitochondria is adversely affected at pH<7.4. ATP production is maximal at 10 M sulfide. The finding of sulfide oxidation coupled to ATP production by killifish mitochondria is unprecedented among vertebrates. In comparison, mitochondria of the specked sanddab oxidize sulfide at a much lower rate. This is the first demonstration of biochemical adaptation to sulfide among coastal marine fishes.Abbreviations ADP adenosine diphosphate - APHA American Public Health Association - ATP adenosine triphosphate - BSA bovine serum albumin - EGTA ethyleneglycol-bis-(-aminoethyl-ether) N,N,N,N-tetraacetic acid - G-6-PDH glucose-6-phosphate dehydrogenase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - HPLC high-performance liquid chromatography; mBBr monobromobimane - NADP nicotinamide adenine dinucleotide phosphate, oxidized form - NADPH reduced form - RCR respiratory control ratio     

Microsatellite primers for the Atlantic coastal killifish,Fundulus heteroclitus,with applicability to related Fundulus species

The mummichog (Fundulus heteroclitus), a common Atlantic coastal killifish, is a model vertebrate species for the study of molecular genetic variation in natural populations and of environmental toxicology. We report the development of a set of 20 microsatellite loci in this species. Average expected heterozygosity across all loci was 0.84 (range: 0.60–0.97), revealing a high level of variability at most loci. A survey of seven additional Fundulus species yielded one or two robust amplification products in over half (63%) of the species–primer combinations tested. Therefore, many of these loci will also prove useful in studies of other members of the genus Fundulus.     

Genetic isolation and evolutionary history of oases populations of the Baja California killifish,Fundulus lima

The Baja California killifish, Fundulus lima, is found in six desert oases of the southern Baja California Peninsula, Mexico. The recent introduction of exotic fishes, particularly redbelly tilapia, have impacted the ecology of Fundulus lima such that it is now endangered. Plans of relocating F. lima to bodies of freshwater that are free of exotics have been proposed, however little is know about the genetic identity of the current populations. In this study, we examined the mitochondrial control region of F. lima samples from 4 oases, and in addition, compared these samples to their sister species, the California killifish F.␣parvipinnis. Using a combination of phylogenetic and coalescent approaches, we were able to determine that the two subspecies of the California killifish, F. p.␣brevis, and F. p. parvipinnis, and F. lima form an unresolved trichotomy that diverged between 200,000 years and 400,000 years ago. The one F. lima individual that we were able to collect in the southernmost oasis grouped with the southern subspecies of the California killifish, F. parvipinnis brevis. In contrast, we found that the 3 northern oases grouped together in a “Fundulus lima” clade. Each oasis is genetically distinct, yet there is no evidence of a␣marked genetic bottleneck in any populations (Haplotype diversity between 0.5 and 0.8). Future relocation plans will therefore need to be done cautiously to preserve the genetic identity of the original populations.     

Brain‐encysting trematodes and altered monoamine activity in naturally infected killifish Fundulus parvipinnis

This paper presents novel evidence to address mechanisms by which trematode parasites effect behavioural changes in naturally infected fish hosts. California killifish Fundulus parvipinnis infected with the brain‐encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours that render them 30 times more likely to be eaten by birds, the parasite's final host. Prevalence of E. californiensis reaches nearly 100% in most F. parvipinnis populations, with parasite biomass constituting almost 2% of F. parvipinnis biomass in some locations. Despite having thousands of cysts on their brains, infected fish grow and mature at rates comparable to those of uninfected populations. The lack of general pathology combined with the specificity of the altered behaviours suggests that the behavioural changes are due to parasite manipulation. The monoamine neurotransmitters serotonin and dopamine, which control locomotion and social behaviour in fishes and other vertebrates, were examined to explore the underlying mechanisms of this behaviour modification. Whereas previous studies were similarly conducted with experimentally infected fish, in this study, brain dopaminergic and serotonergic activity were analysed in naturally infected fish to assess how E. californiensis may alter F. parvipinnis monoamines in a naturally occurring system. A parasite density‐associated decrease in serotonergic activity occurred in the hippocampus of naturally infected fish, as well as a decrease in dopaminergic activity in the raphe nuclei, suggesting that E. californiensis inhibits serotonin and dopamine signaling in naturally infected F. parvipinnis. The neurochemical profile of infected fish is consistent with the hypothesis that E. californiensis affects brain monoaminergic systems in order to induce impulse‐driven, active, and aggressive behaviour in its hosts.     

The relationship between handling time and cortisol release rates changes as a function of brain parasite densities in California killifish Fundulus parvipinnis

This study validated a technique for non‐invasive hormone measurements in California killifish Fundulus parvipinnis, and looked for associations between cortisol (a stress hormone) and 11‐ketotestosterone (KT, an androgen) release rates and the density or intensity of the trematode parasites Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB) in wild‐caught, naturally infected F. parvipinnis. In experiment 1, F. parvipinnis were exposed to an acute stressor by lowering water levels to dorsal‐fin height and repeatedly handling the fish over the course of an hour. Neither parasite was found to influence cortisol release rates in response to this acute stressor. In experiment 2, different F. parvipinnis were exposed on four consecutive days to the procedure for collecting water‐borne hormone levels and release rates of 11‐KT and cortisol were quantified. This design examined whether F. parvipinnis perceived the water‐borne collection procedure to be a stressor, while also exploring how parasites influenced hormone release rates under conditions less stressful than those in experiment 1. No association was found between RENB and hormone release rates, or between EUHA and 11‐KT release rates. The interaction between EUHA density and handling time, however, was an important predictor of cortisol release rates. The relationship between handling time and cortisol release rates was negative for F. parvipinnis harbouring low or intermediate density infections, and became positive for fish harbouring high densities of EUHA.     

Phylogeography of the plains killifish, Fundulus zebrinus

Drainage systems of the Great Plains and western Gulf Slope underwent substantial changes through diversions and stream captures during the Pleistocene, either as the result of the glacial advances or through independent geologic processes. The distributions of a variety of fishes that range across west-central North America, such as the plains killifish (Fundulus zebrinus), are thought to be the product of this Pleistocene influence. We examined the geographic pattern of genetic variation in F. zebrinus using three allozyme loci (n = 793), mitochondrial DNA restriction fragment length polymorphisms (RFLPs, n = 352), and sequencing of the mitochondrial cytochrome oxidase I (COI, n = 23) in an attempt to understand the roles of dispersal and vicariance. The phylogeographic patterns were concordant between the allozyme and mitochondrial data with the exception of the population in the North Canadian River. The populations fell into three geographic assemblages, which we designated as northern, central, and southern. A large phylogenetic break (average Roger's D = 0.702; average sequence divergence in RFLPs = 4.6%; average sequence divergence in COI = 5.5%) separated the northern/central and southern assemblages. The northern region was likely colonized sometime during the mid-Pleistocene. Fish in the Brazos and Pecos Rivers probably reached these drainages through stream captures of the Red River. The large phylogenetic break between the northern/central and southern clades supports previous attempts to recognize two species of plains killifish: F. zebrinus and F. kansae.     

Methylmercury teratogenesis in the killifish, Fundulus heteroclitus.

Exposure of developing eggs of the killifish, Fundulus heteroclitus, to 0.03 or 0.04 mg/l of methylmercuric chloride resulted in a variety of abnormalities. Percentage of axis formation was reduced somewhat, and many embryos developed cyclopia or intermediate conditions leading to cyclopia, reflecting interference with induction of the forebrain. Defects in the cardiovascular system also appeared in the form of failure of the heart to differentiate properly into chambers. The heart was a thin, feebly beating tube, incapable of causing the blood to circulate. Other tissues, however, continued developing fairly normally, and embryos showed spontaneous movement comparable to controls. Embryos with severe cardiovascular or optic defects did not hatch. Upon hatching, some embryos which had previously appeared normal were found to have skeletal malformations in the form of vertebral bends or the inability to uncurl from the position which they had while still inside the chorion. Exposure to the toxicant for shorter periods of time (6, 12, or 24 hours) reduced the incidence of abnormalities. The second day of development was found to be the most sensitive period.     

Effects of heavy metals on development of the killifish, Fundulus heteroclitus

When F. heteroclitus embryos were exposed to inorganic mercury at concentrations of 0.03 or 0.1 mg/1 at the early blastula stage, the percentage of successful axis formation was reduced and a significant proportion of embryos developed cyclopia or intermediate conditions leading to cyclopia. Treatment at the late blastula stage reduced the severity of the defects. Embryos which developed in lead at concentrations of 1 and 10 mg/1 were normal in appearance until hatching, at which time they exhibited lordosis or were unable to uncurl from the position they had while still inside the chorion. No significant effects of cadmium at concentrations up to 10 mg/1 were noted.     

Genetic evidence for female-biased dispersal in the bullfrog, Rana catesbeiana (Ranidae)

Sex-biased dispersal is an important but unexplored area of amphibian ecology. We predicted female-biased dispersal in the bullfrog (Rana catesbeiana) based on aspects of their mating system and tested this prediction using data from seven polymorphic DNA microsatellite loci. Allelic (F-statistics) and genotypic [assignment index, (AIc)] frequencies from nine Ontario populations support our prediction, although significant sex differences in inbreeding and variance of AIc were not detected. The diversity of mating systems found in amphibians represents an important avenue for investigating the relationship between reproductive systems, dispersal and phylogeny.     

Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure

We examined the metabolic responses of the hypoxia-tolerant killifish (Fundulus heteroclitus) to 15 h of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [creatine phospate] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP(free)] and [ATP]/[AMP(free)] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (DeltafG'). The activity of PDH was rapidly (within 3 h) decreased by approximately 50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via posttranslational modification following the accumulation of acetyl-CoA and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD(+)]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia-responsive genes (e.g., lactate dehydrogenase-A or -B) were observed in either muscle or liver.     

Molecular evidence for limited dispersal of vegetative propagules in the epiphytic lichen Lobaria pulmonaria

Propagation, whether sexual or asexual, is a fundamental step in the life cycle of every organism. In lichenized fungi, a great variety of vegetative propagules have evolved in order for the symbiotic partners to disperse simultaneously. For lichens with the ability of sexual and asexual reproduction, the relative contribution of vegetative dispersal is unknown but could, nonetheless, be inferred by studying genotype distribution. The genetic structure of three Lobaria pulmonaria (Lobariaceae) populations from Switzerland was investigated based on the observed variation at six microsatellite loci. All three populations had a clustered distribution of identical genotypes at small spatial scales. The maximum distance between identical genotypes was 230 m. At a distance of 350 m from a source tree, seemingly suitable habitat patches were too far apart to be colonized. Some multilocus genotypes were frequent within local populations but no genotypes were shared among populations. The restricted occurrences of common genotypes as well as the clustered distributions are evidence for a limited dispersal of vegetative propagules in L. pulmonaria. Gene flow among isolated populations will ultimately depend on the capacity of long-distance dispersal and thus probably depend on sexual reproduction.