Development stage-dependent oxidative stress responses to the exposure to roundup original© in a neotropical annual killifish.

Herbicides are the most commonly applied pesticides in Brazil, specifically those based on glyphosate, and are used for different crops, near the habitats of annual killifish. Annual killifish presents a short life cycle with generally restricted geographic distribution. In this context, we evaluated the effect of the Roundup Original© (65, 130 and 260 µg. L-1 of glyphosate) herbicide on different development stages (adult-young and senile) of the annual killifish (Cynopoecilus sp.). We quantified the oxidative balance markers (superoxide dismutase, catalase, glutathione S-transferase, lipid peroxidation levels, and total proteins). We observed that the senile individuals presented 2-fold higher lipid peroxidation levels associated with the maintenance of superoxide dismutase and catalase activity levels even after exposure to the herbicide. However, senile subjects were negatively impacted by the exposure to formulations containing glyphosate, and this was related to a loss of glutathione S-transferase activity. Our research demonstrated that the established physiological markers and this species look promising for toxicology studies.

Biomarkers of oxidative stress in the post-embryonic characterization of the neotropical annual killifish.

Annual killifish are among the most remarkable extremophile species with the shortest vertebrate life span. Few studies have reported on the oxidative balance throughout their life cycle and its association to the natural aging process of these neotropical animals in a natural environment. We standardized and analyzed physiological markers related to the redox balance of the annual killifish (Cynopoecilus fulgens) throughout the post-embryonic life cycle (enzyme activity of Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione S-transferase, as well as the determination of the levels of Lipoperoxidation, Carbonylated Proteins, and Total Proteins). We tested the influence of environmental variables on these biomarkers. Individuals were collected, including juveniles, adults, and seniles, in three sampling units around the Parque Nacional da Lagoa do Peixe, located in the Coastal Plain of Rio Grande do Sul. We observed that males and females used different physiological strategies of their redox balance during their life cycle, and their oxidative balance was influenced by their reproductive period and environmental variables (water temperature, abundance of predators, abundance of another sympatric annual killifish species, and abundance of C. fulgens). The population of each temporary pond presented different physiological responses to the adaptation of their life cycle, and there was an influence of environmental component as a modulator of this cycle. Our study offers reference values that will be useful for comparison in future research with short-lived organisms.

Biomarkers of Aryl-hydrocarbon Receptor Activity in Gulf Killifish (Fundulus grandis) From Northern Gulf of Mexico Marshes Following the Deepwater Horizon Oil Spill.

Following the Deepwater Horizon oil spill, shorelines throughout the Barataria Basin of the northern Gulf of Mexico in Louisiana were heavily oiled for months with Macondo-252 oil, potentially impacting estuarine species. The Gulf killifish (Fundulus grandis) has been identified as a sentinel species for the study of site-specific effects of crude oil contamination on biological function. In November and December 2010, 4-5 months after the Macondo well was plugged and new oil was no longer spilling into the Gulf waters, Gulf killifish were collected across the Barataria Basin from 14 sites with varying degrees of oiling. Fish collected from oiled sites exhibited biological indications of exposure to oil, including increase in cytochrome P4501A (CYP1A) mRNA transcript and protein abundances in liver tissues. Immunohistochemistry revealed increases in gill, head kidney, and intestinal CYP1A protein at heavily oiled sites. Intestinal CYP1A protein was a sensitive indicator of exposure, indicating that intestinal tissue plays a key role in biotransformation of AHR ligands and that ingestion is a probable route of exposure, warranting additional consideration in future studies.

Tissue distribution and depuration kinetics of waterborne 14C-labeled light PAHs in mummichog (Fundulus heteroclitus).

Light polycyclic aromatic hydrocarbons (PAHs) of petrogenic origin are commonly found in estuaries and coastal areas. Though they are known to be toxic to fish, little is known about their uptake and tissue distribution. This paper reports on the results of a study on uptake, elimination, and tissue distribution of three waterborne 14C-labeled PAHs in the mummichog, Fundulus heteroclitus, using whole-body autoradiography. After a 24 h exposure to 1 µCi·L(-1) of 14C-naphthalene, 14C-1-naphthol, and 14C-phenanthrene, fish were transferred to clean water and tissue distribution examined after 0, 1, 3, 7, 14, and 21 days of depuration. All compounds were readily accumulated by fish and were also rapidly eliminated (t0.5 range=1.1 to 3.0 days). Most of the radioactivity in naphthalene- and phenanthrene-treated fish was found in gall bladder≫liver>intestinal lumen. In naphthol-exposed fish, an important labeling of some brain areas was observed. Brain of naphthalene-exposed fish was also labeled after 24 h depuration, indicating that exposure to naphthalene may result in metabolite accumulation in the brain. This is the first study showing that naphthalene, naphthol, and/or unidentified metabolite(s) can accumulate in brain tissues, which may impair normal brain function.

Experimental evaluation of evolution and coevolution as agents of ecosystem change in Trinidadian streams.

Evolution has been shown to be a critical determinant of ecological processes in some systems, but its importance relative to traditional ecological effects is not well known. In addition, almost nothing is known about the role of coevolution in shaping ecosystem function. Here, we experimentally evaluated the relative effects of species invasion (a traditional ecological effect), evolution and coevolution on ecosystem processes in Trinidadian streams. We manipulated the presence and population-of-origin of two common fish species, the guppy (Poecilia reticulata) and the killifish (Rivulus hartii). We measured epilithic algal biomass and accrual, aquatic invertebrate biomass, and detrital decomposition. Our results show that, for some ecosystem responses, the effects of evolution and coevolution were larger than the effects of species invasion. Guppy evolution in response to alternative predation regimes significantly influenced algal biomass and accrual rates. Guppies from a high-predation site caused an increase in algae relative to guppies from a low-predation site; algae effects were probably shaped by observed divergence in rates of nutrient excretion and algae consumption. Rivulus-guppy coevolution significantly influenced the biomass of aquatic invertebrates. Locally coevolved populations reduced invertebrate biomass relative to non-coevolved populations. These results challenge the general assumption that intraspecific diversity is a less critical determinant of ecosystem function than is interspecific diversity. Given existing evidence for contemporary evolution in these fish species, our findings suggest considerable potential for eco-evolutionary feedbacks to operate as populations adapt to natural or anthropogenic perturbations.

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase.

Na(+) and Ca(2+) regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na(+) and Ca(2+), whole body Na(+) and Ca(2+), and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na(+) influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na(+) balance was restored by the time killifish reached 100% SW. Whole body Na(+) did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na(+) influx (50-fold relative to FW values), but not Na(+) efflux between 40 and 60% SW, resulting in a large net loading of Na(+) at higher salinities (60-100% SW), and increases in plasma Na(+) and whole body Na(+) at 100% SW. Killifish were in negative Ca(2+) balance at all salinities, whereas trout were in positive Ca(2+) balance throughout. Ca(2+) influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca(2+) efflux. Ca(2+) flux rates were affected to a larger degree in trout, with twofold increases in Ca(2+) influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca(2+) efflux with salinity, so plasma Ca(2+) concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.

Effects of iodothyronines on the hepatic outer-ring deiodinating pathway in killifish.

Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T2, generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T4, T3, and 3,5-T2 (0.1 microM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T4, T3, and 3,5-T2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T2 can affect both activity and transcription of hepatic deiodinases in teleosts.

The liver of Fundulus heteroclitus expresses deiodinase type 1 mRNA.

The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3(') untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.