Molecular responses differ between sensitive silver carp and tolerant bighead carp and bigmouth buffalo exposed to rotenone.

Some species of fish are more tolerant of rotenone, a commonly used non-specific piscicide, than others. This species-specific tolerance to rotenone has been thought to be associated with the uptake and the efficiency at which the chemical is detoxified. However, rotenone stimulates oxidative stress and superoxides, which are also toxic. Understanding the modes in which fish physiologically respond to rotenone is important in developing improved protocols for its application in controlling aquatic nuisance species. Using a molecular approach, we investigated the physiological and molecular mechanisms of rotenone resistance. Species-specific responses were observed when rotenone-sensitive silver, Hypophthalmichthys molitrix, and both rotenone-resistant bighead carp, Hypophthalmichthys nobilis, and bigmouth buffalo, Ictiobus cyprinellus, were exposed to rotenone. Rotenone levels in plasma were highest 90 min after exposure in both silver carp and bigmouth buffalo, but bigmouth buffalo tolerated over twice the burden (ng mL(-1) g(-1)) than silver carp. Expression of genes related with detoxification (cyp1a and gst) increased in silver carp, but either decreased or remained the same in bighead carp. Genes linked with oxidative stress in the cytosol (gpx, cat and sod1) and hsp70 increased only in silver carp after a 6-h exposure. Expression of genes associated with oxidative stress in the mitochondria (sod2 and ucp2) differed between silver carp and bighead carp. Expression of sod2 changed minimally in bighead carp, but expression of ucp2 linearly increased to nearly 85-fold of the level prior to exposure. Expression of sod2 and ucp2 did not change until 6 h in silver carp. Use of sod1 and sod2 to combat oxidative stress results in hydrogen peroxide production, while use of ucp2 produces nitric oxide, a chemical known to inhibit apoptosis. We conclude that the mechanism at which a fish handles oxidative stress plays an important role in the tolerance to rotenone.

Sexually dimorphic gene expression in the gonad and liver of shovelnose sturgeon (Scaphirhynchus platorynchus).

The development of a biomarker for rapid detection of intersex is desirable to researchers and fisheries managers alike. The first step is to identify a marker that has a dimorphic expression pattern. With a focus on sex-specific genes, we assessed the use of vitellogenin (vtg), forkhead box L2 (foxl2) and doublesex and mab-3-related transcription factor 1 (dmrt1) as molecular biomarkers for the identification of gender in shovelnose sturgeon (Scaphirhynchus platorynchus), a species known to have intersex individuals. A total of 61 shovelnose sturgeons were sampled from the Wabash River, Indiana, in April 2008. Results from our study indicate that least 7.5% of the non-females had testicular oocytes. Expression level of liver vtg was not higher in females than males, nor was gonad dmrt1 expression found to be higher in males. Only the expression of foxl2 was significantly greater in females and was found to be useful for identifying gender. Variation in expression levels of foxl2 in gonads of intersex fish limited its usefulness as a single biomarker for identifying this condition. Instead, the use of foxl2 to dmrt1 (foxl2 transcript abundance/dmrt1 transcript abundance) may be useful in the identification of intersex fish, as this ratio increased with increased feminization. We conclude that foxl2 can be used to identify sex, but not intersex sturgeon.

Using silver and bighead carp cell lines for the identification of a unique metabolite fingerprint from thiram-specific chemical exposure.

Conservation biology often requires the control of invasive species. One method is the development and use of biocides. Identifying new chemicals as part of the biocide registration approval process can require screening millions of compounds. Traditionally, screening new chemicals has been done in vivo using test organisms. Using in vitro (e.g., cell lines) and in silico (e.g., computer models) methods decrease test organism requirements and increase screening speed and efficiency. These methods, however, would be greatly improved by better understanding how individual fish species metabolize selected compounds. We combined cell assays and metabolomics to create a powerful tool to facilitate the identification of new control chemicals. Specifically, we exposed cell lines established from bighead carp and silver carp larvae to thiram (7 concentrations) then completed metabolite profiling to assess the dose-response of the bighead carp and silver carp metabolome to thiram. Forty one of the 700 metabolomic markers identified in bighead carp exhibited a dose-response to thiram exposure compared to silver carp in which 205 of 1590 metabolomic markers exhibited a dose-response. Additionally, we identified 11 statistically significant metabolomic markers based upon volcano plot analysis common between both species. This smaller subset of metabolites formed a thiram-specific metabolomic fingerprint which allowed for the creation of a toxicant specific, rather than a species-specific, metabolomic fingerprint. Metabolomic fingerprints may be used in biocide development and improve our understanding of ecologically significant events, such as mass fish kills.

Detecting the movement and spawning activity of bigheaded carps with environmental DNA.

Bigheaded carps are invasive fishes threatening to invade the Great Lakes basin and establish spawning populations, and have been monitored using environmental DNA (eDNA). Not only does eDNA hold potential for detecting the presence of species, but may also allow for quantitative comparisons like relative abundance of species across time or space. We examined the relationships among bigheaded carp movement, hydrography, spawning and eDNA on the Wabash River, IN, USA. We found positive relationships between eDNA and movement and eDNA and hydrography. We did not find a relationship between eDNA and spawning activity in the form of drifting eggs. Our first finding demonstrates how eDNA may be used to monitor species abundance, whereas our second finding illustrates the need for additional research into eDNA methodologies. Current applications of eDNA are widespread, but the relatively new technology requires further refinement.

Environmental hormones and their impacts on sex differentiation in fathead minnows.

Runoff from lands fertilized with animal manure from concentrated animal feeding operations (CAFOs) is a source of hormones to surface water. In this study we tested the hypothesis that larval fathead minnows exposed to sex steroids singly or in a "typical" CAFO mixture during sex differentiation would respond with changes in the expression of a set of target genes, leading to gonadal abnormalities later in life. In the first experiment, a static daily-renewal system was used to expose larvae during the period of 10-20 days post-hatch (dph) to either 5 ng/L 17ß-trenbolone (17ß-TRB) or 5 ng/L 17α-ethinylestradiol (EE2). In a second experiment, fish were exposed from 0 to 45 dph in a flow-through system to a CAFO mixture composed of steroids and degradates (2-16 ng/L), atrazine and degradates (15-250 ng/L), and nitrate (3-11 mg/L). In the single hormone experiment, expression of genes involved in steroidogenesis (cyp19a, cyp17, and star) was decreased in females. In contrast, no differences in gene expression were observed in fish exposed to the CAFO mixture. However, the majority (84%) of treated males had testes containing an ovarian cavity, indicative of feminization, compared to 0% in the control males. Overall, our results show that: (1) changes in gene expression after single hormone exposures are sex-specific, with females more responsive than males; and (2) phenotypic alterations in testicular development can be elicited by a simulated "CAFO" mixture when fathead minnows are exposed during the first 45 days of development. More research is needed to further discern the complex response of fish to steroid mixtures, especially those associated with runoff from land-applied CAFO waste.

Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data.

The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

Blood chemistry values for shovelnose and lake sturgeon.

Blood chemistry panels are commonly used for assessing the general health of vertebrate animals. Here, we present novel blood chemistry data for two North American sturgeon species, shovelnose sturgeon Scaphirhynchus platorynchus and lake sturgeon Acipenser fulvescens. Measurements were done using a portable chemistry analyzer (VetScan Analyzer; Abaxis). Among the plasma values measured (mean ± SD for shovelnose and lake sturgeon, respectively) were total proteins (3.7 ± 0.9 and 2.8 ± 0.4 g/dL), albumin (2.0 ± 0.5 and 1.1 ± 0.2 g/dL), globulin (1.7 ± 0.7 and 1.7 ± 0.3 g/dL), glucose (107 ± 46 and 62 ± 9.7 mg/dL), sodium (Na(+); 132 ± 3.6 and 150 ± 14 mEq/L), potassium (K(+); 3.5 ± 0.2 and 2.8 ± 1.7 mEq/L), phosphorus (10.4 ± 1.9 and 11.6 ± 3.6 mg/dL), and aspartate aminotransferase (AST; 676 ± 433 and 634 ± 234 IU/L). Higher values for total proteins, albumin, glucose, and Na(+) in shovelnose sturgeon than in lake sturgeon probably are the result of handling stress. In addition, the plasma of male shovelnose sturgeon had higher concentrations of AST, glucose, and globulin than did that of females, whereas the plasma of females had higher concentrations of albumin and K(+) than that of males. This study is the first to report blood chemistry data for shovelnose sturgeon. Robust blood chemistry databases can be used by aquaculturists and fish managers for monitoring sturgeon health.

Dietary carbohydrate level affects transcription factor expression that regulates skeletal muscle myogenesis in rainbow trout.

Understanding the effects of dietary carbohydrates on transcription factors that regulate myogenesis provides insight into the role of nutrient sensing by satellite cells towards myocyte differentiation. We evaluated the influence of dietary carbohydrate level (0, 15, 25 or 35%) on the temporal mRNA expression patterns (4, 8 or 12 weeks) of transcription factors that regulate satellite cell myocyte addition (MA) in rainbow trout (Oncorhynchus mykiss), a vertebrate with indeterminate growth. Relative to the 0% carbohydrate (NC) diet, 15 (IC-15) and 25% (IC-25) carbohydrate containing diets significantly up-regulate MyoD and Myf5, but not Pax7, after 12 weeks of feeding. Simultaneously, the Pax7/MyoD mRNA expression ratio declined significantly with both the IC diets. Myogenin mRNA expression also increased in rainbow trout (RBT) fed the IC-15 diet. The high carbohydrate (HC) diet (35%) attenuated the increased mRNA expression of these transcription factors. It is of note that the 4 and 8 week samples lacked the promyogenic expression patterns. The myogenic gene expression in fish fed the IC-15 diet for 12 weeks indicate a transcriptional signature that reflects increased satellite cell myogenesis. Our results suggest a potential role for satellite cells in the nutrient sensing ability of a vertebrate with indeterminate skeletal muscle growth.