Microsatellite loci for fathead minnow (Pimephales promelas)

We describe the cloning and characterization of seven microsatellite loci (six dinucleotide and one tetranucleotide) from fathead minnow (Pimephales promelas). In a sample of 48 fish from a captive population, polymorphism at these loci ranged from 4 to 11 alleles with expected heterozygosities ranging from 0.59 to 0.82. These loci will be extremely useful to researchers currently studying the ecotoxicology, behaviour and population genetics of this species.     

Development and intensity dependence of Ornithodiplostomum ptychocheilus metacercariae in fathead minnows (Pimephales promelas)

Intensity-dependent development of Ornithodiplostomum ptychocheilus metacercariae was studied in fathead minnows (Pimephales promelas) exposed to 0, 20, or 120 cercariae. Subsamples of hosts were necropsied at 2-wk intervals to monitor parasite recruitment, growth, and time to encystment. The complex development of metacercariae within the cranium of minnows involved growth, encystment, and consolidation phases, each of which were affected by intensity. At the end of the growth phase, metacercariae from low-dose fish were 20% longer than those from high-dose fish and the latter took 2-4 wk longer to encyst. At the end of the postencystment consolidation phase (6-8 wk postinfection), the size of metacercariae decreased by approximately 50%. The rate of consolidation was slower in high-dose fish. Our results show that time, intensity, and temperature affect development of O. ptychocheilus. Because metacercariae development and differentiation are linked to infectivity, events occurring in intermediate hosts can potentially impact the structure and size of trematode suprapopulations.     

Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas)

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.     

Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)

The fathead minnow (Pimephales promelas) has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals (EDCs) on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows (n=70) were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean (+/-SE) spawning intervals for the fish in this study were 3.0 and 3.7+/-0.1 days, respectively. The mean number of eggs produced per spawn was 85+/-2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition (gonadosomatic index [GSI], histopathology) and plasma concentrations of vitellogenin and sex steroids (beta-estradiol, testosterone, 11-ketotestosterone). The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma beta-estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of beta-estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations.     

Intensity-dependent alteration of minnow (Pimephales promelas) behavior by a brain-encysting trematode

We examined the relationship between the numbers of brain-encysting trematodes (Ornithodiplostomum ptychocheilus) and the magnitude of altered behaviors in fathead minnows (Pimephales promelas). Because cysts develop within a brain region that integrates visual stimuli with motor response. we evaluated the standard optomotor response (OMR). Monitoring this task involved recording the time minnows spent following a spinning drum, on which alternating black and white stripes had been painted. Minnows were exposed to 0, 5, 20, 120, and 300 cercariae and then their OMR was evaluated at 2-wk postinfection. Surprisingly, only minnows that had high numbers of parasites (155 +/- 31 worms/fish) or low numbers of parasites (3 +/- 3 worms/ fish) differed significantly in their optomotor performance compared with controls. Reduced OMR of heavily infected minnows was positively correlated with reduction in minnow activity. In contrast, reduced OMR in lightly infected minnows was independent of host activity and was likely associated with the rapid development of parasite larvae within the optic tecta. The nonlinear relationship between parasite intensity and effect on host behavior was consistent with an earlier study, but the underlying mechanisms producing this pattern are unknown.     

Effect of population density and feeding rate on the fathead minnow (Pimephales promelas)

Juvenile fathead minnows ( Pimephales promelas ) were stocked in tanks at different densities and different ration levels, in two experiments, to determine effects on final population density, growth and fecundity. Length and weight of fish increased with increased food availability. Egg size was exponentially related to growth and perhaps directly to ration level. High population density appeared to limit growth and gamete development regardless of food abundance. Additionally, water volume appeared to limit numbers (tolerance density) of fish which can be supported in a specific volume of water.     

Thermal acclimation in selected tissues of the fathead minnow, Pimephales promelas rafinesque

Environmental variables besides temperature such as length, weight, sex, and photoperiod were shown to affect the metabolic rate in gill and white muscle tissue. This required statistical adjustment to account for these factors. Oxygen consumption rates for gill tissue were greater following cold acclimation at all determination temperatures. This suggests thermal compensation had occurred. Metabolic rates for white muscle tissue showed inverse compensation between 7 °C and 15 °C, but little compensation was evident between 15 °C and 23 °C. Both gill and white muscle tissue had Q₁₀ values ranging between 1–00 and 2–00. These are lower than values previously recorded for fathead minnows.     

Microsatellite loci for paternity analysis in the fathead minnow,Pimephales promelas (Teleostei: Cyprinidae)

Many recent studies have employed molecular markers to uncover important aspects of mating systems in teleost fishes. The fathead minnow (Pimephales promelas) is a nest‐building North American cyprinid that spawns multiply and exhibits exclusive male parental care. A battery of microsatellite markers was developed to analyse paternity in this species. The seven characterized loci possess four to 31 alleles and expected heterozygosities of 0.455–0.974. In combination, they elicit an exclusion probability of 0.999, a desirable level for paternity analysis. In addition, cross‐amplifications were conducted to test primer efficacy in 13 other taxa, including two congeners.     

Population dynamics of Ornithodiplostomum ptychocheilus metacercariae in fathead minnows (Pimephales promelas) from four northern-Alberta lakes

Annual, seasonal, and interlake variation in prevalence and intensity of Ornithodiplostomum ptychocheilus (Faust) metacercariae was assessed in populations of fathead minnows (Pimephales promelas) collected from 4 lakes in north-central Alberta. Mean metacercariae intensity in young-of-the-year minnows varied extensively (5-123 metacercariae/host) among year, month, and lakes. In 2 of the lakes, prevalence always reached 100%, and mean intensity always peaked in September or October. The high spatial and annual variation in metacercarial recruitment was partly attributable to variation in host size. but variation in water depth, temperature, snail densities, and bird visitation likely also played a role. A laboratory experiment demonstrated that host and metacercariae survival was intensity-independent during a period of simulated winter. Thus, metacercariae recruited in the fall survive until the following spring.     

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT)

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.     

Temporal characterization of growth of fathead minnow (Pimephales promelas) larvae during sublethal hydrogen cyanide exposure

Growth of fathead minnow yolk sac larvae was characterized from changes in dry weight and total content and concentrations of RNA, DNA and protein in fish exposed to a sublethal level of HCN (58 micrograms/l) and in age matched controls. Cyanide toxicosis occurred within 24 hr of exposure as evidenced by significant reductions in protein and RNA content and RNA/DNA ratio of larvae. After 96 hr exposure to HCN, larvae exhibited the same growth rate and protein synthetic rate (RNA/DNA) as control fish. HCN toxicosis and recovery is rapid and at least partial tolerance to HCN develops within 96 hr of exposure in larval fathead minnows.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.     

Swimming performance and energy homeostasis in juvenile laboratory raised fathead minnow (Pimephales promelas) exposed to uranium mill effluent

Research at the Key Lake uranium mill (Saskatchewan, Canada) suggests effluent discharged from the mill affects energy stores of resident fish, but the mechanisms by which energy homeostasis is affected and the subsequent effects on swimming performance are unknown. In the present study larvae were collected from laboratory raised adult fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). Critical swimming speed (U_crit) was significantly lower in effluent exposed 60 dph fish compared to control fish. Fish used in tests were considered fatigued and compared to fish without swim testing (non-fatigued). There were no differences in whole body glycogen or triglyceride concentrations between effluent exposed versus control fish. However, fatigued fish from both treatments had significantly lower triglycerides, but not glycogen, compared to non-fatigued fish from the same treatment. Whole body β–hydroxyacyl coenzymeA dehydrogenase activity was similar in fish from both treatments, but citrate synthase activity was significantly lower in effluent exposed fish. Our results suggest uranium mill effluent exposure in the laboratory affects aerobic energy metabolism and swimming performance in juvenile fathead minnow, which could affect wild fish survivability.     

Impact of nest substrate limitations on patterns of illegitimacy in the fathead minnow, Pimephales promelas (Cypriniformes: Cyprinidae)

The fathead minnow (Pimephales promelas) is a nest-building North American cyprinid that spawns multiply and exhibits exclusive male parental care. Here, we examined the effects of nest substrate availability on the genetic mating system in 2 natural populations from the glacial pothole region of eastern South Dakota. The sites differed dramatically with respect to the type and density of available nest substrate. Substrate effects were examined by comparing the percentage and distribution of illegitimate offspring between the 2 sites. A total of 1338 embryos from 42 nests (21 from each site) were genotyped at 4 or 5 microsatellite loci. Bimodally distributed frequencies of illegitimate offspring occurred in the nests of guardian males from both sites and indicate the presence of nest takeovers as well as previously undocumented alternative reproductive tactics. As predicted, the rate of usurped nests, a previously documented behavior, was higher in the nest substrate-limited population; furthermore, males were significantly larger here and acquired more mates, suggesting a difference in sexual selection regime. Although these results are based on a single comparison between 2 sites, the results are intriguing and provide a foundation for future studies.     

Histological evaluation of gonadal differentiation in fathead minnows (Pimephales promelas)

The timing of sex determination and the pattern of sex differentiation have not been studied in fathead minnow even though this species of fish are commonly used as a research model for toxicological studies. In this study, the developmental histology of gonadal development was investigated. Fish were cultured in the laboratory conditions and spawning obtained at a photoperiod of 16 h-light and 8 h-dark. Samples were collected from day 7 fish post-spawning (day 7 fps) to day 150 fps and their gonads were processed for histological examination. Developmental histology was assessed by using a light microscopy. The results showed that ovarian differentiation normally occurs at around day 13 fps, while testicular differentiation normally occurs at around day 22 fps.     

Cellular aspects of early development of Ornithodiplostomum ptychocheilus metacercariae in the brain of fathead minnows, Pimephales promelas

Trematode metacercariae typically are regarded as nonfeeding and metabolically inactive. However, the metacercariae of many trematode species undergo complex and prolonged periods of development within their intermediate hosts. In the present study, we used electron microscopy to document chronological changes in development of the tegument of Ornithodiplostomum ptychocheilus metacercariae recovered from the brains of experimentally infected fathead minnows (Pimephales promelas). Commencing at 4 days postinfection (PI), the smooth, thin, syncytial tegument transforms into a complex microlamellar and microvillar system that encircles the entire body surface. The microvilli are oriented in parallel in an extended pattern, reaching directly away from the parasite and toward the receding host tissue. The microvilli disappear at approximately 28 days PI, followed by deposition of the cyst wall and further transformation of the tegument into the spinose, a glandular structure typical of an immature adult. To our knowledge, the progressive disaggregation of host cells at the leading edge of elongating parasite microvilli has not been demonstrated previously for any trematode. These results provide morphological evidence that the metacercariae of some trematode species undergo complex developmental changes associated with feeding in their intermediate host.     

The effects of temperature and dissolved oxygen on antioxidant defences and oxidative damage in the fathead minnow Pimephales promelas

Fathead minnows Pimephales promelas maintained at 25° C for 6 h had significantly higher superoxide dismutase (SOD) activity than fish maintained at 7 or 32° C, but hypoxic conditions (3 mg l^?1 O₂) over the same time period did not affect SOD activity. Fish in better body condition (length‐adjusted mass) had higher SOD activity. In a separate experiment, P. promelas maintained at three water temperatures (7, 23 and 32° C) for 31 days did not differ in liver acrolein, a biomarker of oxidative stress.