An Ultrastructural Study of Eimeria iroquoina Molnar & Fernando, 1974 in Experimentally Infected Fathead Minnows (Pimephales promelas,Cyprinidae) 3. Merogony1

Fathead minnows, Pimephales promelas, raised from eggs in the laboratory, were experimentally infected with oocysts of Eimeria iroquoina from either P. promelas or the common shiner, Notropis cornutus. Within intestinal epithelial cells, trophozoites thought to be derived from the sporozoites contained a prominent electron-dense refractile body. Merozoites dedifferentiated into trophic forms by losing components of their apical complex and pellicle. The inner membrane components of the pellicle appeared discontinuous, and the micronemes became enclosed within vacuoles. Prior to merozoite formation, multinucleate meronts were limited by a single membrane. Golgi complexes were associated with the nuclei of this stage. Merozoites were formed by ectomerogony in one generation and by endomerogony in the final generation. In both forms of merogony the final nuclear division was coupled with the onset of differentiation of the merozoites and featured eccentric mitotic spindles associated with centrocones located within the nuclear envelope and with the precursors of the apical complex. A Golgi complex was closely associated with the nucleus and apical tip of the forming merozoite. Unlike other Eimeria species, the complete pellicle of the merozoites of the final asexual generation of E. iroquoina was formed within the cytoplasm of the meront, without association with the limiting membrane, thus, all pellicular components are synthesized de novo. The inner membranes of the pellicle initially appeared as longitudinal strips, each of which was associated with a pair of the 22–24 subpellicular microtubules. Mature meronts of the final asexual generation averaged 9 μm in diameter and produced 13–16 merozoites. With the exception of the internal completion of the pellicle of the final generation merozoites, the basic processes of merogony in fish Eimeria species are similar to those recorded in terrestrial hosts.     

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.     

Effects of social structure on reproductive activity in male fathead minnows (Pimephales promelas)

The selection of alternative reproductive phenotypes is oftenthought to be the result of physiological state, with smallindividuals forced energetically to postpone the allocationof resources to reproduction. However, for male fathead minnows(Pimephales promelas), we show that seasonal reproductive activityis modulated by social status. In enclosure and pond experiments,small males advanced their reproductive condition, held nesting territories, and spawned earlier in the reproductive seasononly when large males were absent or removed from the population.Since differences in the timing of reproduction among smallmales were not size- or condition-dependent, the common explanationfor the selection of alternative reproductive phenotypes, basedon state-dependence, is insufficient. In the absence of large,socially dominant individuals, small males produced comparablenumbers of offspring as the treatment with large males, although the offspring of these uninhibited small males were smallerat the end of the growing season than the young of large males.Thus, interactions among conspecifics may account for muchof the phenotypic diversity observed within and among naturalfathead minnow populations, through their direct and indirecteffects on growth, recruitment and survival.     

Toxic effects of zinc on fathead minnows Pimephales promelas in soft water

A fathead minnow life-cycle exposure to various zinc concentrations demonstrated that the most sensitive indicators of zinc toxicity were egg adhesiveness and fragility, which were significantly affected at 145 μg Zn 1⁻¹ and above, but were not affected at 78 μg Zn 1⁻¹ and below. These effects occurred shortly after the eggs were spawned (during water hardening) and therefore were not related to effects on the parental fish. Hatchability and survival of larvae were significantly reduced, and deformities at hatching were significantly increased at 295 ug Zn 1⁻¹ and above. Acclimated and unacclimated groups of larvae exposed to identical zinc concentrations for 8 weeks after hatch showed only slight differences in sensitivity.     

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.     

Oxygen consumption in fathead minnows (Pimephales promelas) following acute exposure to water-borne selenium

Following determination of a concentration of selenate-Se lethal during 24-hr exposures at 22 degrees C, routine oxygen consumption was measured in fathead minnows (Pimephales promelas) exposed to various sublethal concentrations of Se for 24 hr. A median lethal concentration for 24-hr exposures (24-hr LC50) of fathead minnows equalled 82 mg Se/l with 95% fiducial limits of 76-89 mg Se/l. Mean weight-specific oxygen consumption rates of minnows exposed to 0, 15, 40 and 60 mg Se/l for 24-hr ranged from 0.43 to 0.49 mg O2/g-1/hr-1 and were not significantly different. These results indicate that acute exposure to Se does not affect oxygen consumption; however, observations during respirometry trials suggest that minnows at the highest Se concentration may have physiologically compensated to maintain oxygen consumption rates.     

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.     

Time dependent cadmium uptake by fathead minnows (Pimephales promelas) during field and laboratory exposure

Fathead minnows (Pimephales promelas) exposed to 2.5 ppb cadmium in an industrially contaminated lake accumulated whole body burdens of 3.19 ppm Cd in 12 hours, while minnows exposed to 48 ppb Cd in a continuous flow laboratory system did not reach comparable levels until the eighth day of exposure. Significant whole body accumulation of Cd (approximately 9 ppm) peaked within 17–20 days of exposure in the laboratory.     

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.     

The role of experience and chemical alarm signalling in predator recognition by fathead minnows, Pimephales promelas

Young-of-the-year, predator-naive fathead minnows, Pimephales promelas , from a pikesympatric population did not respond to chemical stimuli from northern pike, Esox Indus , while wild-caught fish of the same age and size did. These results suggest that chemical predator recognition is a result of previous experience and not genetic factors, Wild young-of-the-year minnows responded to pike odour with a response intensity that was similar to that of older fish, demonstrating that the ability to recognize predators is learned within the first year. The intensity of response of wild minnows which had been maintained in a predator free environment for 1 year was similar to that of recently caught minnows of the same age, suggesting that reinforcement was not required for predator recognition to be retained. Naive minnows that were exposed simultaneously to chemical stimuli from pike (a neutral stimulus) and minnow alarm substance exhibited a fright response upon subsequent exposure to the pike stimulus alone. Predator-naive minnows exposed simultaneously to chemical stimuli from pike and glass-distilled water did not exhibit a fright response to the pike stimulus alone. These results demonstrate that fathead minnows can acquire predator recognition through releaserinduced recognition learning, thus confirming a known mechanism through which alarm substance may benefit the receivers of an alarm signal.     

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.     

Predator-prey behaviour of fathead minnows, Pimephales promelas and largemouth bass, Micropterus salmoides in a model ecosystem

The predator-prey behaviours displayed by largemouth bass, Micropterus salmoides and fathead minnows, Pimephales promelas , were investigated. Fathead PRMOWS were subjected to predation by largemouth bass and observed in a model system that was designed to mimic a natural ecosystem. Anti-predator devices utilized by fathead minnows include schooling, surface swimming, formation of a circular aggregation, and seeking refuge under vegetation.     

Influence of metal concentrations, percent salinity, and length of exposure on the metabolic rate of fathead minnows (Pimephales promelas)

Understanding the effects of chemical toxicants on energetic processes is an important aspect of ecotoxicology. However, the influence of toxicant concentration and time of exposure on metabolism in aquatic organisms is still poorly understood. The purpose of this investigation was to determine the influence of increasing levels of three stressors (Cu, Cd, percent salinity) and exposure time (24 h and 96 h) on the metabolic rate of fathead minnows (Pimephales promelas). In all 24-h exposures, there existed a threshold concentration, above which metabolic rate decreased significantly compared to the control and lower concentrations. In contrast, the metabolic rate of fish exposed for 96 h increased significantly in all concentrations compared to fish from the control. We suggest fathead minnows exhibit a consistent pattern of metabolic response to stressors, regardless of the physiological mechanisms involved, and that this response differs as a function of time of exposure.     

The uptake of Manganese-54 by green algae (Protococcoidal chlorella), Daphnia magna,and fathead minnows (Pimephales promelas)

Concentration factors (CF) of ⁵⁴Mn for three aquatic species: green algae (Protococcoidal chlorella), Daphnia magna, and fathead minnows (Pimephales promelas) were determined following direct exposure to the isotope in solution. The maximum accumulation (CF = 911) in P. chlorella was reached at 48 hours of exposure; the maximum uptake (CF = 65) in Daphnia was reached at 8 hours of exposure; and the maximum accumulation (CF = 22.6) in fathead minnows was at 128 hours of exposure. The data indicate that ⁵⁴Mn accumulation decreases with ascent up a theoretical aquatic food chain when water is the only source of contamination.     

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.     

Effects of acute and chronic waterborne lead exposure on the swimming performance and aerobic scope of fathead minnows (Pimephales promelas)

Fathead minnows were subjected to an incremental velocity test using swim tunnel respirometry for the analysis of aerobic scope and swimming performance, as critical aerobic swim speed (U(crit)), following chronic exposures (33-57 ) to 0.9±0.4, 157±18 or 689±66 nmol L?1 Pb and an acute exposure (24 h) to 672±35 nmol L?1 Pb (mean±SEM). Assessment of Pb-induced anemia and neurological impairment were evaluated by blood hemoglobin (Hb) concentrations and a cost of transport (COT) analysis, respectively. Fish from the acute 672±35 nmol L?1 Pb (24.4±1.2 BL s?1) and chronic 689±66 nmol L?1 Pb (24.6±0.9 BL s?1) treatments exhibited reduced U(crits) compared to control fish (27.6±0.8 BL s?1). Aerobic scope was reduced by acute Pb exposure (8.6±2.6 μmol O? g?1 h?1 vs. 22.6±3.8 μmol O? g?1 h?1 from controls) owing to a decrease in maximum oxygen consumption rate (38.8±0.8 μmol O? g?1 h?1 vs. 54.0±4.2 μmol O? g?1 h?1 from controls). However, no effect on aerobic scope was observed with fish chronically exposed to Pb. Significant differences were not observed for Hb concentrations or COT. These findings suggest that the impaired swimming performances arising from acute and chronic Pb exposures reflect different mechanisms of toxicity.     

Chemical alarm signals increase the survival time of fathead minnows (Pimephales promelas) during encounters with northern pike (Esox Lucius)

We tested the hypothesis that exposure to a conspecific alarmpheromone improves survival of fathead minnows (Pimephales promelas)during staged encounters with an unfamiliar predator (northernpike: Esox luaus). Minnows exposed to the alarm pheromone survived39. 5% longer than controls. This difference in survival timeappeared to result not from direct inhibition of the pike butrather from some aspect of the minnows' antipredator behavior.Minnows exhibited significant increases in both shoaling andshelter use after exposure to the alarm pheromone. For controlminnows, the degree of shoaling was positively correlated withsurvival time, suggesting that increased shoaling is an effectiveantipredator response. This study provides the first directexperimental evidence that chemical alarm signals in fishesimprove survival of receivers.     

Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole

The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and seven genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology.