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.     

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.     

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.     

Fathead minnows (Pimephales promelas) learn to recognize chemical stimuli from high-risk habitats by the presence of alarm substance

We exposed fathead minnows (Pimephales promelas) to water fromone of two distinct habitat types (an open water site or a vegetatedcover site in the same stream) that we mixed with either alarmsubstance or a distilled water control. Upon subsequent exposuresto the habitat waters alone, minnows showed a fright responseto the habitat water that they received in conjunction withalarm substance but not to the other habitat water. These resultsindicate that minnows can learn to recognize high-risk habitatsbased on the association of habitat specific chemical cues withalarm substance. The ability to recognize these habitats couldpotentially lower the minnows' risk of predation. These resultsprovide evidence of a long-term benefit to receivers of a chemicalalarm signal.     

Effects of exposure to predatory cues on territorial behaviour of male fathead minnows

We conducted a laboratory study to determine if male fathead minnows, Pimephales promelas, altered their territorial behaviour associated with reproduction in response to combinations of visual and chemical cues from northern pike, Esox lucius. We introduced the following stimuli to a territorial male: a brick (control), fathead minnow alarm pheromone, a pike fed brook stickleback, Culea inconstans, or a pike fed fathead minnow. The territorial behaviour of males did not change when the control was added. Male minnows experiencing threat from pike fed stickleback significantly reduced the frequency at which they performed three territorial behaviours, but, within 12 h, had returned to pre-exposure activity levels. Male minnows subjected to alarm pheromone alone and to pike fed fathead minnow significantly reduced their territorial behaviour, abandoned their nests, and did not return to pre-exposure levels of activity after 24 h. We suggest that because risk of predation triggers prolonged decreases in territorial defense, it may affect competition between nesting males and female mate choice. We conclude that fathead minnows can assess the severity of predatory threat and adjust their reproductive behaviour accordingly.     

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.     

Effects of northern pike on patterns of nest use and resproductive behavior of male fathead minnows in a boreal lake

We conducted a two-part study to assets predator avoidance byreproductive male fathead minnows (Pimephales promelos) subjectedto predation threat from northern pike (Esox lucius). First,we determined if patterns of nest use by egg-guarding male minnowsin a boreal lake were related to pike densities. We samplednorthern pike and identified four areas of "high pike-density"and three areas of "low pike-density." We censused natural nestsand placed nest boards in these areas. We found eggs on naturalnests more frequently in areas with low densities of pike thanin areas with high densities of pike. However, we could notfully explain the distribution of nests by predation risk. Second,we evaluated the behavioral response of egg-guarding males toa control stimulus (a piece of wood) or a live pike in a wirecage. We used time to return to the nest after a stimulus asa measure of risk taking. Males took different amounts of riskbased on predation threat; males in the predator treatment tooklonger to return to their nests than control males. Risk takingwas not related to the number or age of the eggs but to distanceto nearest egg-guarding neighbor; males with close neighborsreturned sooner than more isolated males. Males in the predatortreatment had lower total activity and egg rubbing than controlmales after they returned to their nests. We conclude that malefathead minnows altered their reproductive behavior in waysthat reduced predation risk, but the cost of predator avoidancemight include egg predation, lost mating opportunities, or usurpationof nests     

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.     

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.     

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.     

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.     

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.     

Ultrastructural observations on fertilization and sporulation in Goussia iroquoina (Molnar and Fernando, 1974) in experimentally infected fathead minnows (Pimephales promelas, Cyprinidae)

The ultrastructural features of fertilization and sporogony of Eimeria iroquoina are described from the intestinal epithelium of experimentally infected fathead minnows (Pimephales promelas). Intact microgametes were observed in the cytoplasm of macrogametes. Within immature macrogametes the microgamete was segregated from the cytoplasm of the former by the plasma membrane of each cell plus additional membranes. Within mature macrogametes, only the plasma membranes separated the gametes. Fertilization by fusion of the limiting membrane of both gametes occurred after the entire microgamete lay within the cytoplasm of the macrogamete. The cytoplasm of the zygote cleaved into sporoblasts within cisternae of endoplasmic reticulum. The sporocystic wall was composed of an outer electron-lucent layer and an inner, thicker layer with periodic striations at right angles to the surface of the sporocyst. The sporocysts were bivalved and joined by a continuous suture. The sporozoites were morphologically similar to sporozoites and merozoites of other Coccidia. Due to the structure of the sporocyst, Eimeria iroquoina Molnar and Fernando, 1974 is amended to Goussia iroquoina (Molnar and Fernando, 1974).     

Fitness trade-offs and the maintenance of alternative male morphs in the bulb mite (Rhizoglyphus robini)

Alternative reproductive phenotypes (ARPs) occur across a wide range of taxa. Most ARPs are conditionally expressed in response to a cue, for example body size, that reliably correlates with the status of the environment: individuals below the (body size) threshold then develop into one morph, and individuals above the threshold develop into the alternative morph. The environmental threshold model provides a theoretical framework to understand the evolution and maintenance of such ARPs, yet no study has examined the underlying fitness functions that are necessary to realize this. Here, we empirically examined fitness functions for the two male morphs of the bulb mite (Rhizoglyphus robini). Fitness functions were derived in relation to male size for solitary males and in relation to female size under competition. In both cases, the fitness functions of the two morphs intersected, and the resulting fitness trade-offs may play a role in the maintenance of this male dimorphism. We furthermore found that competition was strongest between males of the same morph, suggesting that fitness trade-off in relation to male size may persist under competition. Our results are a first step towards unravelling fitness functions of ARPs that are environmentally cued threshold traits, which is essential for understanding their maintenance and in explaining the response to selection against alternative morphs.     

Gene-class analysis of expression patterns induced by psychoactive pharmaceutical exposure in fathead minnow (Pimephales promelas) indicates induction of neuronal systems

Psychoactive pharmaceuticals are among the most frequently prescribed drugs, contributing to persistent measurable concentrations in aquatic systems. Typically, it is assumed that such contaminants have no human health implications because they exist in extremely low concentrations. We exposed juvenile fathead minnows (Pimephales promelas) to three pharmaceuticals, fluoxetine, venlafaxine and carbamazepine, individually and in a mixture, and measured their effect on the induction of gene expression in fish brains using microarray analysis. Gene expression changes were accompanied by behavioral changes and validated by qPCR analysis. Gene Set Enrichment Analysis was used to perform gene-class analysis of gene expression, testing for enrichment of gene sets known to be involved in human neuronal development, regulation and growth. We found significant enrichment of gene sets for each of the treatments, with the largest induction of expression by the mixture treatment. These results suggest that the psychoactive pharmaceuticals are able to alter expression of fish genes associated with development, regulation and differentiation of synapses, neurons and neurotransmitters. The results provide a new perspective for the consideration of potential consequence for human health due to environmental exposure to unmetabolized psychoactive pharmaceuticals.     

An experimental approach to altering mating tactics in male horseshoe crabs (Limulus polyphemus)

Alternative reproductive tactics are often correlated with phenotype, density, environment, or social context. Male horseshoe crabs(Limulus polyphemus) have two mating tactics that are associatedwith phenotype. Males in good condition arrive at the nestingbeach and spawn while attached to females, whereas those inpoorer condition come ashore unattached and crowd around thenesting couples as satellites, fertilizing eggs through sperm competition. The correlation between mating tactic and phenotypemay be due to males choosing tactics based on condition, orit may be that males that have not found a female choose tocome ashore as satellites. To distinguish between these twopossibilities, I conducted an experiment on male horseshoe crabsin the field at Seahorse Key on the northern Gulf coast ofFlorida. I prevented males from attaching to females by placingsmall plastic bags over the claws they use to attach. The resultsshowed that males in poor condition came ashore as satellites,whereas males in good condition remained at sea. This meansthat mating tactics are cued by information about the male'scondition and not about whether he found a female. The evolutionof phenotype-correlated mating tactics can be represented bya model in which the fitness of each tactic changes with conditionand fitness curves cross. I hypothesize that male horseshoecrabs in good condition have higher fitness when attached and that males in poorer condition to better when unattached.