Towards the conservation of the crucian carp in Europe: Prolific hybridization but no evidence for introgression between native and non-native taxa.

Hybridization plays a pivotal role in evolution, influencing local adaptation and speciation. However, it can also reduce biodiversity, which is especially damaging when native and non-native species meet. Hybridization can threaten native species via competition (with vigorous hybrids), reproductive resource wastage and gene introgression. The latter, in particular, could result in increased fitness in invasive species, decreased fitness of natives and compromise reintroduction or recovery conservation practices. In this study, we use a combination of RAD sequencing and microsatellites for a range-wide sample set of 1366 fish to evaluate the potential for hybridization and introgression between native crucian carp (Carassius carassius) and three non-native taxa (Carassius auratus auratus, Carassius auratus gibelio and Cyprinus carpio) in European water bodies. We found hybridization between native and non-native taxa in 82% of populations with non-natives present, highlighting the potential for substantial ecological impacts from hybrids on crucian carp populations. However, despite such high rates of hybridization, we could find no evidence of introgression between these taxa. The presence of triploid backcrosses in at least two populations suggests that the lack of introgression among these taxa is likely due to meiotic dysfunction in hybrids, leading to the production of polyploid offspring which are unable to reproduce sexually. This result is promising for crucian reintroduction programs, as it implies limited risk to the genetic integrity of source populations. Future research should investigate the reproductive potential of triploid hybrids and the ecological pressures hybrids impose on C. carassius.

Divergent Response to the SSRI Citalopram in Male and Female Three-Spine Sticklebacks (Gasterosteus aculeatus).

Selective serotonin reuptake inhibitors (SSRIs) are psychotropic pharmaceuticals used as antidepressants. SSRIs are commonly found in surface waters in populated areas across the globe. They exert their effect by blocking the serotonin re-uptake transporter in the presynaptic nerve ending. The present study examined whether behavioural effects to exposure to SSRI citalopram depend on personality and sex in the stickleback (Gasterosteus aculeatus). Three aspects of stickleback behaviour are examined: feeding behaviour, aggression, and boldness. We exposed sticklebacks to 350-380 ng/l citalopram for 3 weeks. Feeding and aggressive behaviour were recorded before and after exposure, whereas scototaxis behaviour was tested after exposure. The results show treatment effects in feeding and aggressive behaviour. Feeding is suppressed only in the male group (χ2 = 20.4, P < 0.001) but not in the females (χ2 = 0.91, P = 0.339). Aggressive behaviour was significantly affected by treatment (χ2 = 161.9, P < 0.001), sex (χ2 = 86.3, P < 0.001), and baseline value (χ2 = 58.8, P < 0.001). Aggressiveness was suppressed by citalopram treatment. In addition, the fish showed no change in aggression and feeding behaviour over time regardless of sex and treatment, which indicate personality traits. Only females are affected by treatment in the scototaxis test. The exposed females spent significantly (χ2 = 5.02, P = 0.050) less time in the white zone than the female controls.

Exposure to carbamate fungicide iodocarb does not affect reproductive behavior or milt volumes in precocious male brown trout (Salmo trutta L.) parr.

Previous studies with olfactory-disturbing pesticides resulted after exposure in disturbed behavior and physiology in fish. In the present experiment, reproductive behavior and milt volumes of precocious brown trout (Salmo trutta L.) male parr were studied in a large stream aquarium after exposure to the olfactory-disturbing fungicide 15 µg l-1 IPBC (iodocarb; 3-iodo-2-propynyl butyl carbamate) for 96 h. The statistical analyses did not reveal any significant differences for time attending females between controls and IPBC-exposed males. Furthermore, there were no significant differences in milt volumes. However, when taking all fish into consideration, there were significant differences in milt volumes between parr that had been attending females and those had not been attending females. Controls that had attended females had significantly higher milt volumes than controls or IPBC-exposed males that had not attended females. Taking all control and IPBC parr into consideration, there was a statistically significant positive correlation between time attended females and volume of milt and gonadosomatic index (GSI), respectively. In summary, 15 µg l-1 IPBC did not have any significant effects on mature male parr reproductive behavior and milt volumes.

Comparing RADseq and microsatellites to infer complex phylogeographic patterns, an empirical perspective in the Crucian carp, Carassius carassius, L.

The conservation of threatened species must be underpinned by phylogeographic knowledge. This need is epitomized by the freshwater fish Carassius carassius, which is in decline across much of its European range. Restriction site-associated DNA sequencing (RADseq) is increasingly used for such applications; however, RADseq is expensive, and limitations on sample number must be weighed against the benefit of large numbers of markers. This trade-off has previously been examined using simulation studies; however, empirical comparisons between these markers, especially in a phylogeographic context, are lacking. Here, we compare the results from microsatellites and RADseq for the phylogeography of C. carassius to test whether it is more advantageous to genotype fewer markers (microsatellites) in many samples, or many markers (SNPs) in fewer samples. These data sets, along with data from the mitochondrial cytochrome b gene, agree on broad phylogeographic patterns, showing the existence of two previously unidentified C. carassius lineages in Europe: one found throughout northern and central-eastern European drainages and a second almost exclusively confined to the Danubian catchment. These lineages have been isolated for approximately 2.15 m years and should be considered separate conservation units. RADseq recovered finer population structure and stronger patterns of IBD than microsatellites, despite including only 17.6% of samples (38% of populations and 52% of samples per population). RADseq was also used along with approximate Bayesian computation to show that the postglacial colonization routes of C. carassius differ from the general patterns of freshwater fish in Europe, likely as a result of their distinctive ecology.

Sperm from pheromone primed brown trout (Salmo trutta L.) produce more larvae.

Male goldfish (Carassius auratus) exposed to female hormonal pheromones express increased milt volumes and their sperm fertilize more eggs than sperm from unprimed males. Ovulated salmonid females also release odours that increase volumes of strippable milt in males. It is, however, not known if the priming pheromones affect the ability of sperm to fertilize eggs in salmonids. In this study, we compare the proportion of larvae produced from in vitro fertilization tests between primed brown trout (Salmo trutta) males exposed to a mix of female urine and ovarian fluids, and control males exposed only to 0.9 % sodium chloride. We also investigate priming effects on milt yield and sperm motility. Fertilization tests with sperm from single males, as well as sperm from two males (i.e., sperm competition), were performed. Primed males generated more larvae in both the single male and competition fertilization tests. No differences between treatments in milt yield and sperm motility could be established.

Anxiogenic behaviour induced by 17α-ethynylestradiol in male guppies (Poecilia reticulata).

Behaviour studies are used in toxicology research as they are excellent tools to measure physiological end-points caused by exogenous chemicals. In mammals both reproductive and non-reproductive behaviours have been used for a long period of time, whereas in teleost fishes non-reproductive behaviours have received little attention compared to reproductive behaviours. Recent advances in measuring stress related behaviours in zebrafish have provided additional tools to understand behaviour toxicology in fish. One species with well documented reproductive behaviour disturbed by different toxicants is the guppy, which is better suited than zebrafish for reproductive behaviour studies and therefore might be a better model organism for comparative behaviour studies in fish toxicology. Here we report new applications for non-reproductive behaviours in guppy and test these behaviours on males treated with the endocrine disruptor 17α-ethynylestradiol at environmentally relevant concentrations. 17α-ethynylestradiol increased freezing and bottom-dwelling when fish were placed in a non-familiar aquarium, but did not significantly affect shoaling behaviour. These results are similar to the anxiogenic behaviours seen in rats treated perinatally with 17α-ethynylestradiol and add more concern to the impacts of endocrine disruptors on aquatic wildlife.

Effects on guppy brain aromatase activity following short-term steroid and 4-nonylphenol exposures.

Brain estrogen production, performed by the enzyme aromatase, can be disrupted/affected in teleost fish exposed to endocrine disruptors found in polluted aquatic environments. The guppy (Poecilia reticulata) was previously studied and confirmed to suffer negative effects on reproductive behaviors following inhibition of the brain aromatase reaction. Here adult guppies (Poecilia reticulata) of both genders were subjected to known endocrine disruptors: the androgen androstenedione (A), the synthetic estrogen 17alpha-ethinylestradiol (EE(2)), and the estrogenic surfactant 4-nonylphenol (NP), at high (50 microg/L) and at environmentally relevant concentrations (10 ng/L EE(2), 5 microg/L NP, and 0.7 microg/L A) for 2 weeks followed by measurements of brain aromatase activity (bAA). In the adult males, bAA was stimulated by A and EE(2) at 50 microg/L. Female activity was also stimulated by the higher estrogenic treatment. At environmentally relevant concentrations only the EE(2) treatment affected bAA, and only in males. The alkylphenolic substance NP produced no effect in either of the experiments, not on males nor females. The results indicate that short-term steroid treatments have stimulatory effects on guppy brain aromatase even at concentrations that can be found in the environment. We thus suggest bAA of adult guppies to be a suitable bioindicator of endocrine disruptors.

Effects of a pyrethroid pesticide on endocrine responses to female odours and reproductive behaviour in male parr of brown trout (Salmo trutta L.).

Reproductive behaviour of brown trout (Salmo trutta L.) from an anadromous stock was studied in a large stream water aquarium. Four adult males and two ovulated females were placed in the aquarium together with eight mature male parr. Four of the parr were exposed during the previous 4 days to two concentrations (0.1 or 1.0 microgl(-1)) of the pyrethroid pesticide cypermethrin (a disrupter of olfactory receptor function) and four of the parr to the solvent ethanol. The behaviour of all fish was followed for 24h and then blood and milt was collected. Exposure to the higher concentration of cypermethrin disturbed the reproductive behaviour of the parr. They displayed fewer courting events, spent less time near the nesting females and had lower volumes of strippable milt. They also had significantly lower amounts of 11-ketotestosterone (11-KT) in the blood plasma than the control group. The higher cypermethrin group also had significantly lower levels of all these variables than the lower cypermethrin group, apart from strippable milt that showed no significant differences between two groups. No significant differences in non-reproductive behaviours were observed between any of the groups. In the control fish, there were significant positive correlations between (a) the number of courting events and the amount of time spent near the female, (b) blood plasma levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and time spent near the female and (c) plasma levels of 17,20beta-P and the number of courting events. Further, in control fish, higher plasma levels of 17,20beta-P were observed in parr interacting with a female compared to those with no female contacts. A priming experiment confirmed a previous study that cypermethrin damages olfactory reception. Parr exposed to cypermethrin had significantly lower blood plasma levels of 17,20beta-P and 11-KT than control males after exposure to ovarian fluid and urine (known to contain reproductive priming pheromones). When ethanol-exposed males were exposed to ovarian fluid and urine they had significantly higher plasma levels of 17,20beta-P compared to those exposed to water only.

Responses of Acilius sulcatus (Coleoptera: Dytiscidae) to chemical cues from perch (Perca fluviatilis).

In this study we tested the hypothesis that the presence of chemical stimuli from a hungry predator would initiate anti-predator responses, while stimuli from a satiated predator would not. We used chemical stimuli released from starved perch (Perca fluviatilis) and from satiated perch (predator). As prey we used adult Acilius sulcatus (Coleoptera: Dytiscidae). The reaction of the beetles to different predator conditions was tested during daytime. We also tested the reaction to starved perch during the night. A. sulcatus activity decreased when it was exposed to stimuli released from starved perch during daytime when visibility was poor, due to the presence of artificial vegetation. There was, however, no reaction to satiated perch under the same experimental conditions. These results indicate that A. sulcatus can discriminate between chemical cues from hungry and satiated fish predators. When visibility was good and the concentration of chemical cues was constant, the beetles did not react to starved perch in the daytime, but their activity decreased at night in response to stimuli released from starved perch. Visual as well as chemical cues seem to be important for detecting a potential predator. When visibility is good, beetles seem to rely on visual stimuli, while in darkness they seem to use chemical stimuli to detect the presence of predators.

Effects of the SSRI citalopram on behaviours connected to stress and reproduction in Endler guppy, Poecilia wingei.

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1 µg L(-1). In two experiments exposing both sexes to 0.2, 2.3 or 15 µg L(-1) for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15 µg L(-1). They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15 µg L(-1) significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

Reprint of "Effects of the SSRI citalopram on behaviours connected to stress and reproduction in Endler guppy, Poecilia wingei".

Psychoactive drugs, such as selective serotonin reuptake inhibitors (SSRI) have been identified in high levels in effluents from Swedish sewage treatment plants (STP) at concentrations high enough to give pharmacological effects in fish. In humans SSRIs are used in the treatment of depression and they have anxiolytic effects. In the present study we exposed Endler guppy (Poecilia wingei) of both sexes to citalopram that showed the highest concentrations of SSRIs in STP effluents and studied reproductive and non-reproductive behaviour. Male courting behaviours were not affected compared to control fish after 14-28 days exposure to 1 µgL(-1). In two experiments exposing both sexes to 0.2, 2.3 or 15 µgL(-1) for 21 days, fish exposed to the two highest doses showed anxiolytic effects when placed in a novel environment (novel tank diving test, NT). Males were only affected by exposure to 15 µgL(-1). They had significantly longer latency to explore the upper half of the aquarium, more visits and longer time spent in the upper half, and showed less bottom freezing behaviour, all markers of anxiolytic behaviour. In females exposure to 2.3 or 15 µgL(-1) significantly increased freezing behaviour, while no effects on other behaviour variables were observed. No effects on shoaling behaviour could be discerned. These results show that citalopram have anxiolytic effects on guppy fish and thus affect ecologically relevant behaviours of importance to survival of fish.

Inhibition of cytochrome p450 brain aromatase reduces two male specific sexual behaviours in the male Endler guppy (Poecilia reticulata).

In mammalian and avian vertebrate groups, androgens act as controlling agents on male aggression and courtship behaviour by their conversion to oestrogens by cytochrome P450 aromatase in well-defined brain regions. Despite the fact that bony fishes have exceptionally high brain aromatase activity, little is known about it's possible regulatory effects on the reproductive behaviours of teleosts. In this study, Endler guppy males (Poecilia reticulata) were subjected to 26-29 days of 24-h exposure to two different concentrations (15 and 100 microg/L) of the aromatase inhibitor fadrozole in the water. Compared with the control males, two of three courtship activities in males exposed to the higher concentration were reduced when they were paired with receptive stimulus females. Reduction in brain aromatase activity was confirmed in both exposed groups with the use of the tritiated water assay.

Mature atlantic salmon (Salmo salar L.) male parr are attracted to ovulated female urine but not to ovarian fluid.

The behavioral responses to urine and ovarian fluids from conspecific and heterospecific ovulated females were studied in mature Atlantic salmon (Salmo salar) male parr in a two-choice fluviarium. The males reacted differently to the stimulants. They spent more time in water scented by urine from salmon or brown trout (Salmo trutta L.) females compared to the time in water with ovarian fluids from salmon females. Furthermore, the males were attracted to salmon female urine (different from an indifferent reaction). Males exposed to urine of either species had higher plasma concentrations of testosterone (T) compared to unexposed controls. Measurement of the concentrations of prostaglandin F2alpha (PGF2alpha) and its major metabolite 15-ketodihydroprostaglandin F2alpha (15-ketodihydro-PGF2alpha) showed that the concentrations of the substances were higher in ovarian fluids of both species compared to those in urine. PGF2alpha showed a greater difference between ovarian fluid and urine than its major metabolite. The results suggest that urine of both species, in contrast to ovarian fluid, contain substances that attract mature Atlantic salmon male parr and that the active substances are neither PGF2alpha nor 15-keto-PGF2alpha.