Evaluating the Feasibility and Effectiveness of Removing the Globally Invasive Eastern Mosquitofish Gambusia holbrooki from Lotic Environments.

Effective control measures for small-bodied invasive alien species (IAS) in lotic environments are essential for preserving native biodiversity and ecosystem health. This study integrates modeling and field-based removal data to assess the efficacy of electrofishing in controlling populations of the globally invasive Eastern mosquitofish Gambusia holbrooki across four lowland headwater streams in southern Greece over a one-year period. Results indicated significant reductions in mosquitofish post-removal abundance, although natural recruitment persisted, leading to population suppression rather than eradication. Indeed, our pre-removal population viability analysis suggested a temporary suppression of mosquitofish populations, influenced by factors such as the species' life history and reproductive traits. Furthermore, our study suggests modifications of physical control methods expected to enhance effectiveness. Specifically, the narrow implementation timeframe of the removal actions highlights the need for multiple removal campaigns in consecutive years and for long-term population monitoring, thus aligning with past research. The timing of removal efforts is also critical as it must exploit seasonal variability in fish eradication susceptibility, by its implementation within the best "window of opportunity". Finally, addressing knowledge gaps in the ecological impacts of IAS control methods as well as exploration of novel control and monitoring technologies, are also vital for informed management strategies. This study, by proposing modifications to fish removal planning and methodology, contributes to ongoing efforts for optimal IAS control, particularly in regions where such management approaches are underutilized.

Collective order and group structure of shoaling fish subject to differing risk-level treatments with a sympatric predator.

It is imperative for individuals to exhibit flexible behaviour according to ecological context, such as available resources or predation threat. Manipulative studies on responses to threat often focus on behaviour in the presence of a single indicator for the potential of predation, whereas in the wild perception of threat will probably be more nuanced. Here, we examine the collective behaviour of eastern mosquitofish (Gambusia holbrooki) subject to five differing threat scenarios relating to the presence and hunger state of a jade perch (Scortum barcoo). Across threat scenarios, groups exhibit unique behavioural profiles that differ in the durations that particular collective states are maintained, the probability of transitions between states, the size and duration of persistence of spatially defined subgroups, and the patterns of collective order of these subgroups. Under the greatest level of threat, subgroups of consistent membership persist for longer durations. Group-level behaviours, and their differences, are interconnected with differences in estimates of the underlying rules of interaction thought to govern collective motion. The responses of the group are shown to be specific to the details of a potential threat, rather than a binary response to the presence or absence of some form of threat.

Different patterns of coupled predator-prey dynamics when the same species interact in different locations.

Individual predator and prey species exhibit coupled population dynamics in simple laboratory systems and simple natural communities. It is unclear how often such pairwise coupling occurs in more complex communities, in which an individual predator species might feed on several prey species and an individual prey species might be attacked by several predators. To examine this problem, we applied multivariate autoregressive state-space (MARSS) models to 5-year time-series of monthly surveys of a predatory fish, the eastern mosquitofish (Gambusia holbrooki), and its littoral zone prey species, the least killifish (Heterandria formosa), in three locations in north Florida. The MARSS models were consistent with coupled predator-prey dynamics at two of the three locations. In one of these two locations, the estimated densities of the two species displayed classic predator-prey oscillations. In the third location, there was a positive effect of killifish density on mosquitofish density but no detectable effect of mosquitofish density on killifish density. In all three locations, increased submergent vegetation cover was associated with increased prey density but not increased predator density. Eigenvalues analyses for the joint predator-prey dynamics indicated that one of the cyclic locations had more stable dynamics than the other locations. The three different patterns demonstrate that the dynamics of a pairwise predator-prey interaction emerge not only from the characteristics of the prey and the predator, but also those of the habitat and trophic web in which the predator and prey are embedded.

Widespread psychoactive pollutant augments daytime restfulness and disrupts diurnal activity rhythms in fish.

Pharmaceutical pollution is a major driver of global change, with the capacity to alter key behavioural and physiological traits in exposed animals. Antidepressants are among the most commonly detected pharmaceuticals in the environment. Despite well-documented pharmacological effects of antidepressants on sleep in humans and other vertebrates, very little is known about their ecologically relevant impacts as pollutants on non-target wildlife. Accordingly, we investigated the effects of acute 3-day exposure of eastern mosquitofish (Gambusia holbrooki) to field-realistic levels (nominal concentrations: 30 and 300 ng/L) of the widespread psychoactive pollutant, fluoxetine, on diurnal activity patterns and restfulness, as indicators of disruptions to sleep. We show that exposure to fluoxetine disrupted diel activity patterns, which was driven by augmentation of daytime inactivity. Specifically, unexposed control fish were markedly diurnal, swimming farther during the day and exhibiting longer periods and more bouts of inactivity at night. However, in fluoxetine-exposed fish, this natural diel rhythm was eroded, with no differences in activity or restfulness observed between the day and night. As a misalignment in the circadian rhythm has been shown to adversely affect fecundity and lifespan in animals, our findings reveal a potentially serious threat to the survival and reproductive success of pollutant-exposed wildlife.

A forecast effects of climate change and anthropogenic compounds in Gambusia holbrooki: ecotoxicological effects of salinity and metformin.

Due to global warming and extreme weather events, estuarine and coastal ecosystems are facing sudden fluctuations in salinity. These ecosystems are also threatened by organic and inorganic compounds that increase water pollution. Metformin is an antidiabetic drug commonly used by patients with type-2 diabetes, and an increase in environmental concentration has been recorded. To better understand the impacts of these two stressors on aquatic organisms, this study assessed: 1) the acute (96 h) ecotoxicological effects (antioxidant and biotransformation capacity, oxidative damage, energetic reserves, and protein content, neurotoxicity) induced by a range of metformin concentrations in Gambusia holbrooki under different salinities (17, 24, 31 expressed as Practical Salinity Units - PSU); and 2) the same endpoints after chronic exposure (28 d) under a range of metformin concentrations at a salinity of 17. The results obtained from the acute exposure showed interactions between salinity and metformin in G. holbrooki superoxide dismutase (SOD) activity, body protein, and glycogen (GLY) contents. The results revealed that an increase in salinity can modulate the response of G. holbrooki to metformin. Chronically exposed organisms showed that metformin led to a significant decrease in SOD activity at most of the tested concentrations (0.5, 1.0, and 10 µg/L). In addition, glutathione S-transferases increased and glutathione peroxidase activity decreased significantly at concentrations of metformin of 5 and 10 at the µg/L, respectively. Therefore, overall, metformin can lead to potential oxidative stress in G. holbrooki the highest metformin concentrations tested and the GLY content in G. holbrooki increased after exposure to metformin concentrations of 0.5, 1.0 and 5.0 µg/L. Published studies have already shown that metformin alone can lead to oxidative damage in aquatic species, endangering the biodiversity of aquatic ecosystems. Therefore, additional ecotoxicological studies should be performed to characterize if other metformin concentrations combined with salinity, or other climate change-related factors, might impact non-target species. Standard toxicity bioassays may not be predictive of actual pollutants (e.g. metformin) toxicity under variable environmental conditions, and the investigation of a wider range of exposure conditions could improve the accuracy of chemical risk assessments.

The importance of population heterogeneities in detecting social learning as the foundation of animal cultural transmission.

High levels of within-population behavioural variation can have drastic demographic consequences, thus changing the evolutionary fate of populations. A major source of within-population heterogeneity is personality. Nonetheless, it is still relatively rarely accounted for in social learning studies that constitute the most basic process of cultural transmission. Here, we performed in female mosquitofish (Gambusia holbrooki) a social learning experiment in the context of mate choice, a situation called mate copying (MC), and for which there is strong evidence that it can lead to the emergence of persistent traditions of preferring a given male phenotype. When accounting for the global tendency of females to prefer larger males but ignoring differences in personality, we detected no evidence for MC. However, when accounting for the bold-shy dichotomy, we found that bold females did not show any evidence for MC, while shy females showed significant amounts of MC. This illustrates how the presence of variation in personality can hamper our capacity to detect MC. We conclude that MC may be more widespread than we thought because many studies ignored the presence of within-population heterogeneities.

Male size and reproductive performance in three species of livebearing fishes (Gambusia spp.): A systematic review and meta-analysis.

The genus Gambusia represents approximately 45 species of polyandrous livebearing fishes with reversed sexual size dimorphism (i.e. males smaller than females) and with copulation predominantly via male coercion. Male body size has been suggested as an important sexually selected trait, but despite abundant research, evidence for sexual selection on male body size in this genus is mixed. Studies have found that large males have an advantage in both male-male competition and female choice, but that small males perform sneaky copulations better and at higher frequency and thus may sire more offspring in this coercive mating system. Here, we synthesized this inconsistent body of evidence using pre-registered methods and hypotheses. We performed a systematic review and meta-analysis of summary and primary (raw) data combining both published (n = 19 studies, k = 106 effect sizes) and unpublished effect sizes (n = 17, k = 242) to test whether there is overall selection on male body size across studies in Gambusia. We also tested several specific hypotheses to understand the sources of heterogeneity across effects. Meta-analysis revealed an overall positive correlation between male size and reproductive performance (r = 0.23, 95% confidence interval: 0.10-0.35, n = 36, k = 348, 4,514 males, three Gambusia species). Despite high heterogeneity, the large-male advantage appeared robust across all measures studied (i.e. female choice, mating success, paternity, sperm quantity and quality), and was considerably larger for female choice (r = 0.43, 95% confidence interval: 0.28-0.59, n = 14, k = 43). Meta-regressions found several important factors explaining heterogeneity across effects, including type of sperm characteristic, male-to-female ratio, female reproductive status and environmental conditions. We found evidence of publication bias; however, its influence on our estimates was attenuated by including a substantial amount of unpublished effects, highlighting the importance of open primary data for more accurate meta-analytic estimates. In addition to positive selection on male size, our study suggests that we need to rethink the role and form of sexual selection in Gambusia and, more broadly, to consider the ecological factors that affect reproductive behaviour in livebearing fishes.

Unstable intraguild predation causes establishment failure of a globally invasive species.

Biotic resistance is often posited, but rarely known, to be the cause of invasion failure. Competition and predation are the most frequently identified processes that may prevent or limit the establishment of nonnative species. Interactions between nonnative and native species that involve intraguild predation (IGP) are very common in nature, although theory predicts most IGP systems should be unstable and lead to extinction. If this prediction is true, the frequency of invasion failures due to IGP may be underappreciated because of their fleeting nature and, thus, studies of unstable IGP systems are lacking, despite the opportunities they offer for understanding the factors affecting their unstable dynamics. We investigated a failed invasion involving an IGP relationship. In Florida, the guppy (Poecilia reticulata), a worldwide invader, fails to establish in the presence of eastern mosquitofish (Gambusia holbrooki). We tested whether and how resident mosquitofish cause guppy invasion failure using replicated mesocosm and aquarium trials. Both the predator and competitor components of the IGP relationship were strongly asymmetrical, with large impacts on guppies. We identified two effects, direct consumption of neonates and aggressive interference competition, that limited survival and recruitment. The highly unstable nature of this IGP relationship is the primary cause of the failure of the guppy to establish in Florida. Our study shows that the transient nature of an ephemeral IGP relationship can yield important insights into the underlying causes of invasion failure, including the role of strong biotic resistance.

Impacts of wildfires in aquatic organisms: biomarker responses and erythrocyte nuclear abnormalities in Gambusia holbrooki exposed in situ.

Wildfires are an environmental concern due to the loss of forest area and biodiversity, but also because their role as drivers of freshwater systems contamination by metals. In this context, the fish Gambusia holbrooki was used as a model, deployed for in situ exposure in watercourses standing within a recently burnt area and further assessment of toxic effects. The fish were exposed during 4 days at four different sites: one upstream and another downstream the burnt area and two within the burnt area. Biochemical biomarkers for oxidative stress and damage were assessed. The extent of lipoperoxidative damage was monitored by quantifying malondialdehyde and DNA damage evaluated through erythrocyte nuclear abnormalities observation. Chemical analysis revealed higher metal levels within the burnt area, and exposed fish consistently showed pro-oxidative responses therein, particularly an increase of gill glutathione peroxidase and glutathione reductase activity, the records doubling compared to samples from sites in the unburnt area; also the activity of glutathione-S-transferases comparatively increased (by 2-fold in the liver) in samples from the burnt area, and malondialdehyde was produced twice as much therein and in samples downstream the burnt area reflecting oxidative damage. Consistently, the frequency of erythrocyte nuclear abnormalities was higher at sites within and downstream the burnt area. This study supports the use of sensitive oxidative stress and genotoxicity biomarkers for an early detection of potentially noxious ecological effects of wildfires runoff.

Rapid population increase of the threatened Australian amphibian Litoria aurea in response to wetlands constructed as a refuge from chytrid-induced disease and introduced fish.

Amphibians have declined due to multiple impacts including invasive fish and the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Wetland restoration can be used to increase amphibian populations. However the design of created wetlands must account for threats such as Bd and introduced fish. There have been no attempts on a landscape level to manage these threats with habitat design. Here we monitored the green and golden bell frog (Litoria aurea) in 2.6 ha of constructed wetlands designed to enhance breeding and increase survival through passive mitigation of Bd and exotic fish. We compared the fecundity, adult population sizes, introduced fish occupancy, Bd prevalence and survival rates of frogs in created wetlands (CW) to three control sites to determine if and why the habitat design was successful. Monitoring involved weekly capture-recapture during the austral spring and summer for three L. aurea breeding seasons. We hypothesised that (1) if the CWs were successful in passively limiting fish colonisation, a larger number of breeding events would be detected compared to control sites which are known to be widely colonised by introduced fish. (2) If the wetlands were successful in passively mitigating Bd, then we would observe an equal or greater survival rate and equal to or lower Bd prevalence compared to control wetlands. We observed a 3.3-fold increase in adult population size in CW from season 1 to 2, and the population increased further in season 3.We found strong support for hypothesis (1) and weak support for (2). Based on these results, we conclude that this design was beneficial shortly after their formation primarily due to fish exclusion, but further study is required to determine if these benefits extend long-term. Future amphibian restoration studies are needed to improve the design of wetlands to enhance suppression of Bd.

Comparative gut content analysis of invasive mosquitofish from Italy and Spain.

Eastern mosquitofish (Gambusia holbrooki) are among the most widely introduced freshwater species globally. To gain a better understanding of feeding patterns in non-native populations, and which local factors may influence them at the population level, we carried out gut content analysis on 163 specimens from nine invasive populations in Italy and Spain. Based on previous studies, we predicted that (a) mosquitofish are omnivores with a preference for detritus and cladocerans; (b) they display size- and population-specific differences in gut morphologies and diet, with larger fish feeding more intensively over a wider range of prey items; and (c) some of the variation would be associated with differences in local environmental and climatic factors. Our results confirmed our first prediction, because mosquitofish fed on a variety of diet items, among which detritus and Cladocera dominated. However, not a single diet item was shared among all populations. Congruent with our second prediction, we further identified size- and population-specific differences in the occurrence of some diet items and gut morphologies. However, observed patterns in dietary habits did not seem to be driven by the environmental and climatic variables we had quantified. The fairly variable diet likely aids invasion success and helps explain the ubiquity of invasive mosquitofish across Italy and Spain, as mosquitofish seem to be able to rely on whatever a local habitat provides. We further propose that size-specific differences likely capture the substantial sexual size dimorphism (males are smaller than females), while population-specific differences are likely the result of differences in local prey abundance. The lack of an influence of temperature on dietary habits suggests that mosquitofish feeding ecology may be less impacted by rising temperatures than other freshwater fish species. If true, then this suggests climate change-induced effects may further exacerbate the competitive superiority of mosquitofish over native species in the future.

A statistical method for identifying different rules of interaction between individuals in moving animal groups.

The emergent patterns of collective motion are thought to arise from application of individual-level rules that govern how individuals adjust their velocity as a function of the relative position and behaviours of their neighbours. Empirical studies have sought to determine such rules of interaction applied by 'average' individuals by aggregating data from multiple individuals across multiple trajectory sets. In reality, some individuals within a group may interact differently from others, and such individual differences can have an effect on overall group movement. However, comparisons of rules of interaction used by individuals in different contexts have been largely qualitative. Here we introduce a set of randomization methods designed to determine statistical differences in the rules of interaction between individuals. We apply these methods to a case study of leaders and followers in pairs of freely exploring eastern mosquitofish (Gambusia holbrooki). We find that each of the randomization methods is reliable in terms of: repeatability of p-values, consistency in identification of significant differences and similarity between distributions of randomization-based test statistics. We observe convergence of the distributions of randomization-based test statistics across repeat calculations, and resolution of any ambiguities regarding significant differences as the number of randomization iterations increases.

Embryonic Onset of Sexually Dimorphic Heart Rates in the Viviparous Fish, Gambusia holbrooki.

In fish, little is known about sex-specific differences in physiology and performance of the heart and whether these differences manifest during development. Here for the first time, the sex-specific heart rates during embryogenesis of Gambusia holbrooki, from the onset of the heart rates (HRs) to just prior to parturition, was investigated using light cardiogram. The genetic sex of the embryos was post-verified using a sex-specific genetic marker. Results reveal that heart rates and resting time significantly increase (p < 0.05) with progressive embryonic development. Furthermore, both ventricular and atrial frequencies of female embryos were significantly higher (p < 0.05) than those of their male sibs at the corresponding developmental stages and remained so at all later developmental stages (p < 0.05). In concurrence, the heart rate and ventricular size of the adult females were also significantly (p < 0.05) higher and larger respectively than those of males. Collectively, the results suggest that the cardiac sex-dimorphism manifests as early as late-organogenesis and persists through adulthood in this species. These findings suggest that the cardiac measurements can be employed to non-invasively sex the developing embryos, well in advance of when their phenotypic sex is discernible. In addition, G. holbrooki could serve as a better model to study comparative vertebrate cardiovascular development as well as to investigate anthropogenic and climatic impacts on heart physiology of this species, that may be sex influenced.

Multi-species occupancy modeling provides novel insights into amphibian metacommunity structure and wetland restoration.

A fundamental goal of community ecology is to understand species-habitat relationships and how they shape metacommunity structure. Recent advances in occupancy modeling enable habitat relationships to be assessed for both common and rare species within metacommunities using multi-species occupancy models (MSOM). These models account for imperfect species detection and offer considerable advantages over other analytical tools commonly used for community analyses under the elements of metacommunity structure (EMS) framework. Here, we demonstrate that MSOM can be used to infer habitat relationships and test metacommunity theory, using amphibians. Repeated frog surveys were undertaken at 55 wetland sites in southeastern Australia. We detected 11 frog species from three families (Limnodynastidae, Myobatrachidae, and Pelodryadidae). The rarest species was detected at only one site whereas the most common species was detected at 42 sites (naive occupancy rate 0.02-0.76). Two models were assessed representing two competing hypotheses; the best-supported model included the covariates distance to the nearest site (connectivity), wetland area, presence of the non-native eastern mosquitofish (Gambusia holbrooki), proportion cover of emergent vegetation, an interaction term between Gambusia and emergent vegetation cover, and the proportion canopy cover over a site. Hydroperiod played no detectable role in metacommunity structure. We found species-habitat relationships that fit with current metacommunity theory: occupancy increased with wetland area and connectivity. There was a strong negative relationship between occupancy and the presence of predatory Gambusia, and a positive interaction between Gambusia and emergent vegetation. The presence of canopy cover strongly increased occupancy for several tree frog species, highlighting the importance of terrestrial habitat for amphibian community structure. We demonstrated how responses by amphibians to environmental covariates at the species level can be linked to occupancy patterns at the metacommunity scale. Our results have clear management implications: wetland restoration projects for amphibians and likely other taxa should maximize wetland area and connectivity, establish partial canopy cover, and eradicate Gambusia or provide aquatic vegetation to mitigate the impact of this non-native fish. We strongly advocate the use of MSOM to elucidate the habitat drivers behind animal occupancy patterns and to derive unbiased occupancy estimates for monitoring programs.

Off to new shores: Climate niche expansion in invasive mosquitofish (Gambusia spp.).

AIM: Formerly introduced for their presumed value in controlling mosquito-borne diseases, the two mosquitofish Gambusia affinis and G. holbrooki (Poeciliidae) are now among the world's most widespread invasive alien species, negatively impacting aquatic ecosystems around the world. These inconspicuous freshwater fish are, once their presence is noticed, difficult to eradicate. It is, therefore, of utmost importance to assess their geographic potential and to identify their likely ability to persist under novel climatic conditions. LOCATION: Global. METHODS: We build species distribution models using occurrence data from the native and introduced distribution ranges to identify putative niche shifts and further ascertain the areas climatically suitable for the establishment and possible spread of mosquitofish. RESULTS: We found significant niche expansions into climatic regions outside their natural climatic conditions, emphasizing the importance of integrating climatic niches of both native and invasive ranges into projections. In particular, there was a marked shift toward tropical regions in Asia and a clear niche shift of European G. holbrooki. This ecological flexibility partly explains the massive success of the two species, and substantially increases the risk for further range expansion. We also showed that the potential for additional expansion resulting from climate change is enormous-especially in Europe. MAIN CONCLUSIONS: Despite the successful invasion history and ongoing range expansions, many countries still lack proper preventive measures. Thus, we urge policy makers to carefully evaluate the risk both mosquitofish pose to a particular area and to initiate appropriate management strategies.

Combined effects of rearing and testing temperatures on sperm traits.

Temperature experienced during early development can affect a range of adult life-history traits. Animals often show seemingly adaptive developmental plasticity-with animals reared at certain temperatures performing better as adults at those temperatures. The extent to which this type of adaptive response occurs in gonadal tissue that affects sperm traits is, however, poorly studied. We initially reared male mosquito fish (Gambusia holbrooki) at either 18°C or 30°C, and then measured their sperm reserves as adults. We also looked at the velocity of their sperm, at both the matched and mismatched temperatures. Although males reared at 30°C were larger than those initially reared at 18°C, there was no detectable effect of rearing temperature on absolute sperm number. Sperm swam faster at 30°C than 18°C regardless of the male's rearing temperature. Therefore, we found no evidence of adaptive developmental plasticity. Rearing temperature did, however, significantly influence the relationship between male body size and sperm velocity. Larger males had faster sperm when reared at the warmer temperature and slower sperm when reared at the cooler temperature. This suggests that rearing temperature could alter the relationship between pre-copulatory sexual selection and post-copulatory sexual selection as male size affects mating success. Finally, there was a positive correlation between velocities at the two test temperatures, suggesting that temperature experienced during sperm competition is unlikely to affect a male's relative fertilization success.

A century later: Adaptive plasticity and rapid evolution contribute to geographic variation in invasive mosquitofish.

One century after their introduction to Europe, eastern mosquitofish (Gambusia holbrooki) represent a natural experiment to determine the relative contributions of adaptive plasticity and rapid evolutionary change in creating large-scale geographic variation in phenotypes. We evaluated the population-genetic structure and invasion history based on allele length polymorphisms of 15 nuclear microsatellites, which we quantified for N = 660 individuals from 23 populations sampled in 2013 across the invasive range of G. holbrooki in Europe. We analysed body-shape and life-history variation in N = 1331 individuals from 36 populations, sampled in 2013 and 2017, and tested heritability of phenotypic differences in a subset of four populations using a common-garden experiment. The genetic structure of wild-caught individuals suggested a single introduction for all European mosquitofish, which were genetically impoverished compared to their native counterparts. We found some convergent patterns of phenotypic divergence across native and invasive climatic gradients (e.g., increased body size in colder/more northern populations); however, several phenotypic responses were not consistent between sampling years, pointing towards plastic phenotypes. Our analysis of common-garden reared individuals uncovered moderate heritability estimates only for two measures of male body size (intraclass correlation coefficient, ICC = 0.628 and 0.556) and offspring fat content (ICC = 0.734), while suggesting high levels of plasticity in most other phenotypic traits (ICC ≤ 0.407). Our results highlight the importance of phenotypic plasticity in invasive species during range expansions and demonstrate that strong selective pressures-in this case towards increased body size in colder environments-simultaneously promote rapid evolutionary divergence.

An experimental test for body size-dependent effects of male harassment and an elevated copulation rate on female lifetime fecundity and offspring performance.

Many studies investigate the benefits of polyandry, but repeated interactions with males can lower female reproductive success. Interacting with males might even decrease offspring performance if it reduces a female's ability to transfer maternal resources. Male presence can be detrimental for females in two ways: by forcing females to mate at a higher rate and through costs associated with resisting male mating attempts. Teasing apart the relative costs of elevated mating rates from those of greater male harassment is critical to understand the evolution of mating strategies. Furthermore, it is important to test whether a male's phenotype, notably body size, has differential effects on female reproductive success versus the performance of offspring, and whether this is due to male body size affecting the costs of harassment or the actual mating rate. In the eastern mosquitofish Gambusia holbrooki, males vary greatly in body size and continually attempt to inseminate females. We experimentally manipulated male presence (i.e., harassment), male body size and whether males could copulate. Exposure to males had strong detrimental effects on female reproductive output, growth and immune response, independent of male size or whether males could copulate. In contrast, there was a little evidence of a cross-generational effect of male harassment or mating rate on offspring performance. Our results suggest that females housed with males pay direct costs due to reduced condition and offspring production and that these costs are not a consequence of increased mating rates. Furthermore, exposure to males does not affect offspring reproductive traits.

Effects of waterborne exposure to the antidepressant fluoxetine on swimming, shoaling and anxiety behaviours of the mosquitofish Gambusia holbrooki.

Chemical pollution from pharmaceuticals is increasingly recognized as a major hazard to the aquatic biota. Among the wide variety of pharmaceuticals, fluoxetine (FLX) is one of the most widely prescribed antidepressants, and therefore, it is frequently identified in the aquatic environment. As FLX is designed to alter human behaviour and many physiological pathways are conserved across vertebrates, this drug may affect the behaviour of fish living in FLX-polluted environments. Here, we exposed groups of female mosquitofish Gambusia holbrooki to waterborne FLX for 14 days, under semi-static conditions with daily renewal of test solutions. Following exposure, we conducted a set of behavioural assays in individual fish, aimed at assessing the effects of FLX on their locomotor activity and behavioural responses. We found that FLX impaired swimming behaviour at high concentrations (25 µg/L and 50 µg/L) but not at low concentrations close to environmental levels (1 µg/L and 5 µg/L). When swimming activity was assessed 5 min after transfer of the focal fish to the testing tank, 50 µg/L FLX was the only concentration showing significant effects. However, when the same trials were performed 24 h later, 25 µg/L FLX turned out to be an effect concentration in addition to 50 µg/L. Interestingly, these concentrations would elicit fish plasma concentrations comprised within the range of human therapeutic doses. When subjected to a light/dark preference test, fish showed tendency to remain less time in the dark area at high FLX concentrations, thus suggesting an anti-anxiety response. Shoaling behaviour was not affected by FLX exposure. Our study contributes to the growing body of literature evaluating the effects of FLX on animal behaviour. Regarding the experimental design used in behavioural testing, our findings suggest that focal fish should be subjected to long habituation periods, namely of at least a few hours, in order to better assess the effects of drug exposure.

Effects of water pollution and river fragmentation on population genetic structure of invasive mosquitofish.

We analyzed variation at the GPI-2 locus and eleven microsatellite loci of eastern mosquitofish Gambusia holbrooki populations introduced to the Ebro River (Spain), sampling above and below a dam (Flix Reservoir) where severe chronic pollution has been well documented. Allele frequency changes at the GPI-2 locus in the sites nearest to the polluted sediments agree with previous results from studies in mercury-exposed populations of this highly invasive fish. Genetic distinction of the mosquitofish collected close to the polluted sediments was detected at the GPI locus but also at the presumptive neutral microsatellite loci. Recent migration rates estimated from microsatellites indicated that around 30% of fish collected in a specific location were immigrants from upstream and downstream sources. Such high migration rates probably contribute to the mosquitofish's invasive success and suggest that the consequences on the mosquitofish regional genetic structured of high levels of water toxicants could be mediated by immigration from other sites, but the effect of pollutants on local diversity might be higher than observed here.