Mating under the influence: male Siamese fighting fish prefer EE2-exposed females.

Countless pharmaceuticals and endocrine disrupting chemicals (EDCs) exist on the market with more added each day. Many of these compounds are not removed during the wastewater treatment process and enter bodies of water in their active form. EDCs are known to have physiological and behavioral effects in exposed organisms. Exposure to the synthetic estrogen 17α-ethinylestradiol (EE2), a common EDC found in birth control pills, has been found to lead to population collapse after only a few generations in some fish species. Mechanisms identified as potential driving forces for collapse include feminization of males and altered fecundity in both sexes. However, an additional way in which EE2 could lead to population collapse is by altering courtship behavior, which could then change mating preferences and decrease mating opportunities. The current study had the following objectives: determine if exposing female Siamese fighting fish, Betta splendens, to EE2 changes mate choice in males; assess if the dose and duration of female exposure matters; and examine if exposing males to EE2 influences their mating preferences. Both unexposed and exposed males were presented with pairs of females that differed in EE2 dose and exposure duration. The results indicate that males were more responsive to EE2-exposed females than unexposed females, with males being most responsive to females exposed to the low versus high dose. Furthermore, exposed males responded less overall than unexposed males. If EE2 concentration increases in the environment, the likelihood of successful mating could decrease and, therefore, potentially lead to adverse population impacts.

Risky business: Changes in boldness behavior in male Siamese fighting fish, Betta splendens, following exposure to an antiandrogen.

Components of boldness, such as activity level and locomotion, influence an individual's ability to avoid predators and acquire resources, generating fitness consequences. The presence of endocrine disrupting chemicals (EDCs) in the aquatic environment may affect fitness by changing morphology or altering behaviors like courtship and exploration. Most research on EDC-generated behavioral effects has focused on estrogen mimics and reproductive endpoints. Far fewer studies have examined the effects of other types of EDCs or measured non-reproductive behaviors. EDCs with antiandrogenic properties are present in waterways yet we know little about their effects on exposed individuals although they may produce effects similar to those caused by estrogen mimics because they act on the same hormonal pathway. To examine the effects of antiandrogens on boldness, this study exposed male Siamese fighting fish, Betta splendens, to a high or low dose of one of two antiandrogens, vinclozolin or flutamide, and observed behavior in three boldness assays, both before and after exposure. Overall, antiandrogen exposure increased boldness behavior, especially following exposure to the higher dose. Whether or not antiandrogen exposure influenced boldness, as well as the nature and intensity of the effect, was assay-dependent. This demonstrates the importance of studying EDC effects in a range of contexts and, at least within this species, suggests that antiandrogenic compounds may generate distinct physiological effects in different situations. How and why the behavioral effects differ from those caused by exposure to an estrogen mimic, as well as the potential consequences of increased activity levels, are discussed. Exposure to an antiandrogen, regardless of dose, produced elevated activity levels and altered shoaling and exploration in male Siamese fighting fish. These modifications may have fitness consequences.

The bachelorette: Female Siamese fighting fish avoid males exposed to an estrogen mimic.

Due to improper disposal and a lack of removal during the wastewater treatment process, endocrine disrupting chemicals enter aquatic ecosystems where they exert detrimental effects on fish behavior and physiology. Perhaps the most well-studied and prevalent EDC is 17α-ethinylestradiol (EE2), an active ingredient in oral contraceptives, which is known to cause dramatic reductions in male-typical behaviors. While it is likely that alterations in male courtship behavior decrease reproductive fitness, this is rarely explicitly examined. To this end, whether EE2 exposure reduces male attractiveness to female Siamese fighting fish, Betta splendens, was investigated by showing females video images of exposed and unexposed males. Females were randomly assigned to one of two exposure conditions (exposed to EE2, control) and each subject then viewed four different video combinations of male conspecifics (courting exposed+exposed; courting unexposed+unexposed; courting unexposed+exposed; swimming unexposed+exposed). Females, regardless of whether or not they were exposed to EE2, directed markedly less behavior towards exposed males, especially when they viewed an exposed male and an unexposed male simultaneously. These findings demonstrate that EE2 can have significant individual- and population-level consequences on fitness by disrupting sexual selection and, ultimately, the success of exposed males.

Dose-dependent fluoxetine effects on boldness in male Siamese fighting fish.

As the use of pharmaceuticals and personal care products (PPCPs) continues to rise, these compounds enter the environment in increasing frequency. One such PPCP, fluoxetine, has been found in detectable amounts in aquatic ecosystems worldwide, where it may interfere with the behavior of exposed organisms. Fluoxetine exposure has been found to influence boldness and exploration in a range of fish species; however, how it might alter behavior in multiple contexts or over time is rarely examined. To this end, the effects of fluoxetine on boldness over time were studied in male Siamese fighting fish. Three different groups of males (0, 0.5 and 5 µg l(-1) fluoxetine) were tested in multiple boldness assays (empty tank, novel environment and shoal) once a week for 3 weeks to collect baseline measures and then at three different time points post-exposure. The effects of these varying exposure amounts on behavior were then examined for overall response, consistency and across-context correlations. Unexposed males were bolder in all contexts, were more consistent within a context, and had stronger between-context correlations than exposed males. Fluoxetine had dose-dependent effects on behavior, as males that received the higher dose exhibited greater behavioral effects. This study stresses the potential fitness consequences of fluoxetine exposure and suggests that examining behavioral effects of PPCPs under different dosing regimens and in multiple contexts is important to gain an increased understanding of how exposure affects behavior.

Fluoxetine exposure impacts boldness in female Siamese fighting fish, Betta splendens.

The present study examined the effects of the selective serotonin reuptake inhibitor, fluoxetine, on the behavior of female Siamese fighting fish, Betta splendens, in three different boldness assays (Empty Tank, Novel Environment, Social Tendency). When females were unexposed to fluoxetine, boldness was consistent within a context and correlated across assays. Fluoxetine exposure affected behavior within and among individuals on multiple levels. Exposure reduced overall boldness levels, made females behave in a less consistent manner, and significantly reduced correlations over time and across contexts. Fluoxetine exerted its effects on female Betta splendens behavior in a dose-dependent fashion and these effects persisted even after females were housed in clean water. If fluoxetine exposure impacts behaviors such as exploration that are necessary to an individual's success, this may yield evolutionary consequences. In conclusion, the results show that fluoxetine exposure alters behavior beyond the level of overall response and highlights the importance of studying the behavioral effects of inadvertent pharmaceutical exposure in multiple contexts and with different dosing regimes.

Acute exposure to 17α-ethinylestradiol disrupts audience effect on male-female interactions in Betta splendens.

Endocrine disrupting chemicals can negatively impact the morphology and behavior of organisms inhabiting polluted waters. Male-typical behaviors are often reduced after exposure, suggesting that exposure may have population-level effects. One way in which exposure may exert population-level effects is by interfering with communication within a network of individuals. Acute exposure to the estrogen mimic 17α-ethinylestradiol (EE2) disrupts the ability of male Siamese fighting fish, Betta splendens, to modify their behavior during male-male interactions when an audience is present. However, it is unknown whether audience effects during male-female interactions may be similarly altered. To examine this, male-female pairs that were given an acute exposure to EE2 or remained unexposed interacted in the presence of a female, male, or no audience. Sex differences were found between unexposed males and females. More interactant-directed gill flaring was displayed by control males when a male audience was present while control females performed this behavior more in the presence of an audience, regardless of sex. Both males and females in the control group performed more interactant-directed tail beats in the presence of a female audience. EE2 exposure made all audience effects disappear as treated males and females did not differ in their responses between audience types. These results demonstrate that acute exposure to EE2 may disrupt behavioral adjustments to audience type within a social network. This disruption may, in turn, influence population dynamics in this species as both males and females use information obtained from observing interactions in later encounters with the observed individuals.

Acute exposure to 17α-ethinylestradiol alters boldness behavioral syndrome in female Siamese fighting fish.

The role of anthropogenic sources in generating, maintaining, and influencing behavioral syndromes has recently been identified as an important area of future research. Endocrine disrupting chemicals are prevalent and persistent in aquatic ecosystems worldwide. These chemicals are known to have marked effects on the morphology and behavior of exposed individuals and, as such, may serve as a potential influence on behavioral syndromes. However, both the effects of exposure on behaviors beyond courtship and aggression and how exposure might affect behavioral variation at the individual level are understudied. To address this question, we examined boldness behavior in female Siamese fighting fish in three different assays (Novel Environment, Empty Tank, Shoaling) both before and after they were exposed to the estrogen mimic, 17α-ethinylestradiol (EE2). EE2 influences courtship, aggression, and boldness in males of this species but its effects have not been examined in females, to our knowledge. Females were tested multiple times in each assay before and after exposure so that behavioral consistency could be examined. A behavioral syndrome for boldness and activity level occurred across the three assays. The reductions in boldness and loss of the behavioral syndrome that resulted from EE2 exposure were surprising and suggest that the effects of EE2 exposure on female behavior and physiology should be examined more frequently. This study is one of the first to examine the effects of EE2 in females as well as on correlated behaviors and emphasizes the importance of examining the effects of endocrine disrupting chemicals on individual behavioral variation and consistency.

Acute exposure to 17α-ethinylestradiol disrupts audience effects on male-male interactions in Siamese fighting fish, Betta splendens.

Endocrine disrupting chemicals can have profound effects on the behavior of aquatic organisms residing in polluted waters. Males are especially sensitive to the effects of estrogen mimics and both courtship and aggression may be dramatically reduced by chemical exposure. Population-level impacts may occur if these chemicals decrease the ability of males to obtain mates or defend territories. Exposure might also have far-reaching impacts by interfering with information transfer within a network of individuals. For example, males exposed to an endocrine disruptor may be less sensitive to the presence of an audience. Male Siamese fighting fish were used to examine how short-term exposure to 17α-ethinylestradiol (EE2) alters audience effects on male-male interactions. Males either received a nominal dose of EE2 or remained unexposed and then interacted with an opponent in one of three treatments (female, male, or no audience). EE2 altered audience effects in this study. Opponent-directed gill flaring was lower when a female audience was present compared to when there was a male or no audience in both EE2 and control males. The number of opponent-directed tail beats did not differ as a function of audience type in EE2 males. In contrast, unexposed males increased opponent-directed tail beats when a female audience is present. Therefore, EE2 reduces the ability of males to communicate with multiple individuals simultaneously. If this is the case, endocrine disruptor exposure may alter population structure as selection should favor individuals that are able to readily adjust their signaling behavior as a function of social context.

The effects of short-term exposure to an endocrine disrupter on behavioral consistency in male juvenile and adult Siamese fighting fish.

17α-Ethinyloestradiol (EE2) is known to impact courtship and aggression but how exposure affects the consistency with which individuals express these behaviors over time is not commonly addressed. In addition, how juvenile and adult male fish differ in levels of behavioral consistency, both before and after EE2 exposure, is unknown. To examine these questions, juvenile and adult male Siamese fighting fish were presented with a dummy male and dummy female simultaneously both before and after acute exposure to a nominal (15 ng/L) dose of EE2. Multiple trials were conducted to measure how consistent individual differences are affected by age and EE2 exposure. Both female- and male-directed behaviors decreased after short-term exposure to EE2. Juvenile and adult fish differed in the number of female-directed tail beats and bites they performed, with adult fish performing more tail beats and juvenile fish performing more bites. EE2 exposure had a greater effect on consistent individual differences in female-directed than in male-directed behaviors, which may be a byproduct of intense artificial selection for aggression in this species. Repeatability values were lower both before and after exposure in juvenile than in adult fish, suggesting that individuals become more consistent with age. This study suggests that male Siamese fighting fish vary in their sensitivity to short-term EE2 exposure and stresses the importance of examining behavior over multiple time points both within and across age classes to gain a more thorough understanding of the effects of endocrine disruptors on behavior.

Fluoxetine alters behavioral consistency of aggression and courtship in male Siamese fighting fish, Betta splendens.

The detrimental effects of steroid-mimics are well known but investigations on non-steroid pharmaceuticals are less common. In addition, most behavioral studies do not examine the effects at multiple time points. This study examined the effects of fluoxetine, a selective serotonin reuptake inhibitor, on behavior when male Siamese fighting fish encounter female and male dummy conspecifics simultaneously. Thus, how chemical exposure impacts behavioral consistency and whether individuals differ in their sensitivity to exposure was assessed. Overall aggression was reduced after fluoxetine administration while courtship was unaffected. Fluoxetine affected behavioral consistency towards both the male and female, with individuals behaving less consistently to the male and more consistently to the female. In addition, males appeared to differ in their sensitivity to fluoxetine exposure as not all males reduced their aggression after administration. This has important implications for studying the effects of unintended pharmaceutical exposure. Exposure may have evolutionary implications as it may influence both territorial defense and mating success. In sum, these findings demonstrate that pharmaceutical exposure may alter more than just overall level of behavior and stress the importance of examining the effects of exposure on an individual level.

Short-term exposure to an endocrine disruptor affects behavioural consistency in male threespine stickleback.

It has been established that exposure to xenooestrogens can have profound effects on reproductive and aggressive behaviours in aquatic organisms. Studies on the effects of 17α-ethinyloestradiol (EE2) on behaviour, however, rarely examine these effects over multiple trials, across time, or across contexts. In addition, it is possible that individuals vary in their sensitivity to EE2 and individuals may differ in the degree to which their behaviour is affected. The study of consistent individual differences in behaviour has grown in popularity in recent years perhaps because of the evolutionary questions this behavioural variation raises. Adult male threespine stickleback (Gasterosteus aculeatus) from an anadromous population repeatedly received the simultaneous presentation of a dummy male and dummy female conspecific both before and after exposure to a short-term, nominal (15 ng L(-1)) concentration of 17α-ethinyloestradiol (EE2). These before and after measures were then compared against one another as well as against responses of males in a control group that was not exposed to EE2. Courtship, aggression, and nesting behaviours were all affected by EE2 exposure. In addition, behavioural consistency over trials was markedly reduced after EE2 exposure, as shown by lower repeatability values in the after exposure measures and in weaker correlations between the before and after exposure measures in the EE2 group compared to the control group. This suggests that male threespine stickleback vary in their sensitivity to EE2. This study is one of the first to examine the effects of short-term EE2 exposure on behaviour across trials and has important implications for population-level fitness consequences of xenooestrogens.

The effects of stimulus type on consistency of responses to conflicting stimuli in Siamese fighting fish.

Individuals must often decide among a range of behavioral responses in a given situation and individuals will likely vary from one another in the choices they make. A rarely explored example of this is when both a male and a female are present simultaneously and a male must decide to fight or court. Consistent individual differences have been found in decision-making in Siamese fighting fish; however, dummies were used to explore this question and it is possible that individuals respond differently in this situation when faced with live stimuli as a result of feedback. Therefore, this study investigated whether males behave similarly to dummy and live stimuli by exposing them to male and female conspecifics both simultaneously and separately. Subjects were tested first with dummies and then in the same situations using live conspecifics. It was hypothesized that males would respond in the same manner to both the dummy and live fish. The results indicated that individuals behaved similarly to the dummy fish and the live fish in corresponding conditions. In addition, repeatability levels suggest that individuals behaved similarly across trials of a given treatment type while differing from one another in their responses. This study demonstrates that males are consistent in their responses even when receiving feedback from conspecifics and supports the continued use of dummy stimuli as an effective tool.

Male 11-ketotestosterone levels change as a result of being watched in Siamese fighting fish, Betta splendens.

This study investigated the effects of nesting status and the presence of an audience on 11-ketotestosterone (11KT) levels in male Siamese fighting fish, Betta splendens. Prior studies have demonstrated that both nesting status, an indicator of territory-holding power and reproductive state, and the sex of a conspecific audience lead to differences in male behavior during aggressive encounters. Since behavioral changes have already been demonstrated, we chose to investigate whether 11KT levels were also influenced by nesting status and audience presence as 11KT both stimulates, and is stimulated by, reproductive and aggressive behaviors in male teleosts. Male 11KT levels were measured from water samples taken from containers holding fish both before and after interaction. Males interacted under three treatment conditions: no audience, female audience, and male audience. Within these treatments were two nest paradigms: both males had nests or neither male had a nest. 11KT levels varied depending on nesting status and audience type. In general, 11KT levels were lower in interacting males when a female audience was present or when males had nests. Overall, 11KT showed increases or decreases as aggression increased or decreased, as shown by already established behavioral findings [see Dzieweczynski T.L., Green T.M., Earley R.L., Rowland W.J., 2005. Audience effect is context dependent in Siamese fighting fish, Betta splendens. Behav. Ecol. 16, 1025-1030; Doutrelant, C., McGregor, P.K., Oliveira, R.F., 2001. Effect of an audience on intrasexual communication in male Siamese fighting fish (Betta splendens). Behav. Ecol. 12, 283-286.]. Our results suggest that 11KT levels are influenced by reproductive status, as indicated by nest ownership, and audience presence and are most likely modulated by territorial behavior and social environment.