Disentangling genetic, plastic and social learning drivers of sex-specific foraging behaviour in Trinidadian guppies (Poecilia reticulata).

Evolutionary biologists have long been interested in parsing out the roles of genetics, plasticity and their interaction on adaptive trait divergence. Since males and females often have different ecological and reproductive roles, separating how their traits are shaped by interactions between their genes and environment is necessary and important. Here, we disentangle the sex-specific effects of genetic divergence, developmental plasticity, social learning and contextual plasticity on foraging behaviour in Trinidadian guppies (Poecilia reticulata) adapted to high- or low-predation habitats. We reared second-generation siblings from both predation regimes with or without predator chemical cues, and with adult conspecifics from either high- or low-predation habitats. We then quantified their foraging behaviour in water with and without predator chemical cues. We found that high-predation guppies forage more efficiently than low-predation guppies, but this behavioural difference is context-dependent and shaped by different mechanisms in males and females. Higher foraging efficiency in high-predation females is largely genetically determined, and to a smaller extent socially learned from conspecifics. However, in high-predation males, higher foraging efficiency is plastically induced by predator cues during development. Our study demonstrates sex-specific differences in genetic versus plastic responses in foraging behaviour, a trait of significance in organismal fitness and ecosystem dynamics.

Insights into adaptive behavioural plasticity from the guppy model system.

Behavioural plasticity allows organisms to respond to environmental challenges on short time scales. But what are the ecological and evolutionary processes that underlie behavioural plasticity? The answer to this question is complex and requires experimental dissection of the physiological, neural and molecular mechanisms contributing to behavioural plasticity as well as an understanding of the ecological and evolutionary contexts under which behavioural plasticity is adaptive. Here, we discuss key insights that research with Trinidadian guppies has provided on the underpinnings of adaptive behavioural plasticity. First, we present evidence that guppies exhibit contextual, developmental and transgenerational behavioural plasticity. Next, we review work on behavioural plasticity in guppies spanning three ecological contexts (predation, parasitism and turbidity) and three underlying mechanisms (endocrinological, neurobiological and genetic). Finally, we provide three outstanding questions that could leverage guppies further as a study system and give suggestions for how this research could be done. Research on behavioural plasticity in guppies has provided, and will continue to provide, a valuable opportunity to improve understanding of the ecological and evolutionary causes and consequences of behavioural plasticity.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Investigation of extragonadal teratomas in two Poecilia wingei.

Teratoma is a rare tumour in fish consisting of tissues from more than one germ layer, that may be located in either the gonads or extragonadal sites. Teratomas in many fish species remain poorly understood. In this work, we performed the first histological examinations of extragonadal teratomas in Poecilia wingei and also examined the influence of a large teratoma on male sexual activity. The studied teratomas shared general organizational features, but they also had variations in both external and internal features. In teratomas, the most common and highly differentiated tissues were striated muscle and nervous tissue. Despite the tumour, the male P. wingei exhibited normal mating behaviour and retained the ability for successful copulation. The structural features of extragonadal teratomas in guppy fish indicate a possible connection between these tumours and a failure of conserved processes operating in the embryonic germline.

Population Regulation and Density-Dependent Demography in the Trinidadian Guppy.

AbstractClassic theory for density-dependent selection for delayed maturation requires that a population be regulated through some combination of adult fecundity and/or juvenile survival. We tested whether those demographic conditions were met in four experimental populations of Trinidadian guppies in which delayed maturation of males evolved when the densities of those populations became high. We used monthly mark-recapture data to examine population dynamics and demography in these populations. Three of the four populations displayed clear evidence of regulation. In all four populations, monthly adult survival rates were independent of biomass density or actually increased with increased biomass density. Juvenile recruitment, which is a combination of adult fecundity and juvenile survival, decreased as biomass density increased in all four populations. Demography showed marked seasonality, with greater survival and higher recruitment in the dry season than the wet season. Population regulation via juvenile recruitment supports the hypothesis that density-dependent selection was responsible for the evolution of delayed maturity in males. This body of work represents one of the few complete tests of density-dependent selection theory.

Interspecific differences in developmental mode determine early cognitive abilities in teleost fish.

Most studies on developmental variation in cognition have suggested that individuals are born with reduced or absent cognitive abilities, and thereafter, cognitive performance increases with age during early development. However, these studies have been mainly performed in altricial species, such as humans, in which offspring are extremely immature at birth. In this work, we tested the hypothesis that species with other developmental modes might show different patterns of cognitive development. To this end, we analysed inhibitory control performance in two teleost species with different developmental modes, the zebrafish Danio rerio and the guppy Poecilia reticulata, exploiting a simple paradigm based on spontaneous behaviour and therefore applicable to subjects of different ages. Zebrafish hatch as larvae 3 days after fertilisation, and have an immature nervous system, a situation that mirrors extreme altriciality. We found that at the early stages of development, zebrafish displayed no evidence of inhibitory control, which only begun to emerge after one month of life. Conversely, guppies, which are born after approximately one month of gestation as fully developed and independent individuals, solved the inhibitory control task since their first days of life, although performance increased with sexual maturation. Our study suggests that the typical progression described during early ontogeny in humans and other species might not be the only developmental trend for animals' cognition and that a species' developmental mode might determine variation in cognition across subjects of different age.

17ß-Trenbolone activates androgen receptor, upregulates transforming growth factor beta/bone morphogenetic protein and Wnt signaling pathways, and induces masculinization of caudal and anal fins in female guppies (Poecilia reticulata).

Sexually mature female guppies (Poecilia reticulata) were exposed to environmentally relevant concentrations (20, 200, and 2000 ng/L) of 17ß-trenbolone for four weeks. As evidenced by the increased caudal fin index and anal fins developing into gonopodium-like structures, exposed females displayed masculinized secondary sexual characteristics. Differential gene expression and subsequent pathway analysis of mRNA sequencing data revealed that the transcription of transforming growth factor beta/bone morphogenetic protein signaling pathway and Wnt signaling pathway were upregulated following 17ß-trenbolone exposure. Enzyme-linked immunosorbent assays showed that the bone morphogenetic protein 7 protein content was elevated after 17ß-trenbolone exposure. Finally, real-time PCR revealed that 17ß-trenbolone treatment significantly increased androgen receptor mRNA levels, and molecular docking showed potent interaction between 17ß-trenbolone and guppy androgen receptor. Furthermore, 17ß-trenbolone-induced masculinization of caudal and anal fins in female guppies, concomitant to the upregulated expression of differentially expressed genes involved in the above-mentioned two signaling pathways, was significantly inhibited by flutamide (androgen receptor antagonist). These findings demonstrated that 17ß-trenbolone masculinized fins of female guppies by activating the androgen receptor. This study revealed that 17ß-trenbolone could upregulate signaling pathways related to fin growth and differentiation, and eventually cause caudal and anal fin masculinization in female guppies.

Adaptive phenotypic plasticity induces individual variability along a cognitive trade-off.

Animal species, including humans, display patterns of individual variability in cognition that are difficult to explain. For instance, some individuals perform well in certain cognitive tasks but show difficulties in others. We experimentally analysed the contribution of cognitive plasticity to such variability. Theory suggests that diametrically opposed cognitive phenotypes increase individuals' fitness in environments with different conditions such as resource predictability. Therefore, if selection has generated plasticity that matches individuals' cognitive phenotypes to the environment, this might produce remarkable cognitive variability. We found that guppies, Poecilia reticulata, exposed to an environment with high resource predictability (i.e. food available at the same time and in the same location) developed enhanced learning abilities. Conversely, guppies exposed to an environment with low resource predictability (i.e. food available at a random time and location) developed enhanced cognitive flexibility and inhibitory control. These cognitive differences align along a trade-off between functions that favour the acquisition of regularities such as learning and functions that adjust behaviour to changing conditions (cognitive flexibility and inhibitory control). Therefore, adaptive cognitive plasticity in response to resource predictability (and potentially similar factors) is a key determinant of cognitive individual differences.

Sex-biased response of pollution biomarkers in fish: Insights from the killifish Poecilia vivipara.

Copper ions (Cu) are one of the most frequent trace-contaminants found in Brazilian waters and, although considered as an essential element, in high concentrations can accumulate and induce toxicity. Biomarkers are important tools that can be used to assess these impacts, but to be considered trustworthy, they have to be previously tested in target organisms through laboratory studies under controlled conditions. However, many of these experiments are conducted using only males, as it is believed that the hormonal variation of females can bias the results, increasing data variability. Notwithstanding, few studies have actually tested this hypothesis, highlighting the importance of considering and measuring the role of sex in ecotoxicological studies. The aim this study was to evaluate the influence of sex on biomarkers classically used in environmental monitoring programs using the fish Poecilia vivipara as model. For this, females and males were exposed for 96 h to two Cu concentrations (9 and 20 µg/L) and a control group. In liver and gills, Cu accumulation, total antioxidant capacity (TAC) and lipid peroxidation (LPO) were evaluated. In addition, samples of peripheral blood were used for neutrophil to lymphocyte ratio determination, a measure of the onset of secondary stress. Results show that Cu hepatic accumulation did not differ between females and males, but higher levels of this metal were observed in exposed animals compared to control fish. Additionally, interactive effects were observed for hepatic LPO, as males showed elevated oxidative damage in comparison to females. Moreover, Cu exposure elevated hepatic LPO relative to control only in males, but this increase in oxidative damage was not accompanied by changes in liver TAC. On the other hand, differences in branchial Cu accumulation and LPO were not observed. Conversely, control females showed elevated TAC in comparison to control males, but Cu exposure eliminated this difference. Cu exposure also induced an increase in the N:L ratio, indicating the presence of a secondary stress response unrelated to sex. Ultimately, the findings of this study demonstrate that sex can influence the response of biomarkers that are typically used in ecotoxicological investigations in a multifaceted manner. As a result, using animals from a singular sex in such studies may result in consequential outcomes, potentially leading to underestimation or overestimation of results.

Female preference for rare males is maintained by indirect selection in Trinidadian guppies.

When females prefer mates with rare phenotypes, sexual selection can maintain rather than deplete genetic variation. However, there is no consensus on why this widespread and frequently observed preference might evolve and persist. We examine the fitness consequences of female preference for rare male color patterns in a natural population of Trinidadian guppies, using a pedigree that spans 10 generations. We demonstrate (i) a rare male reproductive advantage, (ii) that females that mate with rare males gain an indirect fitness advantage through the mating success of their sons, and (iii) the fitness benefit that females accrue through their "sexy sons" evaporates for their grandsons as their phenotype becomes common. Counter to prevailing theory, we show that female preference can be maintained through indirect selection.

Brain monoaminergic activity during predator inspection in female Trinidadian guppies (Poecilia reticulata).

To understand the processes underpinning social decision-making, we need to determine how internal states respond to information gathered from the social environment. Brain monoamine neurotransmitters are key in the appraisal of the social environment and can reflect the internal state underlying behavioural responses to social stimuli. Here we determined the effects of conspecific partner cooperativeness during predator inspection on brain monoamine metabolic activity in Trinidadian guppies (Poecilia reticulata). We quantified the concentration of dopamine, serotonin and their metabolites across brain sections sampled immediately after ostensibly experiencing cooperation or defection from social partners whilst inspecting a predator model, using a familiar object as a control condition. Our results indicate dopaminergic and serotonergic activity differs with the cooperativeness experienced; these different neurotransmission profiles are likely to affect the expression and regulation of downstream behaviours that ultimately contribute to the patterning of cooperative interactions among individuals in a population.

Compiling forty years of guppy research to investigate the factors contributing to (non)parallel evolution.

Examples of parallel evolution have been crucial for our understanding of adaptation via natural selection. However, strong parallelism is not always observed even in seemingly similar environments where natural selection is expected to favour similar phenotypes. Leveraging this variation in parallelism within well-researched study systems can provide insight into the factors that contribute to variation in adaptive responses. Here we analyse the results of 36 studies reporting 446 average trait values in Trinidadian guppies, Poecilia reticulata, from different predation regimes. We examine how the extent of predator-driven phenotypic parallelism is influenced by six factors: sex, trait type, rearing environment, ecological complexity, evolutionary history, and time since colonization. Analyses show that parallel evolution in guppies is highly variable and weak on average, with only 24.7% of the variation among populations being explained by predation regime. Levels of parallelism appeared to be especially weak for colour traits, and parallelism decreased with increasing complexity of evolutionary history (i.e., when estimates of parallelism from populations within a single drainage were compared to estimates of parallelism from populations pooled between two major drainages). Suggestive - but not significant - trends that warrant further research include interactions between the sexes and different trait categories. Quantifying and accounting for these and other sources of variation among evolutionary 'replicates' can be leveraged to better understand the extent to which seemingly similar environments drive parallel and nonparallel aspects of phenotypic divergence.

Skin bacterial microbiome diversity predicts lower activity levels in female, but not male, guppies, Poecilia reticulata.

While the link between the gut microbiome and host behaviour is well established, how the microbiomes of other organs correlate with behaviour remains unclear. Additionally, behaviour-microbiome correlations are likely sex-specific because of sex differences in behaviour and physiology, but this is rarely tested. Here, we tested whether the skin microbiome of the Trinidadian guppy, Poecilia reticulata, predicts fish activity level and shoaling tendency in a sex-specific manner. High-throughput sequencing revealed that the bacterial community richness on the skin (Faith's phylogenetic diversity) was correlated with both behaviours differently between males and females. Females with richer skin-associated bacterial communities spent less time actively swimming. Activity level was significantly correlated with community membership (unweighted UniFrac), with the relative abundances of 16 bacterial taxa significantly negatively correlated with activity level. We found no association between skin microbiome and behaviours among male fish. This sex-specific relationship between the skin microbiome and host behaviour may indicate sex-specific physiological interactions with the skin microbiome. More broadly, sex specificity in host-microbiome interactions could give insight into the forces shaping the microbiome and its role in the evolutionary ecology of the host.

Alarm cues and alarmed conspecifics: neural activity during social learning from different cues in Trinidadian guppies.

Learning to respond appropriately to novel dangers is often essential to survival and success, but carries risks. Learning about novel threats from others (social learning) can reduce these risks. Many species, including the Trinidadian guppy (Poecilia reticulata), respond defensively to both conspecific chemical alarm cues and conspecific anti-predator behaviours, and in other fish such social information can lead to a learned aversion to novel threats. However, relatively little is known about the neural substrates underlying social learning and the degree to which different forms of learning share similar neural mechanisms. Here, we explored the neural substrates mediating social learning of novel threats from two different conspecific cues (i.e. social cue-based threat learning). We first demonstrated that guppies rapidly learn about threats paired with either alarm cues or with conspecific threat responses (demonstration). Then, focusing on acquisition rather than recall, we discovered that phospho-S6 expression, a marker of neural activity, was elevated in guppies during learning from alarm cues in the putative homologue of the mammalian lateral septum and the preoptic area. Surprisingly, these changes in neural activity were not observed in fish learning from conspecific demonstration. Together, these results implicate forebrain areas in social learning about threat but raise the possibility that circuits contribute to such learning in a stimulus-specific manner.

Live fish learn to anticipate the movement of a fish-like robot.

The ability of an individual to predict the outcome of the actions of others and to change their own behavior adaptively is called anticipation. There are many examples from mammalian species-including humans-that show anticipatory abilities in a social context, however, it is not clear to what extent fishes can anticipate the actions of their interaction partners or what the underlying mechanisms are for that anticipation. To answer these questions, we let live guppies (Poecilia reticulata) interact repeatedly with an open-loop (noninteractive) biomimetic robot that has previously been shown to be an accepted conspecific. The robot always performed the same zigzag trajectory in the experimental tank that ended in one of the corners, giving the live fish the opportunity to learn both the location of the final destination as well as the specific turning movement of the robot over three consecutive trials. The live fish's reactions were categorized into a global anticipation, which we defined as relative time to reach the robot's final corner, and a local anticipation which was the relative time and location of the live fish's turns relative to robofish turns. As a proxy for global anticipation, we found that live fish in the last trial reached the robot's destination corner significantly earlier than the robot. Overall, more than 50% of all fish arrived at the destination before the robot. This is more than a random walk model would predict and significantly more compared to all other equidistant, yet unvisited, corners. As a proxy for local anticipation, we found fish change their turning behavior in response to the robot over the course of the trials. Initially, the fish would turn after the robot, which was reversed in the end, as they began to turn slightly before the robot in the final trial. Our results indicate that live fish are able to anticipate predictably behaving social partners both in regard to final movement locations as well as movement dynamics. Given that fish have been found to exhibit consistent behavioral differences, anticipation in fish could have evolved as a mechanism to adapt to different social interaction partners.

Cascading indirect genetic effects in a clonal vertebrate.

Understanding how individual differences arise and how their effects propagate through groups are fundamental issues in biology. Individual differences can arise from indirect genetic effects (IGE): genetically based variation in the conspecifics with which an individual interacts. Using a clonal species, the Amazon molly (Poecilia formosa), we test the hypothesis that IGE can propagate to influence phenotypes of the individuals that do not experience them firsthand. We tested this by exposing genetically identical Amazon mollies to conspecific social partners of different clonal lineages, and then moving these focal individuals to new social groups in which they were the only member to have experienced the IGE. We found that genetically different social environments resulted in the focal animals experiencing different levels of aggression, and that these IGE carried over into new social groups to influence the behaviour of naive individuals. These data reveal that IGE can cascade beyond the individuals that experience them. Opportunity for cascading IGE is ubiquitous, especially in species with long-distance dispersal or fission-fusion group dynamics. Cascades could amplify (or mitigate) the effects of IGE on trait variation and on evolutionary trajectories. Expansion of the IGE framework to include cascading and other types of carry-over effects will therefore improve understanding of individual variation and social evolution and allow more accurate prediction of population response to changing environments.

Assessing effects of predator density and diversity on neophobia in Trinidadian guppies.

Neophobic predator avoidance, where prey actively avoid novel stimuli, is thought to allow prey to cope with the inability to predict predation risk (i.e. uncertainty) while reducing the costs associated with learning. Recent studies suggest that neophobia is elicited as a response to unpredictable and elevated mean predation risk, and is linked to experience with diverse novel cues. However, no research has disentangled the effects of predator density and diversity on neophobia. We conditioned Trinidadian guppies (Poecilia reticulata) to high- or low-diversity predator model treatments paired with high, intermediate, or low concentrations of conspecific alarm cues as a proxy for predator density. We tested behavioural responses to a novel stimulus vs. a water control to determine differences in neophobia among treatments. We found that neophobic shoaling behaviour was shaped by mean risk (predator density). However both density and diversity shaped neophobic freezing, and to a weaker extent, neophobic area use. Our research suggests that predator diversity might elicit neophobic responses in guppies, but only when mean risk is high enough. The relationship between neophobia and components of predation risk is becoming increasingly relevant as ecological uncertainty becomes more prevalent with increasing climate change, anthropogenic impacts, and invasive species.

Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide promote liver toxicity in guppy (Poecilia reticulata): A histochemical and ultrastructural approach.

Citrate functionalized iron oxide nanoparticles (IONPs) are employed for various purposes-including environmental remediation but the interaction of IONPs with aquatic contaminants is poorly understood. Among those, glyphosate-based herbicides are toxic and affect target organs such as the liver. Evaluations of livers of female Poecilia reticulata by exposures to IONPs at a concentration of 0.3 mg/L were performed with association to: (1) 0.65 mg of glyphosate per litter and (2) 1.3 mg of glyphosate per litter of Roundup Original, and (3) glyphosate P.A at 0.65 mg/L. These associations were carried out progressively, after 7, 14, and 21 days. We detected circulatory disturbances, inflammatory responses, activation of the immune system, regressive changes, and progressive responses with changes in the connective tissue and decreased glycogen reserve from days 14 to 21. Ultrastructural changes in the Disse space and microvilli of hepatocytes indicated decreased contact surface area. In general, the damage was time and concentration dependent, increasing from 7 to 14 days and tending to stabilize from 14 to 21 days. Therefore, herbicide-associated IONPs functioned as xenobiotics inducing intense cellular detoxification processes and activation of hepatic immune responses.

Genetic integration of behavioural and endocrine components of the stress response.

The vertebrate stress response comprises a suite of behavioural and physiological traits that must be functionally integrated to ensure organisms cope adaptively with acute stressors. Natural selection should favour functional integration, leading to a prediction of genetic integration of these traits. Despite the implications of such genetic integration for our understanding of human and animal health, as well as evolutionary responses to natural and anthropogenic stressors, formal quantitative genetic tests of this prediction are lacking. Here, we demonstrate that acute stress response components in Trinidadian guppies are both heritable and integrated on the major axis of genetic covariation. This integration could either facilitate or constrain evolutionary responses to selection, depending upon the alignment of selection with this axis. Such integration also suggests artificial selection on the genetically correlated behavioural responses to stress could offer a viable non-invasive route to the improvement of health and welfare in captive animal populations.

Entomological outcomes of cluster-randomised, community-driven dengue vector-suppression interventions in Kampong Cham province, Cambodia.

Cambodia has one of the highest dengue infection rates in Southeast Asia. Here we report quantitative entomological results of a large-scale cluster-randomised trial assessing the impact on vector populations of a package of vector control interventions including larvivorous guppy fish in household water containers, mosquito trapping with gravid-ovitraps, solid waste management, breeding-container coverage through community education and engagement for behavioural change, particularly through the participation of school children. These activities resulted in major reductions in Container Index, House Index, Breteau Index, Pupal Index and Adult Index (all p-values 0.002 or lower) in the Intervention Arm compared with the Control Arm in a series of household surveys conducted over a follow-up period of more than one year, although the project was not able to measure the longer-term sustainability of the interventions. Despite comparative reductions in Adult Index between the study arms, the Adult Index was higher in the Intervention Arm in the final household survey than in the first household survey. This package of biophysical and community engagement interventions was highly effective in reducing entomological indices for dengue compared with the control group, but caution is required in extrapolating the reduction in household Adult Index to a reduction in the overall population of adult Aedes mosquitoes, and in interpreting the relationship between a reduction in entomological indices and a reduction in the number of dengue cases. The package of interventions should be trialled in other locations.