Female Three-spined Sticklebacks Research Articles

Evaluating Dermal Bone as a Novel Source of Endocrine Information in Ninespine and Threespine Stickleback Fish.

Monitoring the physiology of small aquatic and marine teleost fish presents challenges. Blood samples, often the first choice for endocrinologists, can be difficult or even impossible to obtain and alternative matrices currently used for hormone analyses do not occur in fishes (e.g., hair, feathers etc.) or are not easily collected from small aquatic organisms (e.g., urine and feces). Some teleosts, however, have enlarged bony dermal elements that possibly accumulate and store steroid hormones in physiological relevant concentrations. Both threespine stickleback (Gasterosteus aculeatus) and ninespine stickleback (Pungitius pungitius) have a series of external, lateral bony plates, dorsal spines, and a pair of pelvic spines attached to the pelvic girdle. We investigated if cortisol, the primary circulating glucocorticoid in teleosts, could be extracted from stickleback dermal bone and quantified using a commercially available enzyme immunoassay (EIA). We successfully validated a cortisol EIA for dermal bone extracts, determined that cortisol was detectable in both species, and found that dermal bone cortisol levels significantly correlated with cortisol levels in whole body homogenate. Ninespine stickleback had significantly higher dermal bone cortisol concentrations than threespine stickleback and female threespine stickleback tended to have over twice the mean dermal bone cortisol concentration than males. Because both stickleback species are widely used for ecotoxicological studies, using dermal bone as a source of endocrine information, while leaving the body for contaminant, genomic, histological, and stable isotope analyses, could be a powerful and parsimonious tool. Further investigation and physiological validations are necessary to fully understand the utility of this new sample matrix.

Timing of reproduction modifies transgenerational effects of chronic exposure to stressors in an annual vertebrate.

Stressful environmental conditions can shape both an individual's phenotype and that of its offspring. However, little is known about transgenerational effects of chronic (as opposed to acute) stressors, nor whether these vary across the breeding lifespan of the parent. We exposed adult female (F0 generation) three-spined sticklebacks (Gasterosteus aculeatus) to chronic environmental stressors and compared their reproductive allocation with that of non-exposed controls across early, middle and late clutches produced within the single breeding season of this annual population. There was a seasonal trend (but no treatment difference) in F0 reproductive allocation, with increases in egg mass and fry size in late clutches. We then tested for transgenerational effects in the non-exposed F1 and F2 generations. Exposure of F0 females to stressors resulted in phenotypic change in their offspring and grandoffspring that were produced late in their breeding lifespan: F1 offspring produced from the late-season clutches of stressor-exposed F0 females had higher early life survival, and subsequently produced heavier eggs and F2 fry that were larger at hatching. Changed maternal allocation due to a combination of seasonal factors and environmental stressors can thus have a transgenerational effect by influencing the reproductive allocation of daughters, especially those born late in life.

Habitat change alters the expression and efficiency of a female ornament.

Anthropogenic habitat changes are disrupting the mate choice process in a range of organisms, with consequences for populations and communities. Research has so far focused on male sexually selected traits and female mate choice, given their conspicuousness, whereas effects on female ornaments and male mate choice have been largely overlooked. Yet, females of many species develop ornaments that males use in mate choice. These ornaments can be costly and reduce female fecundity and viability and, hence, influence population growth rate. Thus, attention should be paid to changes in female ornaments and the consequences the changes have for populations. Here, we show that declining visibility in aquatic ecosystems reduces the investment of female three-spined stickleback in a melanin-based ornament in favor of increased mate search activity. The adjustment appears adaptive as males pay less attention to the ornament under poor visibility, and as melanin-based ornaments are generally physiologically costly. It is likely that past fluctuations in visibility have promoted the evolution of environment-dependent plasticity in female ornamentation. More attention should be paid to changes in female ornaments and their adaptive value, across taxa, given the impact that female investment in ornaments can have on fecundity and population growth rate. Environments are changing at an accelerating rate because of human activities and knowledge of the responses of both males and females is needed to evaluate and predict the ultimate impact on populations and biodiversity.

Expression of Opsin Genes in the Retina of Female and Male Three-Spined Sticklebacks Gasterosteus aculeatus L.: Effect of Freshwater Adaptation and Prolactin Administration.

Color vision sensitivity is crucial for fish adaptation during migration and reproduction. Prolactin and prolactin-like hormone are important regulators in both these processes. We hypothesized that prolactin influences the color vision sensitivity during freshwater migrations in fish. We studied the effects of prolactin and freshwater adaptation during the spawning period on the expression of opsin genes (SWS1, SWS2, RH2, LWS) in the retina of female and male three-spined sticklebacks Gasterosteus aculeatus L. Expression of the prolactin gene increased in the brain of females, but not males, while expression of the prolactin-like hormone decreased in the brain of both male and female sticklebacks during freshwater adaptation. Expression of the SWS2 gene decreased in the retina of females and males during freshwater adaptation and after prolactin administration. Expression of the SWS1 gene decreased in the retina of male sticklebacks after prolactin administration, but not during freshwater adaptation. Expression of the RH2 and LWS genes did not depend on prolactin administration in male and female sticklebacks. We conclude that expression of some opsin genes in the retina of sticklebacks is regulated by prolactin and depends on sex and freshwater adaptation. This expands our knowledge of the adaptive effects of prolactin on fish during freshwater migrations.

Changes in Red Color Sensitivity over the Spawning Cycle of Female Three-spined Stickleback (Gasterosteus aculeatus).

Male red nuptial coloration is a primary mating signal for three-spined sticklebacks (Gasterosteus aculeatus), and the retinae of both sexes are especially sensitive to red during the breeding season. Red sensitivity is an important aspect of female mate choice in this species, but only when they are ready to spawn and not over the entire breeding period. Here, we aimed to determine if the red sensitivity of female sticklebacks change over their repeat spawning cycle. To this end, we assessed retinal opsin mRNA levels and behavioral red sensitivity in females over this cycle. Both methods indicated that females were more sensitive to red during spawning than in the inter-spawning intervals. Relative expression levels of red color opsin genes (lws) and optical motor sensitivity were high during spawning, decreased after the spawning period, and then increased again 72-96 h later when they were ready to spawn again. Thus, female sticklebacks altered their color sensitivity according to need, but the underlying mechanism remains unclear.

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.

Personality traits change after an opportunity to mate.

There is growing evidence that personality traits can change throughout the life course in humans and nonhuman animals. However, the proximate and ultimate causes of personality trait change are largely unknown, especially in adults. In a controlled, longitudinal experiment, we tested whether a key life event for adults--mating--can cause personality traits to change in female threespine sticklebacks. We confirmed that there are consistent individual differences in activity, sociability and risk-taking, and then compared these personality traits among three groups of females: (i) control females; (ii) females that had physically mated, and (iii) females that had socially experienced courtship but did not mate. Both the physical experience of mating and the social experience of courtship caused females to become less willing to take risks and less social. To understand the proximate mechanisms underlying these changes, we measured levels of excreted steroids. Both the physical experience of mating and the social experience of courtship caused levels of dihydroxyprogesterone (17α,20β-P) to increase, and females with higher 17α,20β-P were less willing to take risks and less social. These results provide experimental evidence that personality traits and their underlying neuroendocrine correlates are influenced by formative social and life-history experiences well into adulthood.

Dynamics of Prolactin Axis Genes in the Brain of Male and Female Three-spined Stickleback Gasterosteus aculeatus (Gasterostaidae) during Short-Term Freshwater Adaptation

Expression of prolactin axis genes (prolactin Prl1, prolactin-like hormone Prl2, prolactin receptor “a” PrlRa, and prolactin receptor “b” PrlRb) have been studied in the brains of females and males of the three-spined stickleback Gasterosteus aculeatus upon adaptation to fresh water. The expression of the Prl1 gene in females was significantly higher after 24-h adaptation to the freshwater relative to the control group (seawater) and to that in males at the same time point. No changes in Prl2 gene expression in females and Prl1 in males were detected. In males, after 12 and 18 h of adaptation to fresh water, higher expression of the Prl2 gene was observed relative to the control group. The expression of the PrlRa gene in males in seawater is higher than that in females; upon adaptation to fresh water, it does not change in females, and it decreases in males gradually after 3 h of being transferred to fresh water. No differences in the expression of the PrlRb gene between females and males in seawater were detected; however, after 24 h of freshwater adaptation, this parameter was higher in females than in males. Changes in the expression of prolactin axis elements during freshwater adaptation are gender-dependent, and the contribution of prolactin and prolactin-like hormone to adaptation of females and males of the three-spined stickleback to fresh water is significantly different.

Mate assessment behavior is correlated to learning ability in female threespine sticklebacks

In many species, males signal quality with elaborate traits, but females often show inter-individual variation in preference for these traits. Choosing a mate requires multiple cognitive steps; therefore, cognitive style (how an individual processes information) likely influences the perception of sexual signals and ability to choose a high-quality mate. An important component of cognitive style is flexibility; cognitively flexible individuals are more perceptive to shifts in cues. We hypothesized that cognitively flexible individuals would acquire more information about potential mates, better discern between two quality-signaling traits, and thus be more discriminatory. Here, we show that mate assessment is correlated to other cognitive traits. Although we did not detect an effect of cognitive style on mate preference or discrimination, we found that female threespine sticklebacks Gasterosteus aculeatus that spent more time assessing potential mates (more responsive) in a dichotomous mate choice task reached both the initial and reversal learning criterion in a spatial learning task with fewer errors. However, these highly responsive females made more consecutive mistakes immediately at the beginning of the reversal phase, suggesting that they did not quickly adapt to the environmental change but instead rapidly formed strict routines during the learning task that were eventually reversible after repeated errors. Furthermore, we found evidence for condition-dependent mate preference, with larger females preferring the high-quality male. These are among the first results that illustrate how cognitive traits might influence mate choice, which has implications for the strength and direction of sexual selection.

Geographical variation in colour of female threespine stickleback (Gasterosteus aculeatus).

The ecological multifunctionality of colour often results in multiple selective pressures operating on a single trait. Most research on colour evolution focuses on males because they are the most conspicuous sex in most species. This bias can limit inferences about the ecological drivers of colour evolution. For example, little is known about population divergence in colour of female threespine stickleback (Gasterosteus aculeatus), which is among the most intensively-studied model vertebrates in evolution, ecology, and behaviour. In contrast, the evolution and ecology of colour in male stickleback has received considerable attention. One aspect of female colouration that is lacking previous research is non-ornamental body colour. Non-ornamental colour can play defensive and social roles, and indicate other aspects of female stickleback ecology. To remedy this knowledge gap, we measured the colour and brightness of one dorsal and one ventral lateral area on female stickleback from nine lake populations on Vancouver Island. We found that lake populations varied in overall colour brightness and dorso-ventral contrast. In addition, we found that female brightness increased with lake size, indicating potential ecological drivers of these colour differences. Our results demonstrate that there is substantial scope for future research on female colour diversification, which has been overlooked because past researchers focused on dramatic male nuptial colours.

Sex and dose-dependent effects of an estrogen mimic on boldness in threespine stickleback, Gasterosteus aculeatus, from an anadromous population

Endocrine disrupting chemicals (EDCs) like 17α-ethinylestradiol (EE2) are prevalent in aquatic ecosystems worldwide where they have the ability to decrease fitness by altering the ways in which organisms forage, court, and react to predators. Boldness serves as a measure of the risks organisms are willing to take to attain resources and is an important, yet underutilized, measure of fitness-related exposure effects. Additionally, the majority of studies on EDC exposure are conducted on freshwater species despite the fact that coastal environments are also at risk. In this study, sexually mature male and female threespine stickleback, Gasterosteus aculeatus, from an anadromous population were placed into one of three treatment groups: control (no EE2), low dose (5ng/L EE2), or high dose (10ng/L EE2) to determine if EE2 alters boldness. Fish were tested in four different assays (Shoaling Propensity, Empty Tank, Foraging, and Novel Environment) and their behavior was compared over time and across treatment groups. Changes in behavior were seen after as little as one week of exposure to EE2. Both sex differences and dosage effects were found, with these two variables interacting and contingent upon the assay in which they were examined. In general, females were more affected than males and the higher dose of EE2 had a greater effect than the lower dose. These results indicate that EE2, and likely other EDCs, can generate behavioral changes in organisms in estuarine and marine systems in ways that can negatively influence the fitness of these organisms.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-β) and luteinizing hormone (lh-β) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR.Ovarian weights peaked in “ready to spawn” females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-β mRNA levels showed a peak at 48hps, while fsh-β did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Maternal stress has divergent effects on gene expression patterns in the brains of male and female threespine stickleback.

Maternal stress can have long-term effects on neurodevelopment that can influence offspring performance and population evolutionary trajectories. To examine the mechanistic basis for these neurodevelopmental effects of maternal stress, we used RNA-seq to assess differential gene expression across the brain transcriptome of adult male and female threespine stickleback (Gasterosteus aculeatus) from stressed and unstressed mothers. We identified sexually divergent effects of maternal stress on the brain transcriptome. In males, genes that were upregulated by maternal stress were enriched for processes involved in synaptic function and organization and steroid hormone-mediated signalling pathways, whereas in females genes that were upregulated by maternal stress were enriched for processes involved in protein translation and metabolic functions. The expression of several genes involved in the hypothalamic-pituitary-interrenal response to stress and epigenetic processes such as the regulation of DNA methylation patterns and miRNAs increased in males and not in females. These data suggest that maternal stress has markedly different effects on cellular pathways in the brains of male and female offspring of mothers that are exposed to stress, which could have important implications when assessing the long-term ecological and evolutionary impacts of stress across generations.

Erratum: Context-dependent dynamic UV signaling in female threespine sticklebacks.

Scientific Reports 5: Article number: 17474; published online: 10 December 2015; updated: 08 September 2016

Perchlorate Exposure Reduces Primordial Germ Cell Number in Female Threespine Stickleback.

Perchlorate is a common aquatic contaminant that has long been known to affect thyroid function in vertebrates, including humans. More recently perchlorate has been shown to affect primordial sexual differentiation in the aquatic model fishes zebrafish and threespine stickleback, but the mechanism has been unclear. Stickleback exposed to perchlorate from fertilization have increased androgen levels in the embryo and disrupted reproductive morphologies as adults, suggesting that perchlorate could disrupt the earliest stages of primordial sexual differentiation when primordial germ cells (PGCs) begin to form the gonad. Female stickleback have three to four times the number of PGCs as males during the first weeks of development. We hypothesized that perchlorate exposure affects primordial sexual differentiation by reducing the number of germ cells in the gonad during an important window of stickleback sex determination at 14–18 days post fertilization (dpf). We tested this hypothesis by quantifying the number of PGCs at 16 dpf in control and 100 mg/L perchlorate-treated male and female stickleback. Perchlorate exposure from the time of fertilization resulted in significantly reduced PGC number only in genotypic females, suggesting that the masculinizing effects of perchlorate observed in adult stickleback may result from early changes to the number of PGCs at a time critical for sex determination. To our knowledge, this is the first evidence of a connection between an endocrine disruptor and reduction in PGC number prior to the first meiosis during sex determination. These findings suggest that a mode of action of perchlorate on adult reproductive phenotypes in vertebrates, including humans, such as altered fecundity and sex reversal or intersex gonads, may stem from early changes to germ cell development.

Genetic Architecture of Conspicuous Red Ornaments in Female Threespine Stickleback.

Explaining the presence of conspicuous female ornaments that take the form of male-typical traits has been a longstanding challenge in evolutionary biology. Such female ornaments have been proposed to evolve via both adaptive and nonadaptive evolutionary processes. Determining the genetic underpinnings of female ornaments is important for elucidating the mechanisms by which such female traits arise and persist in natural populations, but detailed information about their genetic basis is still scarce. In this study, we investigated the genetic architecture of two ornaments, the orange-red throat and pelvic spine, in the threespine stickleback (Gasterosteus aculeatus). Throat coloration is male-specific in ancestral marine populations but has evolved in females in some derived stream populations, whereas sexual dimorphism in pelvic spine coloration is variable among populations. We find that ornaments share a common genetic architecture between the sexes. At least three independent genomic regions contribute to red throat coloration, and harbor candidate genes related to pigment production and pigment cell differentiation. One of these regions is also associated with spine coloration, indicating that both ornaments might be mediated partly via pleiotropic genetic mechanisms.

Waterborne citalopram has anxiolytic effects and increases locomotor activity in the three-spine stickleback (Gasterosteus aculeatus)

Citalopram is an antidepressant drug, which acts by inhibiting the re-uptake of serotonin from the synaptic cleft into the pre-synaptic nerve ending. It is one of the most common drugs used in treatment of depression, it is highly lipophilic and frequently found in sewage treatment plant effluents and surface waters around the world. Citalopram and other selective serotonin re-uptake inhibitors have, at concentrations that occur in nature, been shown to have behavioural as well as physiological effects on fish and other animals. This study is the result of several different experiments, intended to analyse different aspects of behavioural effects of chronic citalopram exposure in fish. Our model species the three-spine stickleback is common in the entire northern hemisphere and is considered to be a good environmental sentinel species. Female three-spine sticklebacks were exposed to 0, 1.5 and 15μg/l nominal concentrations of citalopram for 21 days and subjected to the novel tank (NT) diving test. In the NT test, the fish exposed to 1.5μg/l, but not the 15μg/l fish made a significantly higher number of transitions to the upper half and stayed there for significantly longer time than the fish exposed to 0μg/l. The 15μg/l group, however, displayed a significantly lower number of freeze bouts and a shorter total freezing time. The test for locomotor activity included in the NT test showed that fish treated with 1.5 and 15μg/l displayed a significantly higher swimming activity than control fish both 5–7 and 15–17min after the start of the experiment. In the next experiment we compared fish exposed to 1.5μg/l and 0.15μg/l to pure water controls with regard to shoaling intensity and found no effect of treatment. In the final experiment the propensity of fish treated with 1.5μg/l to approach an unknown object and aggressive behaviour was investigated using the Novel Object test and a mirror test, respectively. The exposed fish ventured close to the unknown object significantly more often and stayed there for significantly longer time than unexposed fish. The aggression test yielded no statistically significant effects. It is concluded that citalopram changes the behaviour of the three-spine stickleback in a way that is likely to have ecological consequences and that it must not be considered an environmentally safe pharmaceutical.

Context-dependent dynamic UV signaling in female threespine sticklebacks.

Color signals, including ultraviolet (UV) signals, are widespread throughout the animal kingdom and color changes can be influenced by reproductive and motivational state. However, studies on dynamic changes of UV signals are scarce. Three spine sticklebacks (Gasterosteus aculeatus) that show intraspecific UV communication were used to study dynamic UV signaling in females. Reflectance measurements were taken from the distended abdomen, which serves as signal of female fecundity and readiness to spawn for courting males, and the melanized dorsal region. Scans were taken during egg maturation as well as before and after stimulation with a male to investigate context-dependent color changes. We used a physiological model of vision to determine how females might be perceived by conspecifics and quantified chromatic contrasts among both body regions and between body regions and the background for all stages. Females showed a significant increase in abdominal UV intensity during egg maturation and in response to a courting male. Measures of chromatic contrast among body regions (abdomen vs. dorsal region) and against the background (abdomen vs. background) were also increased during egg maturation and in response to the male stimulus (abdomen vs. background). Our results provide evidence for dynamic UV signaling in females in a reproductive context.

No evidence for adjustment of maternal investment under alternative mate availability regimes.

Using treatments that mimic high and low availability of reproductive males, it was found that female three-spined sticklebacks Gasterosteus aculeatus, previously shown to adjust their mate choices when male mates were rare, did not alter their reproductive investment strategies. These results suggest that plasticity in investment is perhaps limited by physiological requirements or dependent on relatively extreme mate availability regimes. The probability of becoming reproductive, number of clutches per season (per female), initial clutch size and mass and the timing of reproduction were all independent of the experience a female had with mate availability. This suggests that pre-copulatory plasticity in reproductive strategies may contribute more to variation in the strength and direction of sexual selection than reproductive investment in offspring.

Life-history plasticity in female threespine stickleback.

The postglacial adaptive radiation of the threespine stickleback fish (Gasterosteus aculeatus) has been widely used to investigate the roles of both adaptive evolution and plasticity in behavioral and morphological divergence from the ancestral condition represented by present-day oceanic stickleback. These phenotypes tend to exhibit high levels of ecotypic differentiation. Population divergence in life history has also been well studied, but in contrast to behavior and morphology, the extent and importance of plasticity has been much less well studied. In this review, we summarize what is known about life-history plasticity in female threespine stickleback, considering four traits intimately associated with reproductive output: age/size at maturation, level of reproductive effort, egg size and clutch size. We envision life-history plasticity in an iterative, ontogenetic framework, in which females may express plasticity repeatedly across each of several time frames. We contrast the results of laboratory and field studies because, for most traits, these approaches give somewhat different answers. We provide ideas on what the cues might be for observed plasticity in each trait and, when possible, we inquire about the relative costs and benefits to expressed plasticity. We end with an example of how we think plasticity may play out in stickleback life history given what we know of plasticity in the ancestor.