Juvenile Sticklebacks Research Articles

Food Selectivity in Juvenile Three-Spined Stickleback Gasterosteus aculeatus L. (Gasterosteidae) at Nursery Grounds in the White Sea

The spatial and temporal variability in the dietary preferences of juvenile three-spined stickleback Gasterosteus aculeatus were studied across the typical coastal habitats of Keret Archipelago, Kandalaksha Bay(the White Sea). The sampling of fish using a beach seine was conducted in the late July–early August period and in late August. Additionally, zooplankton samples were collected to conduct a quantitative assessment of the abundance of potential prey items. The similarity percentage (SIMPER) analysis was applied to the data obtained from the stomach content and revealed the five planktonic taxa most prominently contributing to the diet of juvenile sticklebacks. Among these, the copepod Temora longicornis was the most important prey item at marine sites, while Acartia longiremis dominated the diet of fish in the marine lagoon. The calculation of the selectivity index revealed that some taxa (such as A. longiremis) were always avoided by juvenile fish, whereas the selectivity of some taxa increased in late August. In general, juvenile sticklebacks demonstrated a pronounced individual variation in their selectivity, even when a particular prey item was selected positively during the whole period of study. Our data highlight that despite the predominant consumption of easily available prey by juvenile sticklebacks, they demonstrate taxonomic- and size-specific prey selectivity.

Impacts of Long-Term Exposure to Ocean Acidification and Warming on Three-Spined Stickleback (Gasterosteus aculeatus) Growth and Reproduction

The warming and acidification of surface waters as predicted by the IPCC leads aquatic species to face major multifaceted changes in their environment. Although teleosts have efficient regulatory systems to cope with these changes, such changes clearly have the potential to impact their physiological functions. Hence, it is crucial to estimate the ability of teleost fishes to cope with multi-stresses to predict how they will deal with future environments. In this context, we investigated the joint effect of warming and acidification on three-spined stickleback (Gasterosteus aculeatus) from the juvenile stage to adulthood, focusing on parameters linked to growth, sexual maturation, and reproduction. Juvenile sticklebacks were split in 2 climate scenarios: a “Current” scenario corresponding to the current seasonal physico-chemical parameters of the water of the “Rade de Brest” in France, and a “RCP8.5” scenario with a warming of 3 °C and an acidification of 0.4 pH units. After 7 months, fish in the RCP8.5 scenario reached the same size and mass as those in the Current scenario, but they needed greater amounts of food to reach satiety. Furthermore, the mortality rate over the experiment was higher in the RCP8.5 scenario. Muscle lipid content, an indicator of energy reserves, was lower in females in the RCP8.5 scenario, suggesting an increased need for energy to maintain homeostasis and other physiological functions or a divergence in energy allocation strategy. Moreover, females exhibited lower sexual maturation and egg quality under the RCP8.5 scenario, which could have contributed to the lower fertilisation rate observed. Males were more resilient to the RCP8.5 scenario, exhibiting only a trend for lower kidney somatic index scores. Altogether, these results suggest a delay and/or an inhibition of gametogenesis and maturation in fish in warmed and acidified waters. The analysis of blood sex steroid concentrations, brain gene expression profiles, and physiological indexes did not allow us to discriminate between a delay and an inhibition of maturation in the RCP8.5 scenario. Overall, these findings clearly indicate that there is a long-term global impact of combined acidification and warming on the mortality and reproductive performance of three-spined stickleback.

No signs of behavioral evolution of threespine stickleback following northern pike invasion

AbstractInvasive predators often impose devastating selection pressures on native prey species. However, their effects can be regionally dependent and influenced by the local ecological conditions of their invaded habitats. Evolved behavioral phenotypes are important mechanisms by which prey adapt to the presence of novel predators. Here, we asked how behavior and behavioral plasticity of threespine stickleback (Gasterosteus aculeatus) populations have evolved following the introduction of the invasive predator, northern pike (Esox lucius). We examined the behavior of F1 offspring generated from three pike-free and three pike-invaded populations and measured how stickleback activity and plant use behaviors, and their plasticity, have evolved following pike introduction. To evaluate plasticity, we exposed juvenile stickleback to predator cues during their first year of development and then evaluated how this repeated exposure influenced behavioral responses to an artificial predation event. We found no overarching effect of pike in either evolved behaviors or behavioral plasticity, and no evidence for the presence of developmental plasticity. Furthermore, we found that depending on the phenotype, pike-invaded stickleback populations have either more or less among-population variation than pike-free populations. Our results suggest that evolution in response to invasive predators may be hidden by local adaptation when enough populations are studied.

Predator life history and prey ontogeny limit natural selection on the major armour gene,Eda, in threespine stickleback

AbstractNatural selection shapes the evolution of antipredator traits in prey. However, selection in the wild depends on ecological context, including features of predator and prey populations, making field studies of selection critical to understanding how predators shape selection on prey defences. Threespine stickleback (Gasterosteus aculeatus) is a classic system to study the effects of predators on the natural selection of prey. In lakes and rivers, fish predators have been shown to impose selection against low plated adult stickleback phenotypes and genotypes. We directly measured selection by predatory salmonids on theEctodysplasin‐A(Eda) gene in estuary stickleback from California. Despite previous studies showing a positive correlation between predator presence and frequency of theEda“complete” allele in estuary populations, we found thatEda“low” genotypes were not significantly more frequent in salmonid predator diets. Further, we found no evidence of changes inEdagenotype frequencies across generations that would suggest directional selection driven by predators. Prior selection studies have examined the effects of large resident trout on adult stickleback. In contrast, predators in this study were juvenile anadromous salmonids, which only ate juvenile stickleback whose plate phenotypes had not fully developed. Thus, in this case, predator life history and stickleback ontogeny may preclude strong selection on stickleback armour. Our results underscore the importance of selection studies in the wild for understanding the context‐dependent nature of selection in natural populations.

Long-Lasting Negative Effects of Learning Tasks During Early Life in the Three-Spined Stickleback

Early environment often has profound effects on phenotypic development that last throughout the lifetime. It has been suggested that unpredictable environments may favour cognitive abilities. However, cognitive challenges during development may result in life-history trade-offs, because complex neural reorganization required for coping with these challenges entails stress and energy costs. In this study, we experimentally tested whether early exposure to different cognitive challenges influences cognitive ability, brain size and life-history traits in the three-spined stickleback (Gasterosteus aculeatus). For this, juvenile sticklebacks were challenged over a two-month period to resolve four different learning tasks. Contrary to our predictions, individuals trained in learning tasks later showed a reduced ability to solve a new maze task, suggesting that these learning tasks during early life may impair the ability to cope with new cognitive challenges. Females from the learning task group showed smaller optic tectum volume than the control females. Fish from the learning task treatment reduced their growth during the experiment, then accelerated growth to catch up until the breeding season. Sticklebacks reared in an environment with learning tasks also suffered a greater mortality than the controls both during and after the experiment. For the first time to our knowledge, we demonstrated direct costs of living in environments with cognitive challenges by tracking long-term performances of sticklebacks exposed to dynamic challenges during early life. Our results show that environmental challenges during ontogeny trigger plastic responses, but contrary to the common belief, do not improve the ability to cope with new cognitive challenges.

Spatial heterogeneity and temporal dynamics of protein-degrading activity and life-history traits in threespine stickleback Gasterosteus aculeatus

The paper studies the spatial heterogeneity and temporal dynamics of life-history traits, parasite invasion, and biochemical variables (e.g., tissue protein content and calpain activity) in adult and juvenile threespine stickleback Gasterosteus aculeatus. Samples were collected on three spawning grounds in Kandalaksha Bay, on the White Sea, located within a few kilometers of each other. These sites featured differences in temperature regime, water exchange, feed organism composition, predation load, aquatic vegetation, and spawner density. Whereas we found no parasite-related or size heterogeneity in adult fish on these grounds at the beginning of the spawning season, muscle protein reserves and protease (calpain) activity patterns in adults showed significant spatial heterogeneity, which increased as spawning progressed and was particularly pronounced in females. Sticklebacks expend much energy during spawning and are worn out by the end of the spawning period. To maintain individual viability, skeletal muscle proteins degrade, and the resulting amino acids are oxidized to produce energy. Protein-degrading calpains play a key role in these processes, and also mediate intense protein metabolism in juvenile stickleback, showing higher values in individuals growing under more favorable conditions. Thus, by studying the heterogeneous life-history traits and biochemical characteristics of adult and juvenile stickleback, we discovered the effects of environmental factors on their physiology, biochemical variables, and growth.

Horizontal transmission of the ectoparasite Gyrodactylus arcuatus (Monogenea: Gyrodactylidae) to the next generation of the three-spined stickleback Gasterosteus aculeatus.

In the parthenogenetic monogeneans of the genus Gyrodactylus Nordmann, 1832, the genetic diversity within or between hosts is determined by the relative roles of lateral transmission and clonal propagation. Clonality and limited transmission lead to high-amplitude metapopulation dynamics and strong genetic drift. In Baltic populations of the three-spined stickleback Gasterosteus aculeatus Linnaeus, the local mitochondrial diversity of Gyrodactylus arcuatus Bychowsky, 1933 is very high, and spatial differentiation weak. To understand the transmission dynamics in a single location, the transmission of the parasite from adults to next generation sticklebacks was investigated in a northern Baltic brackish water location. By sequencing 777 nt of cox1, as many as 38 separate mitochondrial haplotypes were identified. In August, the intensity of gyrodactylid infection on adult hosts was high, the haplotype diversity (h) was extreme and differentiation between fish was negligible (total h = 0.926, mean h = 0.938). In October, only 46% of the juvenile sticklebacks carried G. arcuatus. The number of parasites per young fish followed a Poisson distribution 0.92 ± 1.04 (mean ± SD) on October 2, and was clearly overdispersed 2.38 ± 5.00 on October 25. The total haplotype diversity of parasites on juveniles was nearly as high as in adults (h = 0.916), but the mean per fish was only h = 0.364 (FST = 0.60), due to low intensity of infection and rapid clonal propagation of early arrivals. The initial first come first served advantage of the first gyrodactylid colonisers will be lost during the host adulthood via continuous transmission. Nesting and polygamy are suggested as factors maintaining the high genetic diversity of the parasite population. The transmission dynamics and, consequently, the population structure of Baltic G. arcuatus is fundamentally different from that of G. salaris Malmberg, 1957, on the Baltic salmon Salmo salar Linnaeus.

Sex-specific phenotypes and metabolism-related gene expression in juvenile sticklebacks

Sex-specific phenotypes and metabolism-related gene expression in juvenile sticklebacks

Effects of an anthropogenic saltwater inlet on three-spined stickleback (Gasterosteus aculeatus) (Teleostei: Gasterosteidae) and their parasites in an inland brook

In industrialised areas, teleost fish are often exposed to anthropogenic changes of the water quality. These often have negative effects on species with a narrow ecological range. Species with a wider ecological range, such as the three-spined stickleback (Gasterosteus aculeatus Linneaus, 1758), might benefit if water quality alteration reduces interspecific competition and/or parasite infection pressure. In the present study, we investigated sticklebacks in an inland brook, in which the inlet of warm and salty coal mine drainage water increases water temperature and changes the brook from freshwater to brackish (approx. 20 mS cm−1) conditions. We collected sticklebacks up- and downstream of the saltwater inlet (henceforth called freshwater and saltwater sites or habitats) in monthly intervals from April to October 2010, and monitored their body condition parameters and parasite infections. In particular during spring, the water temperature was higher (3.7–4.5°C) in the saltwater habitat and juvenile sticklebacks occurred earlier and grew faster compared to juveniles in the freshwater habitat. In the saltwater habitat, fewer parasite species were detected compared to the freshwater situation (7 vs. 10). Moreover, parasite index, which peaked in young-of-the-year sticklebacks in September, was lower in sticklebacks from the saltwater site. The present study suggests that changes of freshwater conditions by the inlet of warm and salty coal mine drainage water match the adaptive range of three-spined sticklebacks, which grew faster and had lower parasite burden in the altered habitat.

Trophic ecology of introduced populations of Alaska blackfish (Dallia pectoralis) in the Cook Inlet Basin, Alaska

Introduced non-native fishes have the potential to substantially alter aquatic ecology in the introduced range through competition and predation. The Alaska blackfish (Dallia pectoralis) is a freshwater fish endemic to Chukotka and Alaska north of the Alaska Range (Beringia); the species was introduced outside of its native range to the Cook Inlet Basin of Alaska in the 1950s, where it has since become widespread. Here we characterize the diet of Alaska blackfish at three Cook Inlet Basin sites, including a lake, a stream, and a wetland. We analyze stomach plus esophageal contents to assess potential impacts on native species via competition or predation. Alaska blackfish in the Cook Inlet Basin consume a wide range of prey, with major prey consisting of epiphytic/benthic dipteran larvae, gastropods, and ostracods. Diets of the introduced populations of Alaska blackfish are similar in composition to those of native juvenile salmonids and stickleback. Thus, Alaska blackfish may affect native fish populations via competition. Fish ranked third in prey importance for both lake and stream blackfish diets but were of minor importance for wetland blackfish.

Habitat preference of three-spined stickleback juveniles in experimental conditions and in wild eelgrass

Long-term population dynamics of three-spined stickleback Gasterosteus aculeatus in the White Sea during the 20th century has patterns similar to that of eelgrass Zostera marina. In this study we address possible mechanisms of such association through analysis of spatial distribution of juvenile stickleback in the wild and their substrate preferences in experimental conditions. Samples from different habitats (Z. marina, Fucus spp.) in 13 localities of Kandalaksha Bay have shown that the juvenile sticklebacks occurred mainly in the eelgrass beds. Their density was significantly lower in fucoids. In the experiments, carried out in August 2008–2009, the juveniles were offered the following types of substrates: brown macroalgae (dense and scarce), eelgrass and control (no substrate). In the experiments sticklebacks also showed a tendency to prefer eelgrass to fucoids, even if the density of the latter was higher. This may explain their higher densities associated with eelgrass in the wild.

Unsociable juvenile male three-spined sticklebacks grow more attractive

An animal’s personality may be an adaptive behavioral response of individuals to consistent differences in physiology and life-history. Studying the relationship between behavior and other fitness-related traits within the pace-of-life syndrome (POLS) framework, which explains the integration of different traits using the concept of a fast-slow life-history continuum, may thus be a useful approach to understanding the evolution and maintenance of animal personality. According to the POLS hypothesis, for example, fast individuals should be behaviorally more active, bolder or less sociable, and potentially invest more in reproduction than slow individuals. Here, we examined whether the sociability of juvenile sticklebacks (Gasterosteus aculeatus) is linked with their key reproductive traits, carotenoid-based nuptial coloration of males and egg production of females. We also tested whether experimental manipulation of environmental conditions (control vs. warm winter) can change pace-of-life and the link between behavioral and reproductive traits. Male sticklebacks prone to shoaling expressed lower peak red coloration at the reproductive stage, during which they were solitary and territorial. In females, fecundity was not correlated with early social behavior. Experimental sticklebacks reared in warm winter conditions decreased their investment in reproduction, but the negative relationship between sociability and sexual signal of males was maintained. Our results suggest that the POLS could partly explain the maintenance of personality under predation pressure or severe mating competition. In natural environments, any benefit to sociable male sticklebacks may be counterbalanced by the cost of growing less attractive. Why do animals vary in personality, and how is such variation maintained within a population? An interesting approach for understanding this is studying how personality traits are correlated with other fitness-related traits within the pace-of-life framework. For the first time, our results show the link between sociability at the juvenile stage and the expression of sexual ornament during reproduction in male three-spined sticklebacks. Sociable juveniles may benefit from reduced predation risk, but they grow less attractive and this may reduce their mating success during reproduction. Thus, the trade-off between survival and reproduction may contribute to the maintenance of among-individual variation in behavior.

Experimental Evidence of an Eco-evolutionary Feedback during Adaptive Divergence

Differences in how organisms modify their environment can evolve rapidly and might influence adaptive population divergence. In a common garden experiment in aquatic mesocosms, we found that adult stickleback from a recently diverged pair of lake and stream populations had contrasting effects on ecosystem metrics. These modifications were caused by both genetic and plastic differences between populations and were sometimes comparable in magnitude to those caused by the presence/absence of stickleback. Lake and stream fish differentially affected the biomass of zooplankton and phytoplankton, the concentration of phosphorus, and the abundance of several prey (e.g., copepods) and non-prey (e.g., cyanobacteria) species. The adult-mediated effects on mesocosm ecosystems influenced the survival and growth of a subsequent generation of juvenile stickleback reared in the same mesocosms. The prior presence of adults decreased the overall growth rate of juveniles, and the prior presence of stream adults lowered overall juvenile survival. Among the survivors, lake juveniles grew faster than co-occurring stream juveniles, except in mesocosm ecosystems previously modified by adult lake fish that were reared on plankton. Overall, our results provide evidence for reciprocal interactions between ecosystem dynamics and evolutionary change (i.e., eco-evolutionary feedbacks) in the early stages of adaptive population divergence.

Feeding patterns in seagrass beds of three-spined stickleback Gasterosteus aculeatus juveniles at different growth stages

Today, three-spined stickleback Gasterosteus aculeatus are the most abundant fish in the White Sea and are close to their historical maximum. Based on observations from 2011–2013, this study reports quantitative and qualitative characteristics of juvenile stickleback diet during periods of active feeding in coastal Zostera seagrass beds. The following planktonic taxa dominated stomach contents: copepods Temora longicornis and Microsetella norvegica, ciliophora Helicostomella subulata. Benthic organisms such as Oligochaetae and Orthocladiinae also played an important role, whereas the literature suggests they were once rare in marine stickleback diets. Consumption patterns depended on fish size, with the most pronounced diet shift taking place as juveniles reached a length of 15 mm, in late August. In larger juveniles the highest correlation between the abundance of food organisms in stomachs and in the sea was observed for Orthocladiinae, suggesting that they are the preferred food. Overall, changes in diet followed changes in the abundance of available food organisms, but food selectivity analysis of planktonic organisms showed that M. norvegica were actively selected by juveniles.

UV Deprivation Influences Social UV Preference in Juvenile Sticklebacks

Social aggregations occur in many different animal taxa and mainly result from non-random assortment. Investigating factors that shape and maintain the composition of social aggregations are among others a main topic for understanding ecological speciation processes. Aggregation decisions are mediated by olfactory and visual cues, which in many animals are extended into the UV part of the electromagnetic spectrum. Here, we were interested in developmental plasticity of social preferences with respect to UV radiation in aquatic organisms. Specifically, we tested whether different lighting environments with respect to UV wavelengths during early life stages influence the shoaling preference in juvenile threespine sticklebacks (Gasterosteus aculeatus). Family (full-sibling) groups were split and reared under UV-lacking (UV-) and UV-present (UV+) lighting conditions. Subsequent shoal choice experiments, in which test fish from both rearing conditions could simultaneously choose between a shoal seen behind a UV-blocking (UV-) and a shoal seen behind a UV-transmitting (UV+) filter, revealed a significant effect of lighting condition during rearing on association preference. Test fish that had been deprived of UV spent significantly more time near the UVshoal compared to the test fish reared under full-spectrum lighting conditions. The results are discussed with respect to plasticity of the visual system and environmental lighting conditions.

Phenotypic integration between antipredator behavior and camouflage pattern in juvenile sticklebacks.

Predation is a strong selective force that promotes the evolution of antipredator behaviors and camouflage in prey animals. However, the independent evolution of single traits cannot explain how observed phenotypic variations of these traits are maintained within populations. We studied genetic and phenotypic correlations between antipredator behaviors (shoaling and risk-taking) and morphology traits (pigmentation and size) in juvenile three-spined sticklebacks by using pedigree-based quantitative genetic analysis to test phenotypic integration (or complex phenotype) as an evolutionary response to predation risk. Individuals with strongly melanized (i.e., camouflaged) phenotype and genotype were less sociable to conspecifics, but bolder during foraging under predation risk. Individuals with faster growing phenotype and genotype were bolder, and those with lager eyes were more fearful. These phenotypic integrations were not confounded with correlated plastic responses to predation risk because the phenotypes were measured in naïve fish born in the laboratory, but originated from a natural population with predation pressure. Consistent selection for particular combinations of traits under predation pressure or pleiotropic genes might influence the maintenance of the genetic (co)variations and polymorphism in melanin color, growth trajectory, and behavior patterns.

Early exposure to nonlethal predation risk by size‐selective predators increases somatic growth and decreases size at adulthood in threespined sticklebacks

Predation has an important influence on life history traits in many organisms, especially when they are young. When cues of trout were present, juvenile sticklebacks grew faster. The increase in body size as a result of exposure to cues of predators was adaptive because larger individuals were more likely to survive predation. However, sticklebacks that had been exposed to cues of predators were smaller at adulthood. This result is consistent with some life history theory. However, these results prompt an alternative hypothesis, which is that the decreased size at adulthood reflects a deferred cost of early rapid growth. Compared to males, females were more likely to survive predation, but female size at adulthood was more affected by cues of predators than male size at adulthood, suggesting that size at adulthood might be more important to male fitness than to female fitness.

Nutritional state influences shoaling preference for familiars

Preferences for grouping with familiar individuals are shown in many animal species, including the three-spined stickleback ( Gasterosteus aculeatus). Shoaling with familiars is advantageous because of more precise anti-predator behaviours or more stable dominance hierarchies. Additionally, associations with familiar individuals facilitate the evolution of altruistic behaviour. Thus, in situations of increased competition one might expect an increased preference for familiar fish. We gave single juvenile sticklebacks of different nutritional state the choice between shoals composed either of familiar or unfamiliar individuals. Satiated fish preferred to shoal with familiar individuals. A comparative analysis of 8 stickleback studies with 15 different tests using familiars showed that all tests gave similar results, i.e. sticklebacks of all age classes preferred to shoal with familiars in a non-sexual context. In contrast, hungry test fish did not prefer to shoal with familiar fish, but even showed a preference for the unfamiliar group. Because sticklebacks use early-life familiarity to recognize kin, the results suggest the avoidance of competition with relatives. To our knowledge, this is the first study showing an impact of nutritional state on social interactions with familiar individuals.

Impaired Reproduction in Three-Spined Sticklebacks Exposed to Ethinyl Estradiol as Juveniles1

To investigate the population-level effects of exposure to environmental endocrine disrupters, a mesocosm-scale study was carried out in which the reproductive performance of groups of free-spawning three-spined sticklebacks, Gasterosteus aculeatus, exposed as juveniles to a model estrogen, was assessed. Juvenile sticklebacks were exposed to ethinyl estradiol (EE(2)) at measured concentrations of (mean +/- SEM) 1.75 +/- 0.37 ng L(-1) and 27.7 +/- 1.08 ng L(-1) for 4 wk posthatch and then reared thereafter in pristine lake water until they reached adulthood. Exposure to the higher EE(2) concentration resulted in the occurrence of ovotestis among males, whereas no gonadal abnormalities were evident among males exposed to the lower concentration of EE(2). In addition, when spawning was allowed in the mesocosm environment, fewer nests were built per male, and fewer eggs were deposited per nest, in the group exposed to 27.7 ng L(-1). Males from this group also exhibited a less intense nuptial coloration than control males. In the group exposed to 1.75 ng L(-1) EE(2) posthatch, significantly fewer nests were built than in the control group. These results demonstrate that the timing of exposure to estrogenic contaminants, in developmental terms, is critically important. Short-term exposure to estrogens as juveniles can clearly influence reproductive performance as adults, despite all growth and development subsequent to the exposure period taking place in an estrogen-free environment. In addition, these results suggest that reproductive dysfunction can occur even in fish with no gross abnormalities in gonadal structure. This suggests that the absence of gonadal intersex is not a reliable indicator of the reproductive potential, or estrogen-exposure history, of fish populations or the only important factor involved in compromising the reproduction of estrogen-exposed fish.

The juvenile three-spined stickleback (Gasterosteus aculeatus L.) as a model organism for endocrine disruptionI. Sexual differentiation

Juvenile three-spined stickleback ( Gasterosteus aculeatus L.) is introduced as a unique model organism for both androgenic and oestrogenic endocrine action. Intersex is often used as an indicator for disruption of sexual differentiation in fish exposed to different kinds of effluents from human activities. In wild fish it has exclusively been reported in terms of feminisation due to xenoestrogens in the environment. The assumption that the intersex individuals are feminised genetic males can only be proven by genetic sex identification of the intersexual individuals. Intersex and gonadal sex reversal were induced in three-spined sticklebacks by treatment with natural and synthetic steroid hormones. Juvenile sticklebacks were exposed to three nominal concentrations of 17β-oestradiol (E2); i.e. 0.01, 1.0 and 10.0 μg/L; which were administered to the water either continuously from hatching to the end of the experiment (39–58 days post hatch), during the first 2 weeks after hatching only, from 14 days after hatching onwards, or during the chorionated embryo stage until hatching. Other groups were exposed to 17α-ethinylestradiol (EE2) at 0.05 μg/L and 17α-methyltestosterone (MT) at 1.0 μg/L (nominal concentrations). MT was applied continuously, during the first 2 weeks post hatch only, or from 14 days after hatching onwards. Gonad histology was examined and the genetic sex was identified with male sex-linked PCR markers. Treatment with oestrogens caused feminisation at the two highest E2 concentrations and with EE2. Exposure to E2 before hatching had no effect. Intersexual individuals from oestrogen treatments were genetic males. The genetic sex marker identified apparent total reversal of the gonad type of genetic males. Treatment with MT did not reveal a clear picture, since intersex was observed in both genetic females and males. MT also caused severe testis abnormalities, mainly the development of large branched cavities with unidentified origin. The process of sex differentiation is most sensitive to the influence of external steroids during the first 2 weeks after hatching. A lower incidence of intersex could also be induced in sticklebacks exposed from 14 days after hatching by E2 treatment, but not with MT. The combination of gonad histopathology with genetic sex identification in juvenile sticklebacks is suggested as a tool for detecting endocrine disruption in laboratory studies, and might become very useful in field surveys.