The juvenile three-spined stickleback : model organism for the study of estrogenic and androgenic endocrine disruption in laboratory and field

Industrial and domestic sewage effluents have been found to cause reproductive disorders in wild fish, often as a result of the interference of compounds in the effluents with the endocrine system. This thesis describes laboratory-based exposure experiments and a field survey that were conducted with juveniles of the three-spined stickleback, Gasterosteus aculeatus. This small teleost is a common fish in Swedish coastal waters and was chosen as an alternative to non-native test species commonly used in endocrine disruption studies, which allows the comparison of field data with results from laboratory experiments. The aim of this thesis was to elucidate 1) if genetic sex determination and differentiation can be disturbed by natural and synthetic steroid hormones and 2) whether this provides an endpoint for the detection of endocrine disruption, 3) to evaluate the applicability of specific estrogen- and androgen-inducible marker proteins in juvenile three-spined sticklebacks, 4) to investigate whether estrogenic and/or androgenic endocrine disrupting activity can be detected in effluents from Swedish pulp mills and domestic sewage treatment plants and 5) whether such activity can be detected in coastal waters receiving these effluents. Laboratory exposure experiments found juvenile three-spined sticklebacks to be sensitive to water-borne estrogenic and androgenic steroid substances. Intersex – the co-occurrence of ovarian and testicular tissue in gonads – was induced by 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) and 5α-dihydrotestosterone (DHT). The first two weeks after hatching was the phase of highest sensitivity. MT was ambivalent by simultaneously eliciting masculinizing and feminizing effects. When applying a DNA-based method for genetic sex identification, it was found that application of MT only during the first two weeks after hatching caused total and apparently irreversible development of testis in genetic females. E2 caused gonad type reversal from male to female. E2 and EE2 induced vitellogenin - the estrogen-responsive yolk precursor protein, while DHT and MT induced spiggin – the androgen-responsive glue protein of the stickleback. None of the effluents from two pulp mills and two domestic sewage treatment plants had any estrogenic or androgenic activity. Juvenile three-spined sticklebacks were collected during four subsequent summers at the Swedish Baltic Sea coast in recipients of effluents from pulp mills and a domestic sewage treatment plant as well as remote reference sites. No sings of endocrine disruption were observed at any site, when studying gonad development or marker proteins, except for a deviation of sex ratios at a reference site. The three-spined stickleback – with focus on the juvenile stage – was found to be a sensitive species suitable for the study of estrogenic and androgenic endocrine disruption.

Genetic variation and inference of demographic histories in non-model species

Both long-term environmental changes such as those driven by the glacial cycles and more recent anthropogenic impacts have had major effects on the past demography in wild organisms. Within species, these changes are reflected in the amount and distribution of neutral genetic variation. In this thesis, mitochondrial and microsatellite DNA was analysed to investigate how environmental and anthropogenic factors have affected genetic diversity and structure in four ecologically different animal species. Paper I describes the post-glacial recolonisation history of the speckled-wood butterfly (Pararge aegeria) in Northern Europe. A decrease in genetic diversity with latitude and a marked population structure were uncovered, consistent with a hypothesis of repeated founder events during the postglacial recolonisation. Moreover, Approximate Bayesian Computation analyses indicate that the univoltine populations in Scandinavia and Finland originate from recolonisations along two routes, one on each side of the Baltic. Paper II aimed to investigate how past sea-level rises affected the population history of the convict surgeonfish (Acanthurus triostegus) in the Indo-Pacific. Assessment of the species’ demographic history suggested a population expansion that occurred approximately at the end of the last glaciation. Moreover, the results demonstrated an overall lack of phylogeographic structure, probably due to the high dispersal rates associated with the species’ pelagic larval stage. Populations at the species’ eastern range margin were significantly differentiated from other populations, which likely is a consequence of their geographic isolation. In Paper III, we assessed the effect of human impact on the genetic variation of European moose (Alces alces) in Sweden. Genetic analyses revealed a spatial structure with two genetic clusters, one in northern and one in southern Sweden, which were separated by a narrow transition zone. Moreover, demographic inference suggested a recent population bottleneck. The inferred timing of this bottleneck coincided with a known reduction in population size in the 19th and early 20th century due to high hunting pressure. In Paper IV, we examined the effect of an indirect but well-described human impact, via environmental toxic chemicals (PCBs), on the genetic variation of Eurasian otters (Lutra lutra) in Sweden. Genetic clustering assignment revealed differentiation between otters in northern and southern Sweden, but also in the Stockholm region. ABC analyses indicated a decrease in effective population size in both northern and southern Sweden. Moreover, comparative analyses of historical and contemporary samples demonstrated a more severe decline in genetic diversity in southern Sweden compared to northern Sweden, in agreement with the levels of PCBs found.