Centromere identification and inactivation on a neo-Y chromosome fusion in threespine stickleback fish (Gasterosteus aculeatus)

Thesis (Ph.D.)--University of Washington, 2016-05

Healing of skin wounds in the African catfish Clarias gariepinus

The African catfish Clarias gariepinus was used as a model for wound healing and tissue regeneration in a scale-less fish. A temporal framework of histological and cell proliferation markers was established after wound induction in the dorsolateral cranial region, by removing the epidermal and dermal layers, including stratum adiposum (SA). Wound closure and epidermis formation was initiated within 3 h post-procedure (hpp) with migration and concomitant proliferation of epidermal cells from the wound borders. The wound was covered by this primary epidermal front 12 hpp and fusion of the opposing epidermal fronts occurred within 24 hpp. Attachment of the newly formed epidermal layer to the underlying dermis was observed 48 hpp concomitant with a second wave of cell proliferation at the wound edge. Normal epidermal thickness within the wound was achieved 72 hpp. Formation of a basement membrane occurred by 120 hpp with concomitant emergence of the SA from the wound borders. Wound healing in C. gariepinus skin involved closure of the wound and re-epithelization through cell migration with a single wave of early cell proliferation not documented in other species. Furthermore, covering of the wound by epithelium as well as the reappearance of the basement membrane and SA occurred sooner than in other fish species. (C) 2008 The Authors Journal compilation (C) 2008 The Fisheries Society of the British Isles.

A new genus of viviparous gyrodactylid (Monogenea) from the Great Barrier Reef, Australia with descriptions of seven new species

Acanthoplacatus gen. nov., a new genus of viviparous gyrodactylid, is described from the rns and skin of siganid fishes from the Great Barrier Reef, Australia. The genus is characterized by a muscular, tube-like haptor with 16 marginal hooks on the posterior margin. The ventral lobe of the haptor is located anteriorly relative to the dorsal lobe and contains a pair of hamuli and a ventral bar with posteriorly-projecting ventral bar membrane. A dorsal bar is absent. Five pairs of posterior gland cells surround the posterior terminations of the gut. The male copulatory organ is a muscular, non-eversible bulb with several spines around the distal opening. Species of Acanthoplacatus have a bilateral excretory system consisting of six pairs of flame cells and a pair of excretory bladders. Seven new species are described: Acanthoplacatus adlardi sp. nov. and A. amplihamus sp. nov. from Siganus punctatus (Forster, 1801), A. brauni sp. nov. from S. corallinus (Valenciennes, 1835), A. parvihamus sp. nov. from S. vulpinus (Schlegel and Mueller, 1845), A. puelli sp. nov. from S. puellus Schlegel, 1852, A. shieldsi sp. nov. from S. lineatus (Valenciennes, 1835) and A. sigani sp. nov. from S. fuscescens (Houttuyn, 1782). Species can be discriminated by shape and size of the hamuli, marginal hooks and ventral bar and by male copulatory organ sclerite morphology. Three species (A. brauni sp. nov., A. shieldsi sp. nov. and A. sigani sp. nov.) were assessed for seasonal variation of sclerite size. Ten of thirteen morphological characters showed seasonal variation in size for at least one of the species. The characters were longer in winter except dorsal root tissue cap width. Only one character, marginal hook length, showed significant seasonal variation for all three species. Species of Acanthoplacatus were observed to attach using only the marginal hooks and the role of hamuli in attachment is unclear. The dorsal rn of the host is the preferred site for most species but the anal fin, caudal fin and body surfaces are preferred by some species. Prevalences for species range from 57 to 100%.

Assessment of endocrine disrupting activities in the aquatic environment

Hormonstörande ämnen är syntetiska eller naturliga ämnen som stör organismers hormonsystem och bidrar till könsförvirring och sterilitet. Sådana ämnen kommer i ökande takt ut i vattenmiljön genom rester av läkemedel, bekämpningsmedel och industriprodukter. Eftersom det finns många likheter mellan hormonsystemen hos människan och övriga ryggradsdjur kan till exempel fiskar användas som modellsystem för att underöka detta problem. I tidigare undersökningar har man funnit hormonella störningar, bl.a. feminisering och maskulinisering hos fiskar som utsatts för avfallsvatten från kommunala reningsverk eller avfallsvatten från pappersindustrin. Målet med denna avhandling var att undersöka om renat avfallsvatten från kommunala reningsverk längs Finlands kust innehåller hormonstörande ämnen i sådana mängder att de kan försorsaka hormonstörande effekter på fisk. Målet i denna avhandling var också att utveckla cellulära testsystem baserade på fiskceller, eftersom behovet av tillförlitliga och kostnadseffektiva cellbaserade tester för att underlätta riskbedömningen av hormonstörande ämnen är mycket stort för tillfället. Som testsystem har storspiggen använts, som besitter flera användbara biomarkörer för att mäta hormonstörande ämnens påverkan. Resultaten tyder på att problemen med hormonstörande påverkan på fisk inte är lika utbredda i Finland som i många andra europeiska länder. Detta beror troligtvis på att finska reningsverk har effektiva reningstekniker som reducerar mängden hormonstörande ämnen, eller på att utspädningen av avloppen i recipienterna är större än i många andra länder. Dock kan problemen inte helt uteslutas eftersom vissa feminiserande (estrogena) effekter kunde observeras hos fisken i de undersökta recipienterna utanför kommunala reningsverk. I kontrollerade laboratorieförsök där storpsiggar exponerades för kommunalt avloppsvatten uppmättes även här effekter som tyder på förekomst av estrogener i avloppsvattnet. De cell-baserade testsystemen klarade av att förutspå hormonella effekter hos hel fisk och kan därför vara mycket användbara i fortsatta studier av hormonstörande ämnens verkningsmekanismer i preliminära toxicitetsbedömningar.

Anti-predator adaptations in aquatic environments

Predation is an important selective force that has led to the evolution of a variety of fascinating anti-predator adaptations, such as many types of protective coloration and prey behaviours. Because the evolution of life has begun in the aquatic environment and many anti-predator adaptations are found already in relative primitive taxa, it is likely that many of these adaptations evolved initially in the aquatic environment. Yet, there has been surprisingly little research on the mechanisms and function of antipredator adaptations in aquatic systems. To understand the function of anti-predator adaptations and natural selection imposed on prey appearance and behaviour, I have investigated how protective coloration can be used, either as such or together with behavioural adaptations, to manipulate predator behaviour and decrease predation risk. To this end I conducted a series of behaviour ecological laboratory experiments in which I manipulated the visual appearance of artificial backgrounds and prey items. In paper I of this thesis, I investigated background choice as an anti-predator strategy, by observing the habitat choice of the least killifish (Heterandria formosa) between pairs of artificial backgrounds, both in the presence and absence of predation threat. It has been suggested that prey could decrease their risk of being detected by predators either by preferring backgrounds into which they blend or by preferring visually complex backgrounds. The least killifish preferred a background that matched their patterning to a background that mismatched it, showing that they are able to respond to cues of visual similarity between their colour pattern and the surrounding environment. Interestingly however, in female least killifish visual complexity of the background was a more important cue for habitat safety and may override or act together with background matching when searching for a safe habitat. It is possible that in females, preference for visually complex backgrounds is associated with lower opportunity costs than preference for matching backgrounds would be. Generally, the least killifish showed stronger preference while under predation threat, indicating that their background choice behaviour is an antipredator adaptation. Many aquatic prey species have eyespots, which are colour patterns that consist of roughly concentric rings and have received their name because they for humans often resemble the vertebrate eye. I investigated the anti-predator function of eyespots against predation by fish in papers II, III and IV. Some eyespots have been suggested to benefit prey by diverting the strikes of predators away from vital parts of the prey body or towards a direction that facilitates prey escape. Although proposed over a century ago, the divertive effect of eyespots has proven to be difficult to show experimentally. In papers II and III, I tested for divertive effect of eyespots towards attacking fish by presenting artificial prey with eyespots to laboratory reared three-spined sticklebacks (Gasterosteus aculeatus). I found that eyespots strongly influenced the behaviour of attacking sticklebacks and effectively drew their strikes towards the eyespots. To further investigate this divertive effect and whether the specific shape of eyespots is important for it, I tested in paper III the response of fish also to other markings than eyespots. I found that eyespots were generally more effective in diverting the first strikes of attacking fish compared to other prey markings. My findings suggest that the common occurrence of eyespots in aquatic prey species can at least partly be explained by the divertive effect of the eyespot shape, possibly together with the relative simple developmental mechanisms underlying circular colour patterns. An eyebar is a stripe that runs through the eye, and this pattern has been suggested to obscure the real eyes of the prey by visually blending parts of the eyes and head of the prey and by creating false edges. In paper III, I show that an eyebar effectively disrupts an eyelike shape. This suggests that eyebars provide an effective way to conceal the eyes and consequently obstruct detection and recognition of prey. This experiment also demonstrates that through concealment of the eyes, eyebars could be used to enhance the divertive effect of eyespots, which can explain the common occurrence of eyebars in many species of fish that have eyespots. Larger eyespots have been shown to intimidate some terrestrial predators, such as passerine birds, either because they resemble the eyes of the predator’s own enemy or because highly salient features may have an intimidating effect. In papers II and IV, I investigated whether the occurrence of eyespots in some aquatic prey could be explained by their intimidating effect predatory fish. In paper IV, I also investigated the reason for the intimidating effect of eyelike prey marks. In paper II, I found no clear intimidating effect of eyespots, whereas in paper IV, using a different approach, I found that sticklebacks hesitated to attack towards eyelike but not towards non-eyelike marks. Importantly, paper IV therefore presents the first rigorous evidence for the idea that eye mimicry, and not merely conspicuousness, underlies the intimidating effect. It also showed that the hesitation shown by fish towards eyelike marks is partly an innate response that is reinforced by encounters with predators. Collectively, this thesis shows that prey colour pattern and the visual appearance of the habitat influence the behaviour of fish. The results demonstrate that protective coloration provides numerous distinctive ways for aquatic prey to escape predation. Thus, visual perception and behaviour of fish are important factors shaping the appearance and behaviours of aquatic prey.

Molecular Mechanisms of Sexual Dimorphism in Threespine Stickleback

Sexual dimorphism is commonly understood as differences in external features, such as morphological features or coloration. However, it can more broadly encompass behavior and physiology and at the core of these differences is the genetic mechanism – mRNA and protein expression. How, and which, molecular mechanisms influence sexually dimorphic features is not well understood thus far. DNA, RNA and proteins are the template required to create the phenotype of an individual, and they are connected to each other via processes of transcription and translation. As the genome of males and females are almost identical with the exception of the few genes on the sex chromosome or the sex-determining alleles (in the case of organisms without sex chromosomes), it is likely that many of the downstream processes resulting in sexual dimorphism are produced by changes in gene regulation and result from a regulatory cascade and not from a vastly different gene composition. Thus, in this thesis a systems biology approach is used to understand sexual dimorphism at all molecular levels and how different genomic features, e.g. sex chromosome evolution, can affect the interplay of these molecules. The threespine stickleback, Gasterosteus aculeatus, is used as the model to investigate molecular mechanisms of sexual dimorphism. It has well-characterized ecology and behavior, especially in the breeding season when sexual dimorphism is high. Moreover, threespine stickleback has a recently evolved Y chromosome in the early stages of sex chromosome evolution, characterized by a lack of recombination leading to degeneration (i.e. gene loss). The aim of my thesis is to investigate how the genotype links to the molecular phenotype and relates to differences in molecular expression between males and females. Based on previous research on sex differences in mRNA expression, I investigated sex-biased protein expression in adult fish outside the breeding season to see if differences persisted after translation. As sex-biased expression also prevailed in the proteome and previous transcription expression seemed to be related to the sex chromosomes, I investigated the genome level with a particular focus on the sex-chromosomes. I characterized the status of Y chromosome degeneration in the threespine stickleback and its effects on gene function. Furthermore, since the degeneration process leaves genes in a single copy in males, I examined whether the resulting dosage difference of messenger RNA for hemizygous genes is compensated as it is in other organisms. In addition, threespine sticklebacks have wellcharacterized behavioral differences related to the male’s social status during the breeding season. To understand the connection between the genotype and behavior, I examined gene expression patterns related to breeding behavior using dominant and subordinate males as well as female

Diel patterns and tissue-specificity of environmental responses in fish

Humans are profoundly changing aquatic environments through climate change and the release of nutrients and chemicals. To understand the effects of these changes on natural populations, knowledge on individuals’ environmental responses is needed. At the molecular level, the environmental responses are partly mediated by chances in messenger RNA and protein levels. In this thesis I study messenger RNA and protein responses to an assortment of environmental stressors in fish. As daily (diel) rhythms are known to be ubiquitous in different tissues, I particularly focus on diel patterns in the responses. The studied species are the three-spined stickleback (Gasterosteus aculeatus L.) and the Arctic char (Salvelinus alpinus L.), both of which have circumpolar distribution in the Northern hemisphere. In the first two studies, three-spined sticklebacks were exposed to both the non-steroidal anti-inflammatory drug diclofenac and low-oxygen conditions (hypoxia), and their responses measured at separate time points in the liver and gills. The results show how the seemingly unrelated environmental stressors, hypoxia and anti-inflammatory drugs, can have harmful combined effects that differ from the effects of each stressor alone. Moreover, both stressors disturbed natural diel patterns in gene expression. In the third study, I studied the responses of three-spined sticklebacks to two test chemicals: one used in hormonal medicine (17α-ethinyl-oestradiol) and one used as a plasticizer and solvent chemical (di-n-butyl phthalate). The results suggest that the phthalate can affect genes related to spermatogenesis in fish testes, while estrogen-mimicking compounds can lead to numerous disturbances in the endocrine system. In the final study, the temperature-dependence of diel rhythms in messenger RNA levels were evaluated in the liver tissue of the Arctic char, a cold-adapted salmonid. The results show that cold acclimation repressed diel rhythms in gene expression compared to warm-acclimated fish, in which the expression of hundreds of genes was rhythmic, suggesting the circadian clock of the Arctic fish species can be sensitive to temperature. Overall, the results of the thesis indicate that fishes’ responses to abiotic factors interact with their diel rhythms, and more studies on the consequences of these interactions are needed to comprehensively understand human impacts on ecosystems.

Selection and sticklebacks

Over the last decade, there has been increasing circumstantial evidence for the action of natural selection in the genome, arising largely from molecular genetic surveys of large numbers of markers. In nonmodel organisms without densely mapped markers, a frequently used method is to identify loci that have unusually high or low levels of genetic differentiation, or low genetic diversity relative to other populations. The paper by Makinen et al. (2008a) in this issue of Molecular Ecology reports the results of a survey of microsatellite allele frequencies at more than 100 loci in seven populations of the three-spined stickleback (Gasterosteus aculeatus). They show that a microsatellite locus and two indel markers located within the intron of the Eda gene, known to control the number of lateral plates in the stickleback (Fig. 1), tend to be much more highly genetically differentiated than other loci, a finding that is consistent with the action of local selection. They identify a further two independent candidates for local selection, and, most intriguingly, they further suggest that up to 15% of their loci may provide evidence of balancing selection.

Partitioning the relative fitness effects of diet and trophic morphology in the threespine stickleback

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Systematic Variation in the Pattern of Gene Paralog Retention between the Teleost Superorders Ostariophysi and Acanthopterygii

Teleost fish underwent whole-genome duplication around 450 Ma followed by diploidization and loss of 80-85% of the duplicated genes. To identify a deep signature of this teleost-specific whole-genome duplication (TSGD), we searched for duplicated genes that were systematically and uniquely retained in one or other of the superorders Ostariophysi and Acanthopterygii. TSGD paralogs comprised 17-21% of total gene content. Some 2.6% (510) of TSGD paralogs were present as pairs in the Ostariophysi genomes of Danio rerio (Cypriniformes) and Astyanax mexicanus (Characiformes) but not in species from four orders of Acanthopterygii (Gasterosteiformes, Gasterosteus aculeatus; Tetraodontiformes, Tetraodon nigroviridis; Perciformes, Oreochromis niloticus; and Beloniformes, Oryzias latipes) where a single copy was identified. Similarly, 1.3% (418) of total gene number represented cases where TSGD paralogs pairs were systematically retained in the Acanthopterygian but conserved as a single copy in Ostariophysi genomes. We confirmed the generality of these results by phylogenetic and synteny analysis of 40 randomly selected linage-specific paralogs (LSPs) from each superorder and completed with the transcriptomes of three additional Ostariophysi species (Ictalurus punctatus [Siluriformes], Sinocyclocheilus species [Cypriniformes], and Piaractus mesopotamicus [Characiformes]). No chromosome bias was detected in TSGD paralog retention. Gene ontology (GO) analysis revealed significant enrichment of GO terms relative to the human GO SLIM database for growth, Cell differentiation, and Embryo development in Ostariophysi and for Transport, Signal Transduction, and Vesicle mediated transport in Acanthopterygii. The observed patterns of paralog retention are consistent with different diploidization outcomes having contributed to the evolution/diversification of each superorder.

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.

Slow fertilization of stickleback eggs: the result of sexual conflict?

BACKGROUND: The fertilization success in sperm competition in externally fertilizing fish depends on number and quality of sperm. The time delay between sequential ejaculations may further influence the outcome of sperm competition. Such a time interval can load the raffle over fertilization if fertilization takes place very fast. Short fertilization times are generally assumed for externally fertilizing fish such as the three-spined stickleback (Gasterosteus aculeatus). In this pair-spawning fish, territorial males often try to steal fertilizations in nests of neighbouring males. This sneaking behaviour causes sperm competition. Sneakers will only get a share of paternity when eggs are not fertilized immediately after sperm release. Contrary to males, females may be interested in multiple paternity of their clutch of eggs. There thus may be a sexual conflict over the speed of fertilization. RESULTS: In this study we used two different in vitro fertilization experiments to assess how fast eggs are fertilized in sticklebacks. We show that complete fertilization takes more than 5 min which is atypically long for externally fertilizing fishes. CONCLUSION: This result suggests that the time difference does not imply high costs to the second stickleback male to ejaculate. Slow fertilization (and concomitant prolonged longevity of sperm) may be the result of sexual conflict in which females aimed at complete fertilization and/or multiple paternity.

Habitat Choice and Female Preference in a Polymorphic Stickleback Population

Background: A small pond, c. 90 years old, near Bern, Switzerland contains a population of threespine stickleback (Gasterosteus aculeatus) with two distinct male phenotypes. Males of one type are large, and red, and nest in the shallow littoral zone. The males of the other are small and orange, and nest offshore at slightly greater depth. The females in this population are phenotypically highly variable but cannot easily be assigned to either male type. Question: Is the existence of two sympatric male morphs maintained by substrate-associated male nest site choice and facilitated by female mate preferences? Organisms: Male stickleback caught individually at their breeding sites. Females caught with minnow traps. Methods: In experimental tanks, we simulated the slope and substrate of the two nesting habitats. We then placed individual males in a tank and observed in which habitat the male would build his nest. In a simultaneous two-stimulus choice design, we gave females the choice between a large, red male and a small, orange one. We measured female morphology and used linear mixed effect models to determine whether female preference correlated with female morphology. Results: Both red and orange males preferred nesting in the habitat that simulated the slightly deeper offshore condition. This is the habitat occupied by the small, orange males in the pond itself. The proportion of females that chose a small orange male was similar to that which chose a large red male. Several aspects of female phenotype correlated with the male type that a female preferred.