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Differences in selection pressure in nature and labs have profound effects on zebrafish strains. The widely used AB strain of zebrafish has been domesticated over several decades. Recently, there has been an upsurge in the availability of genetically modified lines, e.g., the spiegeldanio (spd), which has a mutation in the fibroblast growth factor receptor 1a (fgfr1a) gene. This mutant strain (fgfr1a) has previously been reported to be bolder than fish of the Tubingen strain, from which it was generated. Our knowledge on behavioral differences between different zebrafish strains, relative to wild-caught zebrafish, is limited. In the present study we compare behaviors related to interpretation of boldness in male and female offspring (F1) of wild-caught fish, AB and fgfr1a(-/-) zebrafish. A second aim of the study was to compare the behavior of fish from these strains when tested in different behavioral assays, i.e., shelter seeking, novel tank diving and scototaxis tests. The results demonstrate that behavioral variation exists both within and between the strains, but interpretation of boldness reveals a complex pattern in which behavior differs between strains but is also related to sex and test. Therefore, a careful assessment of various strains of fish using both males and females is warranted in order to strengthen interpretation of results. This is especially important in studies where zebrafish are used as model organisms for human conditions as well as studies evaluating the effects of pharmacological or toxicological substances on behavior.

This thesis investigates if boldness is reflected in the function of brain histaminergic system in zebrafish (Danio rerio). Moreover, behavioural differences in AB line, spiegeldanio (spd) line and wild caught strain of zebrafish have also been explored apart from the winner-loser effect in AB and spd fish. This thesis also includes studies on the effect of progestins on reproductive behaviour in zebrafish and regulation of leptinergic system on sexual maturation in male Atlantic salmon (Salmo salar L.).

Boldness is reflected in higher expression of histamine receptor 1 (hrh1) in the telencephalon and diencephalon of male zebrafish and dominance by an elevated expression of hrh1 in the optic tectum. In female zebrafish boldness is also associated with lower expression of histamine receptor 3 (hrh3) in the optic tectum and dominance by lower expression of hrh3 in the telencephalon. Comparison of behavioural traits of zebrafish of AB, spd and wild type shows that wild type strain is most shy and shows no gender difference. AB is bolder than spd in the open field test while spd is bolder AB in the novel tank dive test. Similarly results for aggression are also test dependent since the spd is more aggressive than AB in the mirror test, however no difference is measured during dyadic fight test. A typical loser effect and activation of serotonergic system is observed in both AB and spd fish. Further, both levonorgestrel (LNG) and progesterone (P4) cause an early puberty in male zebrafish. However only levonorgestrel causes males biased population at environmental concentrations. In male Atlantic salmon, during early spring, both leptin paralogues, lepa1 and lepa2 in the liver and leptin receptor (lepr) in the brain are downregulated in non-maturing control group. At final maturational stage both hepatic lepa1 and lepa2 are upregulated 7.7 times and 49 times respectively in maturing control males. A significant upregulation of lepr is also measured from mid to late spermatogenesis.  

This thesis elucidates that an elevated brain histaminergic tone is associated with boldness and dominance and in both sexes changes at gene level are orchestrated by different brain region. Boldness is a contextual trait as it depends on strain, line, sex and test. The loser effect after losing a fight is present in both AB and spd line, however it has been shown for the first time in spd line here. Only androgenic progestin causes male biased population but both androgenic and anti androgenic progestin cause early puberty in zebrafish. The expression of leptinergic system is significantly affected during early sexual maturation in parr stage of salmon. Moreover, depleted fat stores are associated with low leptin levels and feed restriction is association with an elevated leptinergic tone in liver and pituitary. This thesis not only emphasizes that strain vs line difference exists and should be an important criterion before designing any experiment, but it also indicates an important role histaminergic system, progestins and leptinergic system in divergent behaviour profiles, puberty and sexual maturation, respectively of teleosts and contributes to our understanding of it.

Zebrafish which carries a mutation in the fibroblast growth factor receptor 1A (fgfr1a), also known as spiegeldanio (spd), has previously been reported to be bolder and more aggressive than wildtype (AB) zebrafish. However, in previous studies aggression has been quantified in mirror tests. In dyadic fights the behavior of the combatants is modified by the behavior of their opponent, and fighting a mirror has been reported to have different effects on brain gene expression and brain monoaminergic systems. In the present study aggression was quantified in fgfr1a mutants and AB zebrafish using a mirror test after which the fish were allowed to interact in pairs, either consisting of two fgfr1a mutants or one AB and one fgfr1a mutant fish. Following dyadic interaction aggressive behavior was again quantified in individual fish in a second mirror test after which the fish were sacrificed and brain tissue analyzed for monoamines and monoamine metabolites. The results confirm that fgfr1a mutants are more aggressive than AB zebrafish in mirror tests. However, fgfr1a mutant fish did not have any advantage in fights for social dominance, and agonistic behavior of fgfr1a mutants did not differ from that of AB fish during dyadic interactions. Moreover, as the AB fish, fgfr1a mutant fish losing dyadic interactions showed a typical loser effect and social subordination resulted in an activation of the brain serotonergic system in fgfr1a mutants as well as in AB fish. Overall the effects of dyadic interaction were similar in fgfr1a mutant fish and zebrafish of the AB strain.

Introduction

Aggression is a competition based survival strategy. The spiegeldanio (spd) strain of zebrafish (Danio rerio), which has a mutation in the fibroblast growth factor receptor 1a, is bolder and more aggressive than the wild type fish [1]. Usually a socially dominant fish has preferential access to food, mate and shelter, and shows very characteristic postures like erection of the fins. It is also aggressive frequently biting, striking and chasing the subordinate fish as well as threatening its own mirror image in mirror tests [2]. However, what happens when an already known bold and dominant fish like spiegeldanio loses a dyadic fight. Spd fish are more aggressive in mirror tests, attacking their mirror image more frequently than wild type conspecifics. However, are they more aggressive in dyadic fights? Do they show an inhibition of aggressive behaviour when losing fights, the typical loser effect? The behavioural inhibition observed in animals losing fights for dominance is at least in part believed to be mediated by an activation of the brain serotonin (5-hydroxytryptamine, 5-HT) system. Do spd fish show a typical increase in brain 5-HT activity in response to social subordination? Dopamine (DA), on the other hand, is associated with aggression and social dominance. What are the effects of winning and losing fights for social dominance in spd fish? In the present study these questions were addressed in an attempt to increase or understanding of the control of agonistic behaviour and social stress.

Animals and Methods

The Spd strain of zebrafish were raised and reared at 27°C in an Aquaneering Zebrafish system at Uppsala University Biomedical Center. The animals were kept at a 14:10 h of light-dark photoperiod. The water used in the fish tanks was Uppsala municipal tap water (pH 7.2-7.6) of which 10% was exchanged daily. Fish were fed twice daily with Tropical energy food (Aquatic Nature, Belgium) and Artemia (Platinum Grade 0, Argentemia, Argent, Aquaculture, Redmond, USA). The use of animals was approved by the Uppsala Animal Ethical Committee (permit Dnr 55/13) and followed the guidelines of the Swedish Legislation on Animal Experimentation (Animal Welfare Act SFS1998:56), and the European Union Directive on the Protection of Animals Used for Scientific Purposes (Directive 2010/63/EU). The fish were transferred to the individual compartments of dimension 29 x 7.5 x 20 cm (length x breadth x height) in experimental tanks used for dyadic interaction and allowed to recover in isolation overnight. These experimental tanks were made from poly methyl methacrylate plastic and each tank was equipped with a submerged pump with filter (Eheim, typ 2006020, pumping capacity 1/h180, made in China), a heater (Sera aquarium, 25W, made in EU) and an air stone, all of which were placed at the back of the tank separated from the fish by a white perforated PVC screen (Figure 1). The setup of the arena was such that the two fish (1 dyadic pair) had an olfactory but not any visual cue of each other before the dyadic interaction. In the mirror test the fish were made to fight against the mirror image that was displayed in the mirror which was pasted on the wall of the arena. Prior to the beginning of the dyadic contest the mirror was covered with a black plexiglas slide cover. The experiment was carried out in the following sequence: The fishes were netted out and placed in the arena in the compartments A and B (Figure 1) and separated from each other by a partition. The cover of the mirror (opaque black PVC partition, Figure 1) was then removed and fish were made to interact with their own mirror image for 10 minutes. Then the slide covering the mirror was pulled down and the middle separating partition was pulled out and the fish were given an opportunity to fight. Dyadic fight was recorded two times, morning and evening on day one with the help of a video filming camera. Then next day in the morning the dyadic fight was again recorded. During the dyadic interaction the two fishes indulged in mutual display of aggressive behaviour which was followed by chasing and biting attacks performed by the dominant fish over the subordinate fish. Then middle partition was introduced again. Fish were given 6 minutes to habituate and the cover from the mirror was removed and fishes were again allowed to interact with their mirror image. Again the mirror was covered and the fish was allowed to get involved in the dyadic fight.  Then each fish was taken out from the compartment at the same time and sacrificed for sampling of brain tissue.

The three dimensional model of tank used in the behavioural tests I) Tank used for mirror test and for dyadic fight later on. It consists of two compartments, A and B. The movable partition separating the two compartments would be removed during the dyadic fight test. Compartment C is located at the back and is separated from the compartment A and B with the help of white coloured opaque perforated partition. It contains an air stone (for diffusion of air bubbles), heater (27°C), water pump (for circulation of water) and a drainage tube to exchange the water. II) Diagram of the settings used for dyadic interactions. The mirrors are covered with the help of a black PVC slide and the middle partition is pulled out. This allows the fish to interact.

Brain dissection and analysis of monaoamines and monoamine metabolites

Brains were divided into forebrain (telencephalon and diencephalon), optic tectum and the rest (here denoted brain stem). The frozen brains were homogenised in 4% (w/v) ice-cold perchloric acid containing 100 ng/ml 3, 4-dihydroxybenzylamine (DHBA, the internal standard) using a Sonifier cell disruptor B-30 (Branson Ultrasonics, Danbury, CT, USA) and were immediately put on dry ice. Subsequently, the homogenised samples were thawed and centrifuged at 15,000 rpm for 10 min at 4^o C. The supernatant was used for high performance liquid chromatography with electrochemical detection (HPLC-EC), analysing the monoamines dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) as well as the DA metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), as described by Øverli et al. [3]. In short, the HPLC-EC system consisted of a solvent delivery system model 582 (ESA, Bedford, MA, USA), an autoinjector Midas type 830 (Spark Holland, Emmen, the Netherlands), a reverse phase column (Reprosil-Pur C18-AQ 3 µm, 100 mm × 4 mm column, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) kept at 40° C and an ESA 5200 Coulochem II EC detector (ESA, Bedford, MA, USA) with two electrodes at reducing and oxidizing potentials of -40 mV and +320 mV. A guarding electrode with a potential of +450 mV was employed before the analytical electrodes to oxidize any contaminants. The mobile phase consisted of 75 mM sodium phosphate, 1.4 mM sodium octyl sulphate and 10 µM EDTA in deionised water containing 7 % acetonitrile brought to pH 3.1 with phosphoric acid. The quantification of samples was done by comparing it with standard solutions of known concentrations. DHBA was used as an internal standard to correct for recovery with the help of HPLC software ClarityTM (Data Apex Ltd, Czech Republic). The serotonergic and dopaminergic activity was measured as the ratio of 5-HIAA/5-HT and DOPAC/DA respectively. The brain monoamines were normalized with respect to brain protein weights which were determined with Bicinchoninic acid protein determination kit (Sigma Aldrich, Sweden). The assay was read at a wavelength of 570 nm with the help of a plate reader (Labsystems multiskan 352, Labsystems Thermo Fisher Scientific).

Results

A clear dominant subordinate hierarchy was established within 30 minutes of dyadic interaction. The number of aggressive acts (bites, strikes and chases) performed by the looser fish decreased significantly from the first dyadic fight to the last (i.e. the fourth) dyadic fight. For the winner fish the number of aggressive acts performed against a mirror during the second mirror test increased or remained same as before after winning a dyadic fight, whereas for the looser fish it decreased significantly. The results from the present study indicate that subordinate fish have higher 5-HIAA/5-HT ratio in the optic tectum as compared to the dominants. More results from this study would be presented at the conference.

References

1. Norton W, Bally-Cuif L (2010) Adult zebrafish as a model organism for behavioural genetics. BMC Neurosci. 11:90.

2. Rowland WJ (1999) Studying visual cues in fish behaviour: a review of ethological techniques. Env Biol Fishes. 56:285-305.

3. Øverli Ø, Harris CA, Winberg S (1999) Short-term effects of fights for social dominance and the establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow trout. Brain Behav Evol. 54:263-275.

Background: Female threespine sticklebacks, Gasterosteus aculeatus, are batch spawners. As in most teleosts, the ovulated eggs are kept in the ovarian cavity until spawning. If spawning or spontaneous release of the eggs does not take place, they can become overripe and harden, and in most cases remain in the ovary. The overripe eggs are lost for reproduction and also block further spawnings. Reproductive hormones regulate egg production and may be involved in the mechanism of overripening. Question: What are the reproductive endocrinological parameters characterizing overripening of ovulated eggs in the threespine stickleback? Organism: Wild-caught adult threespine sticklebacks from the southern Baltic at Skare in southern Sweden and the island of Asko in northwestern Baltic Proper in Sweden. Experiments: We collected blood samples for hormone measurements, as well as pituitaries and brains for measurement of mRNA from both sexually mature non-overripe (non-ovulated and/or ovulated) and overripe (egg-bound) females. For the Skare fish, sexual maturation was induced under laboratory conditions by exposure to a long photoperiod and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. The Asko fish were sampled directly from nature, during the natural summer breeding season and we compared the non-overripe (including non-ovulated, with oocytes in different maturing or ripening stages, and ovulated females) with the overripe females. Methods: In the fish collected from Skare, we used radioimmunoassay to measure the plasma levels of four steroids: testosterone, estradiol, 17,20 beta-dihydroxypregn-4-en-3-one (17,20 beta-P), and 17,20 beta, 21-trihydroxypregn-4-en-3-one (17,20 beta,21-P). We also measured the mRNA levels of gonadotropins [GTHs: follicle-stimulating hormone (fsh-beta) and luteinizing hormone (lh-beta)] in the pituitary, and of gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain by real-time quantitative PCR. In the fish collected from Asko, we measured only progestogens (17,20 beta-P and 17,20 beta,21-P). Results: In the fish from Skare, overripe female sticklebacks had significantly lower levels of circulating plasma steroid hormones (testosterone, estradiol, 17,20 beta-P), as well as of pituitary lh-beta and brain kiss2 and gpr54 mRNA than the non-overripe females. In the fish caught from Asko, overripe females had lower 17,20 beta-P levels than the non-overripe non-ovulated females, but there was no difference between the non-overripe ovulated and the overripe females. The 17,20 beta,21-P plasma levels were under the limit of detection in all groups.

In mammals, leptin acts as an adiposity signal and is a crucial link between nutritional status and the reproductive axis. So far the link between leptin and energy balance during sexual maturation in teleosts has been poorly investigated. In this study, seasonal gene expression changes in two leptin genes (lepa1 andlepa2) and the leptin receptor were investigated during early sexual maturation in male Atlantic salmon parr under fully fed (control) and feed restricted conditions from April through September. Both Atlantic salmonlepa1 and lepa2 in the liver and lepr in the brain were significantly down-regulated in non-maturing control males in early spring, coinciding with the start of the growth and fat accumulation. In maturing control males, hepatic leptin expression increased during mid-spermatogenesis and lepa1 and lepa2 mRNA levels were up-regulated by 7.7 and 49 times respectively during final maturation. For the first time in a fish species, a significant up-regulation of lepr expression was observed in the testis throughout mid to late spermatogenesis. Feed restriction decreased the incidence of sexual maturation by 53% and highly up-regulated both leptin genes in the liver and the leptin receptor in the pituitary. This study shows that hepaticlepa1 and lepa2 expression and lepr expression in the testis is affected by early sexual maturation in male Atlantic salmon parr. Fast growth and high fat stores are associated with low leptin levels while feed restriction has a stimulatory effect on hepatic leptin and leptin receptor gene expression in the pituitary, suggesting a role for leptin other than that as an adiposity signal.