探討Kryptolebias marmoratus獲得輸贏經驗後體內雄性激素、雌性激素、皮質醇與血清素受器基因表現量逐時變化情形

全文

(1)國立臺灣師範大學生命科學系碩士論文. 探討 Kryptolebias marmoratus 獲得輸贏經驗後體內雄性激素、雌性激素、皮質醇與血清素受器基因表現量逐時變化情形 Temporal Changes in Androgen, Estrogen, Cortisol and 5-HT1A Receptor Gene Expression after Wins and Losses in Kryptolebias marmoratus. 研究生：李誠裕 Cheng-Yu Li. 指導教授：許鈺鸚博士 Yuying Hsu Ph. D.. 中華民國九十九年十二月.

(2) 謝辭隨著論文付梓，兩年半的碩士研究生涯即將劃下句點。這段時間內，幸有許多人的支持與協助，本研究才能順利完成。首先我要感謝指導教授許鈺鸚老師，老師總是花許多時間在學生身上，不厭其煩的在我遇到困難時給我鼓勵和指導，也支持我接受挑戰；無數次的口頭報告預講，實驗討論和論文修改，多虧老師的耐心提點，讓我從中獲益良多，也很慶幸可以在老師的門下學習。這本論文的順利完成，特別還要感謝Alan在英文上的潤飾與修改，讓論文能清楚呈現實驗精髓。再來我要感謝Dr. Ryan Earley與淑萍學姐。Dr. Earley不僅在本研究中提供實驗儀器設備、實驗技術上指導與協助，更帶領我認識了國外實驗室研究風氣，鼓勵我再繼續往學術路上深造；而淑萍學姐全程協助我所有實驗進行，有她的幫忙才可以在短短四個月內有效率的收集全部實驗數據。此外，也要感謝三位口試委員，包括台灣師範大學生命科學系李壽先博士以及東海大學生命科學系卓逸民博士，對我的論文提出許多寶貴的建議與指教。在此也要感謝行政院國家科學委員會補助研究經費（計畫編號：NSC97–2621–B–003–005–MY3）。由衷感謝我實驗室夥伴怡婷、張靖、郁雲、家怡和明義。有你們分擔疲累與苦悶、給予我許多課業上或生活上的建議與關懷，讓這段研究生歲月在忙碌以外，充滿了許多樂趣。我也要感謝Adam、Amanda、Boopathy、Mark與Stephanie，學長姐宗楷、依涵，學弟妹儆寰、昱儒、暐霖, 韋稔對這份研究提出的寶貴意見。感謝我最愛的家人們，也許你們不了解我的研究，卻是支持我向前的最大動力。最後，謝謝一直默默支持我的郁欣，每次的開心榮耀都有你分享，每次的挫折沮喪也都有你陪伴，謝謝妳一路以來的鼓勵與信任。. i .

(3) 摘要許多動物的打鬥呈現出一個共同的現象，亦即前次打鬥之勝、負經驗會提升或降低個體在後續打鬥中的攻擊性與獲勝機率，稱之為勝者、敗者效應。由於動物的攻擊性被發現與神經內分泌系統有密切的關係，因而勝者、敗者效應也被認為應受到神經內分泌系統之調控。先前的研究顯示 Kryptolebias marmoratus 之打鬥行為呈現出明顯的勝敗者效應，而且其打鬥行為與睪固酮（testosterone）和皮質醇（cortisol）分泌量有顯著的關聯；然而，此魚之勝敗者效應卻非經由這些荷爾蒙或雌性激素（estrogen）、睪丸硬甾酮（11-ketotestosterone）等之分泌量所調控形成。由於荷爾蒙除了分泌量的變化外，其受器蛋白的表現量多寡亦會影響個體之攻擊性與打鬥行為，因此我於本研究中探討個體在獲得勝、敗或者控制經驗後，其腦部與生殖腺體之雄性激素受器（androgen receptor）、雌性激素 α/β 受器（estrogen α/β receptor）、血清素 1A 受器（5-HT1A receptor）與葡萄糖皮質素受器（glucocorticoid receptor）等受器基因表現量是否有所差異。實驗結果顯示，擁有三次落敗經驗的個體，其腦部之雄性激素受器基因與生殖腺之雌性激素 α 受器基因表現量顯著低於擁有一次控制經驗的個體；而在其他受器基因表現量上，各經驗處理組間並無顯著差異。由於贏的經驗與一次落敗經驗並未顯著的影響任何受器基因表現量，本研究結果顯示這些受器基因表現量應非此魚之勝、敗者效應之主要調控機制。然而本研究之其他分析顯示，接受落敗經驗處理的個體，其基礎睪固酮分泌量與個體在接受第一次落敗經驗訓練時的攻擊性行為（先開鰓蓋展示機率與打鬥持續時間）呈現顯著的正相關；而腦部受器基因表現量（雄性激素受器除外）也與個體在接受第一次經驗訓練時之攻擊性行為（先展示機率，先開鰓蓋展示機率，打鬥持續時間）呈現顯著正相關。這些結果顯示此魚之攻擊性仍與荷爾蒙以及荷爾蒙/血清素受器有密切的關係。因此，整體而言，本研究顯示荷爾蒙以及荷爾蒙/血清素受器雖然與此魚之攻擊性有密切的關係，卻非此魚展現勝、敗者效之主要生理機制。關鍵字：勝者／敗者效應、荷爾蒙受器、血清素受器、打鬥行為、紅樹林鱂魚 . ii .

(4) Abstract Recent winning/losing experiences can increase/decrease an individual’s aggressiveness and its probability of winning subsequent contests, phenomena referred to as winner/loser effects. Winner and loser effects are probably mediated by the neuroendocrine system because animals’ aggression is closely associated with this system. Kryptolebias marmoratus displays winner and loser effects and its contest behaviors are associated with testosterone and cortisol levels. It appears, however, that neither winner nor loser effects are mediated by changes in the levels of testosterone or cortisol in this fish. Because hormone receptors could also modulate the effect of hormones on behavior, in this study I investigated associations in this fish between androgen receptor, estrogen α/β receptor, 5-HT1A receptor and glucocorticoid receptor gene expression levels and winning and losing experiences. The results showed significantly reduced expression levels of androgen receptor genes in the brains and estrogen α receptor genes in the gonads of individuals with three losing experiences. None of the experience treatments led to differences in brain or gonad estrogen β receptor, 5-HT1A receptor or glucocorticoid receptor gene expression levels. These results did not establish hormone/serotonin receptors to be the primary physical mechanism underlying winner and loser effects. Nonetheless, in those individuals assigned losing experience(s), the baseline hormone (testosterone) levels and the receptor gene expressions levels (except androgen receptor) were positively correlated with aggressive behavior (initiating displays, initiating gill displays and contest duration) in the 1st experience training. These results did demonstrate a relationship between fighting experience and hormones and hormone/serotonin receptor gene expressions. I concluded from these findings that although hormones and hormone/serotonin receptors have important associations with the fish’s aggressiveness, they are not the major physiological mechanisms underlying winner or loser effects in this fish. Keyword: winner/loser effect, hormone receptor, 5-HT1A receptor, contest behavior, Kryptolebias marmoratus. iii .

(5) Contents Introduction…………………………………….……..……………………..…...01 1.1 1.2 1.3 1.4 1.5. Animal Contest and Winner/Loser Effects………..…………..………………….…………………01 Contest Behavior and Neuroendocrine System…………………...…………….…….……...……..02 Winner/Loser Effects and Neuroendocrine System…………….…...………………...……......…...07 Previous Studies of Kryptolebias marmoratus…………….………..……………...………....…….08 Objectives……………………………………………………………...……...…….………………09. Materials and Methods……………………………………..…………..…….….11 2.1 2.2 2.3 2.4. Study Organism…………………………………………………………...………….....…………...11 Experimental Design and Procedures……………………………………..…………..…………….12 Providing a Losing/Winning Experience…………………………………..………….…………….14 Definitions of Contest Behaviors in Experience Training………...….……..….…….…...…….16. 2.5 Hormone Extraction and Assay…………………………………………….........……….…………16 2.6 Quantifying Receptor-Gene Expression………………………………………...………....………..17 2.7 Data Analysis…………………………………………………………………..………….………...20. Results…………………………………………………….…….…..……….……23 3.1 The Relationships between Baseline Hormone Levels and the Contest Behaviors in the 1st Experience Training……………………………………...………..……………...……………....…23. 3.2 Effect of Experience Type and Decay Time on Post-Experience Receptor-Gene Expression Levels………………………………………………………………………………..……………....24. 3.3 Relationships between the Contest Behaviors in the 1st Experience Training and Post-experience Receptor-Gene Expression Levels in Brain Tissue.……………………………………..………..…25. 3.4 The Relationships between Baseline Hormone Levels and Post-Experience Receptor-Gene Expression Levels………………………………………………………………………...................26. Discussion…………………………………………………………….…...……...27 4.1 Effect of Experience Type on Post-Experience Receptor-Gene Expression Levels………..……....27 4.2 The Relevance of the Fish’s Baseline Hormone levels and Post-Experience Receptor-Gene Expression Levels in Brain to the Fish’s Contest Behaviors……………………………………......31. 4.3 Other Possible Mechanisms Mediating Winner and Loser Effects…………..………..….………...34 4.4 Conclusions……………………………………………………………………………..….………..35. References………………………………………………..……………..….……..37 Tables and Figures……………………………………….……….…….…..……46 Supplemental Data……………………………………..…………………...…...57 iv .

(6) . Introduction. 1.1. Animal Contest and Winner/Loser Effects Animals often fight with each other over access to limited resources (mates, breeding sites, food, shelters etc.). As fighting is potentially costly to contestants (e.g. in time and energy, Neat et al. 1998; higher predation risk, Brick 1999; physical injuries, Austad 1983), it is beneficial for animals to evaluate the potential costs and benefits associated with a contest and adjust their fighting strategy accordingly (Smith & Price 1973; Smith & Parker 1976). Many studies have shown that animals can assess the quality and quantity of a resource: individuals usually fight longer and more intensely for more valuable resources. For example, male jumping spiders (Euophrys parvula) were more willing to escalate fights when a model female was presented (Wells 1988). Individuals’ assessments of resources can also be influenced by their internal states. For instance, hungrier hermit crabs were more aggressive and more likely to fight for food than less hungry ones (Laidre & Elwood 2008). That potential contest costs can influence animals’ contest decisions has also been demonstrated in numerous studies. Male cichlid fish (Nannacara anomala), for example, used lower-intensity contests and took longer to escalate into mouth wrestling when a model predator was presented, because intensifying a contest would compromise an individual’s ability to monitor predators (Brick 1999). These examples show that animals’ contest behavior is sensitive to the costs and benefits associated with the contest. Past contest experience has also been found to influence individuals’ behavior in subsequent contests. Individuals with a recent winning experience often become more aggressive, are more likely to initiate future contests and to win again (winner effect),. 1 .

(7) . while individuals with a recent losing experience tend to display the opposite behavior and lose again (loser effect). (See Hsu et al. 2006 for a review.) Winner and loser effects are usually hypothesized to result from prior winning and losing experiences affecting an individual’s assessment of its own fighting ability and therefore its estimation of contest costs (Whitehouse 1997). Winner or loser effects have been reported for animals of all different taxa, such as burying beetle (Nicrophorus humator, Otronen 1990), crab spider (Misumenoides formosipes, Hoefler 2002), crayfish (Orconectes rusticus, Bergman et al. 2003), Siamese fighting fish (Betta splendens, Baenninger 1970), copperhead snake (Agkistrodon contortrix, Schuett 1997) and blue-footed booby (Sula nebouxii, Drummond & Canales 1998). Despite the prevalence of winner/loser effects, the physiological mechanisms underlying them are still unclear.. 1.2 Contest Behavior and the Neuroendocrine System Contest behavior is modulated by the neuroendocrine system; steroid hormones (e.g. androgens, estrogens and glucocorticoids) and neurotransmitters (e.g. serotonin, dopamine and norepinephrine) are frequently explored because of their associations. with aggressiveness and dominance status (see Nelson & Chiavegatto 2001 for a review). There have been many studies of the relationship between contest behavior and steroid hormones and/or neurotransmitters and many of their results are contradictory. Their major findings are summarized below.. 1.2.1 Steroid Hormones and Contest Behavior. Androgens. 2 .

(8) . Androgens are steroid hormones which mediate the development and maintenance of male sex organs and secondary sex characteristics in vertebrates by binding to androgen receptors. In vertebrates, androgens are mainly produced by the gonads, but the adrenal cortex also secretes low levels of them. Contest interaction may directly affect gonadotropin-releasing hormone neurons (GnRH neurons) to activate the hypothalamic-pituitary-gonad (HPG) axis and stimulate the secretion of androgens (Francis et al. 1993). While testosterone is the primary androgen in reptiles, birds and mammals, 11-ketotestosterone is the primary androgen in fish (Borg 1994; Vermeulen et al. 1994). Androgen levels and receptor expression levels are frequently shown to affect an individual’s contest behavior and social status. For instance, individuals with higher (natural or exogenous) testosterone levels tend to behave more aggressively and/or achieve higher social status in mice (Mus musculus, Zielinski & Vandenbergh 1993) and lambs (Ovis aries, Ruiz-de-la-torre & Manteca 1999); male mice exhibiting a spontaneous mutation that fails to generate the long form of the androgen receptor are less aggressive than normal individuals (Olsen 1983; Maxson 2000). Contest interaction and social status, interestingly, also affect an individual’s androgen levels. Defeated rhesus monkeys (Macaca mulatta), rodents and birds usually have depressed plasma testosterone levels (Harding 1983; Leshner 1983; Huhman et al. 1991), and repeated winning experiences significantly increased testosterone levels in California mice (Peromyscus californicus, Oyegbile & Marler 2005, 2006).. Estrogens Estrogens are a group of steroid compounds which promote the development of female secondary sexual characteristics and are involved in regulating the menstrual. 3 .

(9) . cycle. In vertebrates, estrogens are mainly produced in the ovaries by the developing follicles and the corpus luteum. The adrenal cortex and the mammary glands, however, also secrete low levels of them (Goodman 2008). Research indicates that levels of both estrogen and estrogen receptor gene expression affect an individual’s contest behavior and aggressiveness. Injection with estradiol promotes male-like aggressive behavior and increases the probability of winning contests in female mice (Simon & Gandelman 1978). Of three kinds of estrogen receptors (α-, β- and γ-isoform, Giguère et al. 1988; Hong et al. 1999), estrogen α and β receptors were most well-studied and associated with contest behavior and aggressiveness. After a targeted knockout of the estrogen-α receptor gene (ERαKO), male mice exhibit decreased levels of aggression in resident-intruder paradigm tests, while, after a knockout of the estrogen-β receptor (ERβKO), mice display normal or increased levels of aggression. ERαKO female mice, however, exhibit increased aggression towards other intruder females mice relative to wild-type females (Ogawa et al. 1997; Ogawa et al. 1998; Ogawa et al. 1999; Ogawa et al. 2000). These results revealed that α- and β-isoforms of estrogen receptor might mediate aggression in opposite directions, and that the effects of the α-isoform on aggression might differ between male and female mice. Contest interaction and social status also affect an individual’s estrogen-receptor expression; for example, subordinate or defeated male African cichlids (Astatotilapia burtoni) have lower ERβa and ERβb levels in the anterior brain than dominants or winning males (Burmeister et al. 2007).. Glucocorticoids Fighting can induce short-term but strong stress responses in animals (Miczek et. 4 .

(10) . al. 2002). In vertervrates, the stress response is mainly mediated by the neuroendocrine system. In mammals, stress can induce changes in the hypothalamic-pituitary-adrenal axis (HPA axis) which activates the adrenal cortex to release. glucocorticoids;. in. fish,. stress. acts. through. the. hypothalamic-pituitary-interrenal axis (HPI axis) activating the interrenal gland to release glucocorticoids. Glucocorticoids increase an individual’s ability to face a stressful environment (McEwen 2000) by supressing the function of insulin, decreasing the synthesis of glycogen and elevating the level of blood glucose (Genuth 1993). The primary glucocorticoid is corticosterone in amphibians, reptiles, birds and rodents and cortisol in fish and primates (Wendelaar Bonga et al. 1995). Glucocorticoid levels and receptor-expression levels affect an individual’s contest behavior and aggressiveness. For example, long-term cortisol treatment in rainbow trout (Oncorhynchus mykiss) inhibits aggressive behavior, while short-term cortisol treatment does not (Øverli et al. 2002). In lizards (Anolis carolinensis), blocking cortisol receptors with mifepristone reduces aggressive attacks/displays during the early stages of aggressive interaction (Summers et al. 2005). An individual’s glucocorticoid levels are influenced by contest interaction: losing a fight often induces an increase in glucocorticoid levels (Sakakura et al. 1998; Schuett & Grober 2000; Øverli et al. 2004) and a loser’s post-fight glucocorticoid level correlates with the amount of aggression it was subjected to in the fight (Winberg & Lepage 1998; Elofsson et al. 2000; Sloman et al. 2001; Earley & Hsu 2008).. 1.2.2 Neurotransmitters and Contest Behavior Several neurotransmitters (e.g. serotonin, NO, dopamine) are closely associated with animal aggression. Among these, serotonin (5-hydroxytryptamine; 5-HT) has. 5 .

(11) . been associated not only with aggressive behavior (Edwards & Kravitz 1997; Weiger 1997) but also with dominance status (Winberg & Nilsson 1993). Serotonin is synthesized from L-tryptophan via tryptophan hydroxylase (Aldegunde et al. 2000). Neurons producing and storing serotonin are located primarily in the raphe nuclei of the brainstem. In mammals the projections of the raphe nuclei extend to various brain regions, including the hippocampus, cerebral cortex, amygdala, hypothalamus and pituitary gland. Once released into synapses, serotonin binds to various receptors (Hoyer et al. 2002). Serotonin is mainly metabolized to 5-hydroxyindoleacetic acid (5-HIAA) by monoamine oxidase (MAO). The activity of the serotonergic system or the turnover of serotonin is often reported as the ratio of 5-HIAA: 5-HT (Winberg et al. 1992; Koutoku et al. 2003; Dias & Crews 2006). Serotonin and related receptor-expression levels influence an individual’s contest behavior and aggressiveness. Acute serotonin treatment, for example, decreases aggressive behavior (latency to attack and duration of attacks) in Siamese fighting fish (Betta splendens, Clotfelter et al. 2007). And, blocking the serotonin reuptake receptors (i.e. facilitating chronic serotonin elevation) of dominant individuals causes these individuals to become less aggressive (fewer attacks and displays) and lose to previous losers in the green anole lizard (Anolis carolinensis, Larson & Summers 2001). Activating 5-HT1A receptors reduces aggressive behavior: acute treatment with a 5-HT1A receptor agonist decreases aggressive behavior in Siamese fighting fish (Clotfelter et al. 2007). Not surprisingly, contest interaction in turn affects an individual’s serotonin level. For instance, subordinate or defeated male lizards (Anolis carolinensis) have significantly increased 5-HIAA/5-HT ratios in the hippocampal cortex and the nucleus accumbens after a fight, ratios which gradually decrease over a week (Summers et al. 1998). Because of the close association between the neuroendocrine system and an. 6 .

(12) . individual’s contest decisions, the influence of a recent winning/losing experience on an individual’s contest decisions is probably mediated through this circuit. My research project therefore aimed to explore this possibility.. 1.3 Winner/Loser Effects and the Neuroendocrine System If winner and loser effects are mediated by the neuroendocrine system, winning and losing experiences may temporarily alter an individual’s hormone or receptor-expression levels which may then affect its subsequent contest behavior. Several studies have investigated the relationship between the winner effect and testosterone levels, while there is no literature regarding the physiological mechanism underlying the loser effect. In a study of California mice (Trainor et al. 2004), castrated male individuals were given a forced winning experience and then injected with testosterone. (Members of the control group were injected with saline.) The result was that individuals injected with testosterone displayed a significant winner effect, while the control individuals did not. This study indicated that a winner effect might accompany a temporary increase in testosterone. It is also possible, however, that the behavior changes simply because of the exogenous testosterone rather than because of a winner effect. In another study of male California mice, the authors compared effects of three, two, one or zero winning experiences on the probability of winning a subsequent encounter; testosterone and corticosterone were also measured after the final encounter (Oyegbile & Marler 2005). The results showed that individuals with more winning experiences had a significantly higher probability of winning a subsequent encounter (winner effect) and had significantly higher post-encounter testosterone. However, because the hormone levels immediately after winning experiences were. 7 .

(13) . not measured, these results could not demonstrate a direct linkage between winning experiences and the circulating testosterone levels. One further study by Fuxjager et al. (2010) also investigated the relationship between the winner effect and androgen-receptor gene expression. The author provided male California mice with three winning experiences in either their home cage or unfamiliar cages, and then compared the probability of winning a subsequent encounter in their home cage or unfamiliar cages; androgen- and progestin- receptor expression levels in various brain regions were also measured after the final encounter. The results showed that individuals with winning experiences and a subsequent encounter in thei home cage had significantly higher androgen-receptor gene/protein expression in the nucleus accumbens and ventral tegmental area brain regions. This study indicated that winning experiences in combination with a contest in home cage increased androgen receptor gene/protein expression in specific brain regions (NAcc & VTA regions). However, because individuals which received three winning experiences in their home cage displayed higher aggression in subsequent contests and the expression levels were measured after the contests (but not after winning experiences), it is possible that the androgen receptor gene/protein expressions increased because of the elevated aggressive interaction in the home-cage contests, rather than because of the winning experiences. The results of the study, unfortunately, could therefore not demonstrate a direct link between winning experiences and the elevated androgen receptor gene/protein expression. Overall, whether winner/loser effects are mediated by hormone levels or receptor levels remains unclear although both hormones levels and receptor expression levels are highly correlated with contest behavior.. 1.4 Previous Studies of Kryptolebias marmoratus. 8 .

(14) . Kryptolebias marmoratus is a species of mangrove killifish. It is an ideal organism for examining the behavioral and the neuroendocrine mechanisms underlying winner and loser effects, because it displays both winner and loser effects and because these effects last at least 48 hours (Hsu & Wolf 1999, 2001). Previous studies also demonstrated that the contest behavior in this fish is highly correlated with hormone levels (Earley & Hsu 2008). Pre-fight cortisol related negatively and pre-fight testosterone related positively to contest initiation and winning probability, particularly in the smaller opponent. In pairs where the larger fish won the contest, winners with higher pre-fight testosterone and lower pre-fight cortisol attacked the losers more frequently. Losers that escalated with winners had significantly higher post-fight levels of all three hormones than losers that retreated without escalation. Also, winners that attacked losers at higher frequency had higher levels of post-fight cortisol (Earley & Hsu 2008). In this fish, however, levels of testosterone, 11-ketotestosterone, estradiol and cortisol did not change after (randomly assigned) wins or losses (Lee 2009; Lu 2010), which indicated that winner and loser effects are not mediated by the circulating levels of these hormones. Because receptors could also modulate these hormones’ effect on behavior, I further explored the relationship between winner/loser effect and receptors in this fish by investigating the changes in receptors after the fish received randomly assigned winning or losing experiences.. 1.5 Objectives The primary objective of this study was to use K. marmoratus to explore the roles of hormone and serotonin receptors in mediating winner and loser effects. To do this, I examined the temporal changes (0 hr, 3 hr, 48 hr after experience training) in glucocorticoid receptor (GR), androgen receptor (AR), estrogen receptor α (ERα),. 9 .

(15) . estrogen receptor β (ERβ) and 5-HT1A receptor (5-HT1AR) gene expression levels after both wins and losses. Furthermore, the fish were given either one or three winning, losing or no recent contest experiences to examine whether a different number of experiences would have a different effect on these receptors. If winner and loser effects are mediated by the changes in receptor expressions, I would expect to discover that: (1) For those individuals with winning experience(s), AR and ERα gene expression levels increase in 0 hr and 3 hr after experience training, then gradually decrease in 48 hr, and/or ERβ, 5-HT1AR and GR decrease in 0 hr and 3 hr after experience training, then gradually increase in 48 hr. (2) For those individuals with losing experience(s), AR and ERα gene expression levels decrease in 0 hr and 3 hr after experience training, then gradually increase in 48 hr, and/or ERβ, 5-HT1AR and GR gene expression levels increase in 0 hr and 3 hr after experience training, then decrease in 48 hr.. 10 .

(16) . Materials and Methods. 2.1 Study Organism The mangrove killifish K. marmoratus is one of two vertebrate species known to reproduce by self-fertilization (Costa et al. 2010). It inhabits mangrove areas ranging from south-eastern Brazil, Venezuela, much of the Caribbean, the Bahamas and Yucatan to southern Florida (Kallman & Harrington 1964; Harrington & Kallman 1968). Most populations of the fish exist in nature as isogenic, homozygous strains, although outcrossing heterozygous populations have been discovered in Twin Cays, Belize (Taylor et al. 2001; Mackiewicz et al. 2006a; Mackiewicz et al. 2006b). This fish is aggressive in both the field and the laboratory (Taylor 1990). Because it displays both winner and loser effects (Hsu & Wolf 1999, 2001; Hsu et al. 2009) and the contest behavior in this fish is highly correlated with hormone levels (Earley & Hsu 2008), it is an ideal organism for exploring these effects’ underlying physiological mechanisms. This study used individuals of three strains of K. marmoratus from different geographical areas (DAN2K: Dangria, Belize, collected in 2000; RHL: San Salvador, Bahamas, collected in 2001; SLC8E: St. Lucie County, Florida, U.S.A., collected in 1995) which were originally collected from the field by Dr. D. Scott Taylor, (Florida, U.S.A.). Fish were kept individually in a 13 × 13 × 10 cm translucent plastic container (maintenance container). Every container was filled with 800 ml of approximately 25 ppt synthetic sea water (Instant Ocean® powder) and labeled with a unique code for individual identification. Two holes were drilled close to the upper edges of each container for feeding and aeration. Because the fish is capable of respiring through its skin (Grizzle & Thiyagarajah 1987), no extra aeration was needed. Fish were maintained at 25 ± 2 °C on a 12:12 h light:dark cycle and fed. 11 .

(17) . newly hatched brine shrimp (Artemia) nauplii at 3:00 pm every day. The fish can be less than 2 cm in standard length (from the tip of the snout to the end of the caudal peduncle) when they first reach maturity. For ease of handling, only fish larger than 26 mm in standard length (> 6 months old) were used.. 2.2 Experimental Design and Procedures. 2.2.1. Experimental Design This study aimed to examine changes in the expression levels of steroid hormone and serotonin receptor genes after one or three recent wins/losses and whether these changes decay with the passage of time. Before they were exposed to any of the treatments, individual fishes’ levels of testosterone (T) and cortisol (CORT) were measured as indicators of their baseline physical condition. A total of six experience types (three winning experiences, ET3W; three losing experiences, ET3L; a control (for ET3W and ET3L) with three no-contest experiences, ET3N; one winning experience, ET1W; one losing experience, ET1L; a control (for ETW and ETL) with one no-contest experience, ET1N) and three time-decay treatments (0 hour delay, 0h; 3 hours delay, 3h and 48 hours delay, 48h) were implemented for this study, for a total of 18 treatment combinations (Table 1). Results from previous studies (Rosa et al. 2002; Mohanan et al. 2005) indicated that a sample size of 4-6 for each treatment should be enough to detect the differences. A total of 298 fish were available for this study and were randomly assigned to the 18 treatments (n = 16 or 17 for each of the treatments).. 2.2.2. Experimental Procedures (Fig. 1). 12 .

(18) . Baseline Hormone Collection On the first day of the experiment (Day 1), I measured each individual’s baseline hormone levels, following the procedures for hormone sample collection and analysis from Earley & Hsu (2008). To minimize the impact of the daily cycle which is likely to exist for steroid hormones (Emata et al. 1991), I collected all baseline hormone samples at the same time of day. At 9:30 am, each focal individual was placed in a glass beaker with 400 ml clean 25 ppt synthetic sea water and left in the beaker for 1 hour. After 1 hour, the focal individuals were removed from the beakers and returned to their maintenance containers. Hormones were then extracted from the water samples using C18 solid phase extraction columns (Lichrolut RP-18, 500 mg, 3.0 ml; Merck, NJ, USA) fitted to a 24-port manifold. Columns were first primed with 2 consecutive washes with 2 ml HPLC grade methanol (MeOH) followed by 2 consecutive washes with 2 ml ultrapure water. Tubing then was fastened securely to the top of each column and placed into the water sample collected from the fish. A vacuum was engaged and the water sample passed through the tubing into the column. Columns then were frozen (-20 ºC) until further processing. Freeze storage of water samples and columns has been determined not to impact steroid concentrations (Ellis et al. 2004).. Experience Training On Days 2 to 4, the focal individuals pre-assigned to have three experiences (ET3W, ET3L and ET3N individuals) received one winning, losing or no experience per day for three consecutive days. The standard methods for providing winning and losing experiences to the fish are described below. The focal individuals pre-assigned to receive one experience (ET1W, ET1L, and ET1N individuals) received. 13 .

(19) . their single experience on Day 2.. Fish Dissection and Tissue Collection Immediately after the completion of the experience training, individuals assigned to the 0 hour delay treatment were decapitated and their heads and gonads preserved in RNAlater® (Applied Biosystems/Ambion Inc., TX, USA) and stored in a freezer at -80 ºC for subsequent examination of the gene expressions of steroid hormone receptors and serotonin receptors (section 2.6). Three hours and 48 hours after the completion of the experience training, individuals assigned to the 3 hour and 48 hour decay treatments were decapitated and their heads and gonads were preserved as above.. 2.3 Providing a Losing/Winning Experience To ensure that focal individuals lost or won, I fought them against much larger/smaller (difference > 2 mm) standard winners/losers from the same clone. I staged contests among several large fish and the one that defeated all others was designated a standard winner, to provide losing experiences to the focal individuals of its strain. Small fish were fought among themselves and the one that lost to all others was used as a standard loser to provide winning experiences to the focal individuals of its strain. Each focal individual assigned to receive three contest experiences (ET3L & ET3W) was fought against three different standard winners/losers to provide its 3 consecutive losing/winning experiences. For experience training, standard aquaria (12 × 8 × 20 cm3, containing water 13 cm deep and 2cm of gravel) were divided by opaque partitions into two, symmetrical compartments. To receive its pre-assigned losing/winning experience, the focal. 14 .

(20) . individual was placed in one compartment (randomly selected) with a standard winner/loser in the other. All fish were given a 30-min acclimation period before the partition was removed and the focal individual allowed to interact with its trainer. A losing experience was successful if the focal individual retreated and swam away immediately without retaliation when attacked by the standard winner. A winning experience was successful if the focal individual attacked and chased the standard loser without retaliation. The opaque partition was replaced in the aquarium to separate the focal fish and its trainer as soon as the focal fish received its pre-assigned experience. All fish were returned to their maintenance containers after experience training. Most focal individuals received their pre-assigned winning or losing experience quickly (Three-experiences treatment: mean = 89.12 s, 78.02 s, 62.68 s for the 1st, 2nd, and 3rd losing experience respectively; mean = 109.98 s, 71.00 s, 53.04 s for the 1st, 2nd, and 3rd winning experience respectively; One-experience treatment: mean = 126.04 s for losing experience; mean = 112.94 s for winning experience). Twelve individuals assigned to receive winning experiences, however, did not attack the standard losers within 2 hours and were replaced in their maintenance containers. They were exposed to standard losers again one month later and successfully attacked the standard losers and obtained their winning experience. The entire training was video-taped for future behavioral analysis. ET1N/ET3N individuals received no real experience training for 1 day or 3 consecutive days but received the same amount of handling and disturbance as the ET1L/ET3L or the ET1W/ET3W individuals. The ET1N and ET3N individuals were placed in one of the compartments (randomly selected) in a standard aquarium with no opponent in the other. After a 30-min acclimation period, the partition was removed to permit the focal individual to swim around in the aquarium for the same amount of time as those receiving winning or losing training.. 15 .

(21) . 2.4 Definitions of the Contest Behaviors in Experience Training During experience training, I recorded following behavioral measurements (Hsu et al. 2009) for future behavioral analysis: (1) Move initiator: When the partition was lifted, the two individuals usually rapidly swam towards the bottom of the aquarium and stayed still on, or close to the gravel. The individual that first resumed activities was the “move initiator”. (2) Display initiator: the individual that first oriented its head towards its opponent and/or swam directly towards its opponent was the “display initiator”. (3) Gill (opercular) display initiator: the individual that first erected its gill was the “gill display initiator”. (4) Attack initiator: the individual that first started physical interactions by swimming rapidly towards and pushing or biting its opponent was the “attack initiator”. (5) Latency to move: the time interval between removing the partition and the first move was defined as “latency to move”. (6) Latency to display: the time interval between removing the partition and the first display was defined as “latency to displays”. (7) Latency to gill display: the time interval between removing the partition and the first gill display was defined as “latency to gill display” (8) Latency to attack: the time interval between removing the partition and the first attack was defined as “latency to attack” (9) Contest duration: the time interval between removing the partition and the loser’s first retreat. (10) Escalation: contests in which contestants engaged in mutual attacks were “escalated”; contests those were resolved after only displays or after a single attack were “non-escalated”.. 2.5 Hormone Extraction and Assay When extracting hormones, the columns were thawed and purged with 2 × 2 ml washes of distilled water. Hormone was eluted from the columns into 12 × 75 mm (6 ml) borosilicate vials by 2 consecutive 2 ml washes with HPLC grade MeOH. The 4. 16 .

(22) . ml of eluted solvent was evaporated at 37 ºC (water bath) with a gentle stream of nitrogen (~10 psi), which was passed over the samples through an evaporating manifold. The resulting hormone pellet was then re-suspended in 800 μl of enzyme-immunoassay (EIA) buffer supplied with the kits and the samples stored at -20 ºC until assay. Cayman Chemicals Inc. EIA kits were used for quantifying testosterone and cortisol. Each of the hormones was assayed for each individual in duplicate. Briefly, 50 μl of each sample was pipetted into a well on a 96 well plate coated with mouse anti-rabbit IgG followed by 50 μl of acetylcholinesterase tracer and 50 μl of antiserum. Testosterone plates were incubated for 2 hour at room temperature and cortisol plates were incubated overnight (18 h) at 4 ºC on an orbital shaker. The plates then were washed five times with wash buffer (provided with the kits), blotted dry and 200 μl of Ellman’s reagent added to each well. The plate was wrapped in aluminum foil and placed on an orbital shaker for 60-120 min depending on the assay. Plates were read at 405nm on an Absorbance Microplate Reader (ELx808TM, BioTek, VT, USA). 50 µl was taken off the top of each sample (from 298 individuals) to give about 15 ml of pool; all K. marmoratus pooled samples run in duplicate were used as controls on each plate. All the hormone levels data are presented as pg/ml. Intra-assay coefficients of variation were (assay plate from 1 to 10) testosterone (7.3%, 5.0%, 5.8%, 4.5%, 2.8%, 7.7%, 5.1%, 10.0%, 5.5%, and 5.7%) and cortisol (9.0%, 5.4%, 4.7%, 3.6%, 3.9%, 0.9%, 5.7%, 1.9%, 3.1%, and 2.7%). Inter-assay coefficients of variation were testosterone (7.9%) and cortisol (4.8%). Because there two individuals (one for cortisol and one for testosterone) gave exceptionally high levels, their hormone values were not included in the final data analysis.. 2.6 Quantifying Receptor-Gene Expression. 17 .

(23) . 2.6.1 Identifying the Receptor Genes We developed nested sets of primers for the receptor genes by downloading known sequences from the NCBI website (www.ncbi.nih.gov). These sequences were aligned using Clustal X, and primers were designed from highly homologous regions of the alignment. The forward and reverse primers that I used in traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR) are listed in Tables 2 and 3. All primer pairs had similar annealing temperatures (range: 61-65 ºC) to facilitate interchangeable usage during traditional PCR. Traditional PCR was conducted with an Eppendorf Mastercycler® Gradient, using the 5 PRIME HotMaster Mix (5 PRIME Inc., MD, USA) and temperature gradient feature to maximize amplification and efficiency. Ten individuals (5 DAN2K and 5 RHL) of K. marmoratus were decapitated and their brains and gonads were removed by microdissection then transferred directly to RNAlater® for preservation of RNA and stored at -80 ºC. The brain and gonad tissues from the 10 fish were subsequently pooled and transferred to 400 μl (brain tissues) and 800 μl (gonad tissues) cold TRIzol (Sigma-Aldrich® Co, MO, USA) and homogenized for 40 sec. Following homogenization, 200 μl of chloroform was added and the samples were vortexed and incubated at room temperature for 2-3 min. Samples were centrifuged and the aqueous phase was transferred to another tube containing 500 μl of isopropyl alcohol, vortexed and incubated at room temperature for 10 min. The supernatant was removed and 1 ml of 75% ethanol was added to precipitate total RNA. The RNA pellet was dissolved in 50 μl ultrapure water by repeat pipetting. Then, cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., CA, USA). PCR amplification of K. marmoratus AR, 5-HT1AR, ERα, ERβ and GR cDNA was carried out with the. 18 .

(24) . following conditions: 40 cycles of 94 ºC for 15 sec, 61 ºC for 50 sec and 72 ºC for 30 sec, and a final 10 min at 72 ºC. The annealing temperature was adjusted depending on the different primer sets (Table 2). I targeted a 120-300 bp region of the receptor genes. The PCR product generated from K. marmoratus tissue cDNA was purified using QIAquick® PCR purification kits, QIAquick® Gel Extraction Kits or MinElute PCR Purification Kits (QIAGEN Inc., CA, USA), run on 1.5% agarose gels, and visualized with ethidium bromide for a basic assessment of whether I was successful at amplifying the target region. The PCR product was sent off for sequencing by using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., CA, USA), and the sequence was subject to a BLAST search to ensure that the product aligns with the receptor genes.. 2.6.2 Quantitative PCR I quantified gene expression using qPCR performed on the Mastercycler® ep realplex System with SYBR green (KapaTM Biosystems, Inc., MA, USA) according to the manufacturer’s instructions. The study individuals’ brain and gonad tissues stored at -80 ºC were transferred to 400 μl (brain tissues) and 800 μl (gonad tissues) cold TRIzol and homogenized for 40 sec. Following homogenization, total RNA was extracted as previously described. Each RNA sample was quantified by using a NanoDrop-1000 Spectrophotometer, and adjusted to a final concentration of 50 ng/μl. cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., CA, USA). To run qPCR, 2 μl of ds DNA standards (for AR, 5-HT1AR, ERβ and GR gene, concentration of standards were 1, 0.1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 pg/μl; for ERα and RPL8 gene, concentration of standards were 10, 1, 0.1, 10-2, 10-3, 10-4, 10-5. 19 .

(25) . and 10-6 pg/μl) and samples were pipetted in duplicate into 96-well PCR plates (twin tec. PCR Plate 96, semi-skirted, wells colorless, Eppendorf, NY, USA). Then, 8 μl mixture (5 μl SYBR® FAST Master Mix 2X Universal, 0.2 μl forward and 0.2 μl reverse 10 μM primers, and 2.6 μl ultrapure water) was pipetted into each well containing standards or samples with a multiple channel pipette. qPCR cycles were as follows: 95 ºC for 20 sec, and 40 cycles of 95 ºC for 1 sec and 60 ºC for 20 sec. All the qPCR primer pairs had similar (± 1ºC; range: 61-65ºC) annealing temperatures, and the size of PCR products were between 90-230 bp (Table 3). Melting curve analysis using Mastercycler® ep realplex System software (Eppendorf, NY, USA) was performed to confirm the primer efficiency. The RPL8 gene was used as a control gene to normalize expression levels between samples. All data were expressed relative to RPL8 gene to normalize for any difference in reverse transcriptase efficiency. Threshold cycle (Ct) value (the PCR cycle number at which fluorescence was detected above threshold and decreased with increasing input target quantity) was obtained from Mastercycler® ep realplex System software (Eppendorf, NY, USA) and used to calculate ΔCt values (ΔCt = Cttarget. gene-Ctcontrol gene). of each sample (Schmittgen & Livak 2008). ΔCt value is. negatively correlated with relative gene expression: the higher ΔCt value indicates lower receptor-gene expressions levels. One receptor-gene expression data of gonad was not obtained because the gonad tissue was lost during dissection process, and one set of brain receptor-gene expression data was excluded because that individual’s RPL8 gene expression level was abnormally lower than all the other individuals.. 2.7 Data Analysis Pairwise correlations were used to measure the relationships between hormone. 20 .

(26) . levels, between receptor-gene expression levels and between hormone levels and receptor-gene expression levels in the fishes’ brains and gonads. The hormone levels were log transformed to fit the normal distribution. To avoid an inflated chance of Type 1 error, the α level for this analysis was adjusted to be more conservative (P < 0.0024) by using the Bonferroni adjustment. Multiple linear regressions and logistic regressions were used to examine the relationships between hormone levels (CORT & T) and contest behavior (latency to movement, initiating displays, latency to display, initiating gill displays, latency to gill display, initiating attacks, latency to attack, escalation and contest duration) in the 1st experience training. The hormone levels, latency to movement, latency to display, latency to gill display, latency to attack and contest duration were log transformed to fit the normal distribution. Because individuals with a first winning/losing experience would become more/less aggressive in subsequent contests (ET3W always initiated contests in their second and third experience training; ET3L never initiated contest in their second and third experience training), I only used contest behavior in the first experience training. I also analyzed the winning and losing individuals’ behaviors separately, because they faced different opponents (STW/STL) and were expected to behave differently. The focal fish’s standard length, last contest experience, strain type and standard length of trainer were included in the model as control factors. Multiple linear regressions were also used to examine the influence of the type of contest experience and decay time on receptor-gene expression levels. Log transformed hormone levels (CORT & T), fish’s standard length, last contest experience and strain types were include in the model as control factors. Multiple linear regressions were used to examine the relationships between contest behaviors in the 1st experience training and receptor gene expression levels. Because the contest behaviors in experience trainings were highly correlated with. 21 .

(27) . each other, including them in the models at the same time would result in a multicollinearity problem. The importance of each of these behaviors was therefore tested separately. The correlation between contest behaviors and receptor-gene expression was tested, and standard length, last contest experience and strain type were included in the model as control factors. JMP (v. 5.0.1; SAS Institute Inc., Cary, NC, U.S.A.), a commercial statistical package, was used for all the statistical analyses in this study.. 22 .

(28) . Results. A total of 298 individuals were used in this study. The mean value of ΔCt of each receptor genes were as follows: In brain: AR (10.26±1.01), ERα (1.60±1.39), ERβ (1.41±1.41), GR (2.29±1.12), 5-HT1AR (0.95±1.32); In gonad: AR (3.52±0.90), ERα (3.24±0.60), ERβ (2.91±0.42), GR (5.35±0.57), 5-HT1AR (7.12±0.58). The mean value of hormone levels was T (942.77±823.96) and CORT (151.78±135.49). The receptor-gene expression levels and hormone levels for each treatment are shown in supplemental data (Table 1-6).. 3.1 The Relationship between Baseline Hormone Levels and Contest Behaviors in the 1st Experience Training Baseline CORT and T levels did not appear to have a significant influence on the contest behavior in the 1st experience training of those fish allocated to receive winning experiences (ET1W`& ET3W, matched with standard losers), (Table 4). Strain type, however, did have a significant effect on their contest behavior: the SLC8E strain took less time than average to initiate moves (P = 0.039), displays (P = 0.015) and attacks (P = 0.044). Individuals whose last contest experience was a win took longer to initiate gill displays (P = 0.019). For those fish allocated to receive losing experiences (ET1L & ET3L, matched with standard winners), individuals with higher baseline T levels were more likely to initiate gill displays (P = 0.014) and persisted longer before eventually retreating (P = 0.019), although CORT levels did not have a significant relationship with any of the behaviors examined (Table 5). Larger individuals were also more likely to initiate gill displays (P = 0.050). Individuals whose last contest experience was a win were more. 23 .

(29) . likely to escalate a contest (P = 0.032).. 3.2 Effect of Experience Type and Decay Time on Post-Experience Receptor-Gene Expression Levels. 3.2.1 Brain Tissue Receptor-Gene Expression Levels Experience type had a significant effect on AR gene expression levels in the individual’s brains (P = 0.036, Table 6). Individuals with three losing experiences had significantly lower AR gene expression levels than the control group, which received one no-experience (P = 0.007). In addition, decay time effects were discovered in AR, ERα, ERβ and 5-HT1AR gene expression levels. Because the regression model’s interaction terms relating experience and time decay (experience × time-decay) to these receptor gene expression levels were not significant (all interaction terms P ≥ 0.718), it may be that the decay-time effect was caused by handling disturbance, rather than the experimental treatments. The results also showed that the receptor-gene expression levels related negatively with baseline CORT levels (P ≤ 0.017; except the relationship between CORT and AR, P = 0.114, and the relationship between CORT and GR, P = 0.051) but positively with baseline T levels (P ≤ 0.017).. 3.2.2 Gonad Tissue Receptor-Gene Expression Levels Experience type had a significantly affect on ERα gene expression levels in the individual’s gonads (P = 0.047, Table 7). Individuals with three losing experiences had significantly lower ERα gene expression levels than the control group which received one no-experience (P = 0.006). Decay time effects were also discovered in AR and ERα gene expression levels, but, as with the fishes’ brains, the fact that the. 24 .

(30) . regression model’s interaction terms relating experience and time decay to these receptor gene expression levels were not significant (all interaction term P ≥ 0.353) suggests that this decay-time effect might also be caused by handling disturbance. The analysis also showed that the receptor-gene expression levels related negatively with CORT levels (P ≤ 0.014; except the relationship between CORT and AR, P = 0.131 and the relationship between CORT and ERα, P = 0.075) but positively with T levels (P ≤ 0.001; except the negative relationship between T and AR, P < 0.001, and the relationship between T and ERβ, P = 0.177). 3.3 Relationship between the Contest Behaviors in the 1st Experience Training and Post-Experience Receptor-Gene Expression Levels in Brain Tissue Among the individuals allocated to receive winning experiences (ET1W`& ET3W, matched with standard losers), those which took longer to initiate moves and displays had significantly lower GR gene expression levels (initiate moves, P = 0.033; initiate displays, P = 0.043, Table 8). Interestingly, among the individuals allocated to receive losing experiences (ET1L & ET3L, matched with standard winners), those which were more likely to initiate displays, gill displays and took longer to retreat from contests had significant higher ERα (initiating displays, P < 0.001; initiating gill displays, P < 0.001; contest duration, P = 0.013), ERβ (initiating displays, P < 0.001; initiating gill displays, P < 0.001), GR (initiating displays, P < 0.001; initiating gill displays, P = 0.036) and 5-HT1AR (initiating displays, P < 0.001; initiating gill displays, P = 0.016) gene expression levels (Table 9). The relationships between contest behavior and AR gene expression levels, however, were not significant, although losing experiences did affect AR gene expression levels in brain tissue.. 25 .

(31) . 3.4 The Relationships between Baseline Hormone Levels and Post-Experience Receptor-Gene Expression Levels Table 10 shows that receptor-gene expression levels are highly correlated with other receptor-gene expression levels. The gene expression levels of ERα, ERβ, GR and 5-HT1AR were positively correlated in both brain and gonad tissue (P ≤ 0.002; except the relationship between ERβ and 5-HT1AR in brain, P = 0.008). The relationships between AR and other receptor-gene expression levels were often negative in gonad (P < 0.001; except ERβ in gonad; r = 0.140, P = 0.016) and were more significant in gonad tissue (P < 0.001) than in brain tissue (P ≤ 0.639). CORT levels did not have a strong relationship with the gene expression level of the receptors examined (P ≥ 0.035), expect for its negative relationship with ERβ in gonad (P < 0.001). T levels, on the other hand, were closely related with the gene expression level of the receptors (exceptions: AR, GR and 5-HT1AR in brain, P ≥ 0.005; ERβ in gonad, P = 0.906). The significant relationships between T levels and receptor-gene expression levels were mainly positive (P < 0.001), except for the negative relationship between T and AR in gonad tissue (P < 0.001). In addition, T levels were positively correlated with CORT levels (P < 0.001).. 26 .

(32) . Discussion My results show that individuals with three losing experiences had significantly lower AR (in brain tissue) and ERα (in gonad tissue) gene expression levels than individuals in the control group, with one no-experience. Also, baseline hormone levels (T) and receptor-gene expression levels (ERα, ERβ, GR and 5-HT1AR) were positively correlated with individuals’ contest behavior in the 1st experience training, particularly in those individuals given losing experience(s). The results do not, however, clearly establish that hormone/serotonin receptors might be the physical mechanism underlying winner and loser effects: neither one losing experience nor either of the winning experience treatments had a significant effect on hormone/serotonin receptor-gene expression levels. The following paragraphs discuss the results, the possible reasons for them and ideas for future research in more detail.. 4.1 Effect of Experience Type on Post-Experience Receptor-Gene Expression Levels My study discovered that neither one losing experience nor either of the winning experience treatments had a significant effect on hormone/serotonin receptor-gene expression levels. However, recent studies (e.g. Marini et al. 2006; Fuxjager et al. 2010) indicated that not only circulating hormone levels, but also receptor-gene expression levels are highly associated with contest experience. For instance, in California mouse, Fuxjager et al. (2010) found that three winning experiences significantly increased the expression levels of the AR gene in the medial anterior bed nucleus of the stria terminalis (BNSTma region), nucleus accumbens (NAcc) and ventral tegmental area (VTA); this region is a key brain area that controls social. 27 .

(33) . aggression. While in Sprague-Dawley rats, Marini et al. (2006) discovered that GR gene expression levels were found significantly increasing in rat hippocampus 30 hr after receiving a forced losing experience. These results suggested that winning or losing experiences may affect receptor-gene expression in some brain regions; however, in K. marmoratus, we did not find any significant changes of brain receptor gene expression levels in ET1L or ET3W. These different results might be a consequence of difference in study animals, difference in target brain regions or differences in experimental design: (1) Differences in experimental design. Experimental design might be an important factor behind the different results. In my study, the focal individuals were divided from their trainer immediately after the contest was resolved. While in Marini’s study (2006), the focal individuals were left in visual, auditory and olfactory contact with the dominant individuals for 30 min after the contest was resolved; this might not only have enhanced the magnitude of social defeat stress for the focal individuals but also possibly had a different effect on brain receptor-gene expression levels. For instance, research on European starling (Sturnus vulgaris) showed that long-term stress reduced glucocorticoid receptor-gene expression in the hypothalamic paraventricular nucleus and mineralocorticoid receptor-gene expression in the hippocampus (Dickens et al. 2009), while short-term stress did not. It suggested that the changes of GR gene expression might be not only caused by losing experience, but also by long-term social defeat stress. Comparison with Fuxjager’s study (2010), I measured “post-experience” receptor gene expressions levels, while they measured “post-encounter” (post-fight) receptor gene expression levels. For this reason, the changes of AR gene expression levels in their study may be caused by winning experience or contest interaction in the subsequent encounter. It is possible that the AR gene/protein expressions increased because of the elevated aggressive interaction. 28 .

(34) . in contests (encounter stage), rather than because of the winning experiences. The results of the study, unfortunately, could not demonstrate a direct link between contest experiences and the changes of receptor gene/protein expression levels. (2) Differences in target region (whole brain & specific brain regions). Different brain regions might mediate different behaviors and respond differently to contest experience. Fuxjager et al. (2010), for instance, focused on the brain regions that control the output of antagonistic behavior and social aggression in mice (e.g. nucleus accumbens, lateral septum, medial anterior bed nucleus of the stria terminalis, medial amygdale, anterior hypothalamus, ventrolateral subnucleus of the ventromedial hypothalamus, ventral premammillary nucleus, ventral tegmental area, dorsal periaqueductal gray), while Marini et al. (2006) focused on the brain region (e.g. hippocampus) that was described as particularly sensitive to the effects of social defeat stress and stress hormones in rats. My study, however, checked gene expression levels in the whole brain rather than in a specific brain region, because the brain of K. marmoratus is too small to allow separation into different regions. If different brain regions respond differently to winning/losing experiences, the effect of contest experiences on receptor gene expression levels across the whole brain would be diluted and become hard to detect. Although my study did not discover any effect of a losing contest experience or three winning contest experiences on receptor-gene expression, the results still revealed significant relationships between receptor-gene expression levels and aggressive behavior, which suggests that the effect of contest experience on receptor gene expression may not be diluted even when measured over the whole brain. (3) Differences in study animal (rodents & fish). Another difference between my study and those quoted above is the study organism: the previous studies all used rodents (California mice & Sprague-Dawley rats) whereas I used a hermaphroditic. 29 .

(35) . fish, K. marmoratus. Because the physiological structures of rodents and fish are totally different (especially their brains and gonads), they may respond differently to the same experiences. For instance, androgen (methyl-testosterone) would efficiently change the sex of K. marmoratus, from hermaphrodite to male (Kanamori et al. 2006); for this reason, circulating androgen levels or AR sensitivity in this fish’s gonads must be restricted below a specific threshold in order to prevent sex change, while the rodent’s physiological condition might be not restricted in this way. The result that individuals with three losing experience had significant lower AR gene expression levels may be not caused by losing experience, because individuals with one losing experience did not have significant lower AR gene expression (or a non-significant trend) than individuals in the control group. This result may be caused by social defeat stress. Contest interaction is a kind of stress for contestants, especially for losing indivuduals (social defeat stress), and it induces animals’ quick but strong stress response (Miczek et al. 2002). This suggests that individuals with three losing experiences might be under more stress than those with one losing experience. Past studies have shown that social defeat stress may cause changes in receptor gene expression in brain tissue (e.g. Marini et al. 2006). Although the effect of social defeat stress on AR gene expression in brain tissue remains unclear, there are some in vitro data revealing a relationship between stress response and AR gene expression. For instance, Burnstein et al. (1995) discovered that stress hormone, glucocorticoids, down-regulated AR mRNA expression levels and decreased AR protein levels in COS 1 cell lines. Based on this finding, it is possible that the reduction in AR gene expression levels in those individuals with three losing experiences was caused by their strong stress response rather than their losing experience. My study also showed significantly lower ERα gene expression in the gonads of. 30 .

(36) . individuals given three losing experiences than in those of the control group. There is little published research about the relationship between winning/losing experiences and receptor gene expression levels in gonads; studies have, however, revealed that social defeat stresses caused by agonistic contests suppress gonad function or reproductive ability (e.g. Tilbrook et al. 2000). This result suggests that the stress induced by serial losing experiences affects gonad function and also possibly suppresses gonad receptor-gene expression levels, although no direct linkage between losing experiences and gonad ERα gene expression levels has been discovered.. 4.2 The Relevance of the Fish’s Baseline Hormone Levels and Post-Experience Receptor-Gene Expression Levels in Brain to the Fish’s Contest Behaviors My results showed that baseline hormone levels were highly correlated with the fish’s contest decisions when facing a much larger opponent (i.e. standard winner), in training for losing experience: individuals with higher baseline T levels were more likely to initiate gill displays and persisted longer before retreating. This result is similar to that of Earley & Hsu (2008), who found that pre-fight T levels related positively to contest initiation in the smaller opponent. Positive associations between T levels and aggression are also reported for other animal species (e.g. in rats, Caldwell et al. 1984; in sparrows, Wingfield & Hahn 1994; in humans, Archer 2006) and in studies using castration or exogenous hormone injections (Francis et al. 1992; Ruiz-de-la-torre & Manteca 1999). Social mixing male lambs with testosterone enantate injections, for instance, were more aggressive than control individuals (Ruiz-de-la-torre & Manteca 1999); castrated male cichlid fish (Haplochromis burtoni; Francis et al. 1992) with an accompanying reduction in T levels had significantly. 31 .

(37) . lower aggression than sham-operated males. In my results, however, no positive correlations were discovered between T levels and aggressive behavior in individuals facing smaller, submissive opponents (i.e. standard loser) when in training for winning experience. This may be because standard losers behaved submissively and usually quickly retreated from the experience training even without focal individuals initiating gill displays or launching attacks. Therefore the behavior in training of focal individuals might not precisely reflect the endocrine states. My study also indicated that not only baseline hormones but also hormone/serotonin receptor-gene expression levels (ERα, ERβ, GR and 5-HT1AR) in the fish’s brains were positively correlated with those individuals’ aggressive behavior in experience training, particularly in individuals given losing experiences (fighting with larger habitual winners). Research in rodents has discovered that ERα expression levels are positively correlated with aggressive behavior, while ERβ expression levels are negatively or non-correlated with aggressive behavior. For instance, Trainor et al. (2007) found that increases in aggressiveness in short-day treatment mice accompanied increased ERα gene expressions and decreased ERβ gene expressions in LS/BNST brain regions. Previous studies also indicated that ERα gene knockout male mice were less aggressive, whereas ERβ gene knockout male mice exhibited normal or increased aggression (Ogawa et al. 2000). In my results, ERα gene expression was positively correlated with individuals’ aggressive behavior as in the previous studies, while ERβ gene expression, unlike the previous research, was also positively correlated with individuals’ aggressive bahavior. Most of above research, however, focused on aggressive behavior in rodents; whether the affect of ERα and ERβ aggression in this fish is the same as in rodents is still unclear. It is possible that both ERα and ERβ gene expression levels have a positive effect on aggressive behavior in. 32 .

(38) . this fish. 5-HT1AR is another receptor which has been found to be associated with aggressiveness. Classified by the expression location on neuron cells, there are two kinds of 5-HT1AR. One type is located on postsynaptic cells; the other type, located on presynaptic cells are called autoreceptors. Previous research indicated that the expression level of postsynaptic 5-HT1AR might be negatively correlated with aggressiveness, while autoreceptor expression levels might be positively correlated with aggressiveness. For instance, Caramaschi et al. (2007) discovered that the postsynaptic 5-HT1AR agonist increases receptor sensitivity, and decreases aggressiveness in rodents; the autoreceptor agonist decreases the firing rate of presynaptic cells and suppresses 5-HT synthesis and thereby increases aggressiveness in rodents. In my results, the 5-H1A receptor gene expression levels were positively correlated with individuals’ aggressiveness. Although I could not distinguish between the expression of these two kinds of receptor genes, it may be that the more aggressive individuals had higher autoreceptor gene expression levels in presynaptic cells than the less aggressive individuals. In my results the expression of GRs genes was positively correlated with individuals’ aggressive behavior. GRs have usually been found to correlate with social stress response rather than with aggressiveness (e.g. Marini et al. 2006; Dickens et al. 2009); however, some studies suggest a positive relationships between GRs expression and aggressiveness. For example, in research on lizards (Anolis carolinensis), blocking GR by mifepristone reduced aggressive attacks/displays in the early stages of fighting (Summers et al. 2005). This result demonstrates that GR gene expression levels might be positively correlated with aggressiveness, which is similar to the relationships observed in my study.. 33 .

(39) . 4.3 Other Possible Mechanisms Mediating Winner and Loser Effects In addition to changes in levels of hormone/serotonin receptor gene expression, there are other mechanisms which might mediate winner/loser effects. Winning and losing experience might change individuals’ behavior by learning and memory processes, which would be mediated by a different set of physiological mechanisms. The capability of fish to change its aggressiveness or willingness to fight as a result of learning has been demonstrated (e.g. Hollis 1984; Hollis 1999; Carpenter & Summers 2009). Memory formation is involved in learning processes. According to recent research, winner and loser effects in K. marmoratus can last for one month (Lan 2010), which suggests that not only short-term but also long-term memory should be involved in mediating these effects. In the process of long-term memory formation, 5-HT (in Aplysia model) or dopamine (in mouse model), released in vivo during sensitization, binds to a cell surface receptor on the sensory neurons that activate the enzyme adenylyl cyclase to convert ATP to the second-messenger, cAMP, and then activates the cAMP-dependent protein kinase (PKA). PKA recruits MAP kinase and translocate into the cell nucleus. PKA activates gene expression by phosphorylating the transcription factors that bind to the cAMP-responsive element (CRE), the CRE binding protein (CREB). This process also activates the synthesis of new proteins, changes synapse connections and initiates synaptic growth to increase synaptic efficacy (Abel & Kandel 1998; Bailey et al. 2004). In three experimental systems (Aplysia, Drosophila and mice), the cyclic AMP-dependent protein kinase PKA pathway and the CREB protein have been demonstrated to be involved in the consolidation of short-term changes in neuronal activity into long-term memory storage (Abel & Kandel 1998). In addition to CREB protein, N-methyl-D-aspartate (NMDA) receptors. 34 .