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
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.
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
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
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
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
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
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
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.
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 1
stExperience 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
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
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 1
stExperience 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.
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).
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
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
however, in K. marmoratus, we did not find any significant changes of brain receptor