Tank setup for observatoins:
All behavior data was collected using 
RNA was isolated using a starndard 
RNA amplification was performed previously by Molly Schumer using Ambion 
All qPCR was performed using immomix and syber green detected in real time using the DNA engine Opticon from MJ Research.
Renn Lab Thesis Project
2010 Reed Graduate
KAVITA KRISHNAKANT
Analysis of the genetic basis of aggression in Julidochromis marlieri and Julidochromis
transcriptus using real-time quantitative PCR
Although there are varying patterns of behavior across species, the underlying molecular mechanisms have just begun to be recently understood. This study investigated the genes regulating territorial aggression and dominance in a sex-role conventional and a sex-role reversed cichlid species belonging to the genus Julidochromis.
J. transcriptus is classified
as a sex-role conventional species because males are typically larger and engage in
territorial behavior more often than the females. |
J. marlieri, a sister species, exhibits a behavioral pattern is reversed and is therefore referred to as "sex-role reversed". Females in J. marlieri are larger and
more aggressive than males and usually dominate their mates. |
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Since the opposite sex in either
the species engages in territorial and dominance behavior, this is an ideal system in which to study
whether the genes regulating this behavior are independent of gonadal sex. Previous microarray
studies (Schumer 2009) have shown that there are certain gene expression patterns that are associated with
sex-roles independent of gonadal sex in these two species.
There were two aims in this study:
1) To
verify the sex-specific behaviors in the two species
2) to validate the gene expression pattern
associated with sex-specific territorial behavior by RT-qPCR using. For this second aim, validation was done at three levels
level 1: technical replicate, using the same amplified RNA samples that had been applied to the microarray results.
level 2: technical replicate, using unamplified RNA from the same individuals tested by microarray.
level 3: biological replicate, using RNA samples from the new individuals from the current study.
The behavioral data confirmed the species differences in behavioral patterns.
| Difference in counts of attack-mate behavior in male and female J. transcriptus pairs | Difference in counts of attack-mate behavior in male and female J. marlieri pairs |
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| Error bars indicate plus or minus standard error. Red indicates female and blue indicates male. | |
RT-qPCR was performed on aRNA and RNA samples collected by Schumer (2009) to verify her microarray findings that there is a core set of genes responsible for aggressive behavior independent of gonadal sex in J. transcriptus and J. marlieri. These data were then compared to her microarray data. RT-qPCR data using aRNA and RNA were also compared to each other to ensure that the sex-role biased gene expression underlying aggressive behavior was not a factor of RNA amplification.
In validation of past results, RT-qPCR on the RNA and aRNA samples collected by Schumer (2009) demonstrated that relative expression of Parvalbumin was higher in the aggressive individuals independent of the gonadal sex. Also as expected, relative expression of Isotocin-Precursor as determined by RT-qPCR on RNA was found to be male biased in J. transcriptus however according to aRNA data the expression of Isotocin-Precursor was female biased in J. transcriptus. This irregularity is peculiar since microarrays were performed using aRNA and thus, the relationship between data obtained by performing RT-qPCR on aRNA and microarrays should be stronger than data obtained using RNA. While MAP1a did not show a sex biased expression in J. transcriptus for RT-qPCR using RNA and aRNA from Schumer (2009), the relative expression of MAP1a was, as expected female biased in J. marlieri by all methods of measurement. In general, when the RT-qPCR results were validated, there was good correlation between the different measures at the level of the individual.
J. transcriptus correlation statistics:
J. marlieri correlation statistics:
RT-qPCR was also performed on unamplified RNA collected from some of the aimals observed in the current study. Again, the genes of interest were Parvalbumin, Isotocin Precursor and Microtubule-Associated Protein 1a (MAP 1a) and GAPDH was used as the normalizing gene. The average relative gene expression trends show that all three genes are upregulated in the aggressive females belonging to the sex-role reversed species J. marlieri. While statistical analyses were not performed due to a small sample size these results constitute a biological validation of previous microarray results.
Comparison between expression of three different genes -Parvalbumin, Isotocin
Precursor and MAP1a in male and female J. marlieri fish as measured by RT-qPCR
using RNA.
Over all the results demonstrate an increase in the expression of Parvalbumin and Isotocin-Precursor in the brains of individuals exhibiting territorial behavior regardless of the gonadal sex.
This research was supported in part by a grant from the Reed College Biology Undergraduate Research Project Program (B.U.R.P.)
