2. Differential regulation of the foraging gene associated with task behaviors inharvester ants By Jonathan Kahn, Andrew Hoadley, and Claire Spitzer
3. Summary This paper is a first time series analysis of foraging gene expression in harvester ants. It shows that the task-specific expression patterns of foraging are aligned with the circadian rhythm of the ants. This data underlines the importance that time-related expression studies are extremely important and thus can be useful in explaining mechanisms that influence behaviour; such as foraging.
4. Background The individual behavior of a harvester ant is one thatchanges over their lifetime; Transition from nest workers to foragers as they age. This social organization is highly implicated(?associated with?) by a genetic pathway that involves the cGMP-activated protein kinase gene, foraging.
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6. Background Continued This experiment looked to compare the amino acid sequence of foraging across social insects and observed whether the differential regulation of this gene is directly associated with task-related behaviours.
7. Methods The major techniques utilized during this experiment include: Deep freeze storage of samples qPCR/RT-PCR DNA sequencing/analysis
8. Deep Freeze Storage Upon collection of individual ants, specimens were immersed in liquid nitrogen and stored at -70°C until dissection This step is essential for the accuracy of the study and preservation of target RNA Deep freeze storage of samples ensures: Desired expression of RNA given time of sampling Prevention of degradation of “foraging” RNA
9. qPCR Otherwise known as quantitative PCR (also abbreviated RT-PCR) This technique was utilized in order to quantify the expression of the 130bp foraging gene at given intervals for which mRNA was extracted What is normal PCR?
10. qPCR (Traditional PCR) Once mRNA is extracted from the specimen, designed primers and DNApolare added to the extraction to create cDNA (complimentary DNA) of the target gene With PCR (polymerase chain reaction), primers and a high-temperature optimum DNApol are added to the extraction above to create DNA This mixture is then heated and cooled repeatedly to amplify the DNA
12. qPCR (Traditional PCR) However, this technique usually takes 20-40 cycles and can be used as a semi-quantitative tool at best since attachment of primers to DNA is not consistent So What is the difference between PCR and qPCR?
13. qPCR With qPCR, a fluorescent probe is used As DNApol elongates the target gene during cycles, the probe fluoresces and this is detected by a machine After multiple cycles, the machine’s log of relative fluorescence detection can be analyzed to obtain an accurate idea of the mRNA that was originally present
15. qPCR Traditional PCR reveals results at the end which are unhelpful in wanting to quantify gene expression qPCR, however, provides real time results which may be traced backwards to yield the level of mRNA that was originally present, essential to the evaluation of forgaing expression at a given time for this experiment
16. DNA Sequencing/Analysis A machine reads the target DNA that was developed by using Sanger sequencing principles Using ddNTPs (A, T, G, and C nucleosides) which both fluoresce (by differing color) and terminate elongation, the machine can assemble the overlapping fragments of newly synthesized DNA according to fluorescence to reveal the sequence This is then further analyzed by inputting the sequence into a global database, found at www.nih.gov
In this phylogenetic tree, branch length indicates degree of difference between ammino acid sequence. The black bar indicates the hymenoptera (the group od insects containing both social and non-social wasps bees and ants).Comparative analysis of ammino acid sequence of foraging shows a considerable degree of conservation among ants and other highly social insects, regardless of their tendancy to form social groups.For example Harpegnathossaltator, the Indian jumping ant, and Camponotusfloridanus, a carpenter ant, group with Nasoniavitripennis, a non-social wasp group in an unresolved cluster. These three sequences are too similar to be distinguished with confidence, despite the fact that these species are evolutionarily very distant.
The foraging gene is a cGMP-dependent kinase (cGPK) that contains four conserved domains:Two CAP effector DomainsA catalytic domainAnd a a protein kinase C terminal domain
Sequencing results indicate that both the protein sequence of foraging and the associationof the foraging gene with food-related behavior are conserved across hymenopteran species. This suggests that differential regulation of gene expression, rather than a change in protein structure is most likely mechanism by which foraging influences behavior within species This implicates changes in the pathways responsible for the regulation of foraging, rather than foraging protein sequences, in the the variability in the expression patterns of the this gene across species
Mixed model repeated measures ANOVA show gene expressionvaries significantly across time points (F=5.33, p<0.001). As predicted, this change in gene expression across time was significantly different across tasks.The highest mRNA levels for foragers are recorded at midday. At this time, foragers have more than three times the level of foraging mRNA than nest workers (t=2.56, p=0.05) This correlates with the forager’s peak foraging activity.
