Differential regulation of the foraging gene associated with

task behaviors inharvester ants

By Jonathan Kahn, Andrew Hoadley, and Claire Spitzer

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.

Background

• The individual behavior of a harvester ant is one that changes 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.

cGMP-activated Protein Kinase Gene

• This gene is associated with behaviour in the harvester ant (also many other species-used as an example) and is conserved amoung species although the expression patterns and functions of the gene vary across taxa.

Background Continued

• Many species such as the honeybee and the fruit fly provide examples as to how important the foraging gene is for behaviour and implications on the species structure; such as division of labour.

• The foraging gene becomes much more interesting to look at after the discovery of task-specific expression in alignment with the circadian clock(?).

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.

MethodsThe major techniques utilized during this experiment include:• Deep freeze storage of samples• qPCR/RT-PCR• DNA sequencing/analysis

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

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?

qPCR (Traditional PCR)

• Once mRNA is extracted from the specimen, designed primers and DNApol are 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

qPCR (Traditional PCR)

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?

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

qPCR

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

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

Results

Effector Catalytic Domain

Terminal Domain

Foraging Protein Sequence

Workers

Foragers