title: RNA-Seq of Leptothorax acervoum: adult female brain
design: The experiment started on the 5th of August 2020 by preparing experimental colonies. For this purpose, we selected in total ten colonies of L. acervorum, five colonies of L. muscorum as well as five colonies of H. sublaevis, and each colony was assigned to one of five replicates. Subsequently, each replicate was processed by starting with one of the L. acervorum colonies and individually blocking in a dry sponge and marking 30 adult workers with a red metal wire around the abdomen (0.02 mm diameter, Elektrisola, red) without further anaesthesia. Marked ants were then placed into a new nest box containing a fresh slide nest together with the queen. This helped to distinguish old from newly emerged workers after the experimental period. To keep colony composition as natural as possible we first marked and transferred 15 ants that were outside of the nest or which moved outside after little disturbance and afterwards transferred the same number of ants to the top of the brood pile. Moreover, we added 15 small brood items to the new experimental colony. Additionally, from this first colony per replicate, 24 pupae were removed and depleted with large larvae in case, not enough pupae were present in the nest. Six of those pupae were transferred into the new experimental colony which originated from the same colony (from now on referred to as original treatment). We repeated the procedure described above for the other L. acervorum colony of the same replicate, but instead transferred six pupae from the original colony into this new experimental colony (from now on referred to as conspecific treatment) as well as for the L. muscorum colony (from now on referred to as heterospecific treatment). For the H. sublaevis colony, we standardized the number of host workers as described above and additionally added all ants of H. sublaevis (18.4  13.56 individuals), as these do not contribute to the usual colony chores and thus do not represent real workers (from now on referred to as parasitic). Afterwards, the remaining replicates were treated the same way. About 10 weeks (69 days) after the emergence of the first worker, colonies were transferred to 22C and the red foil was removed to allow workers to adapt to light. A day thereafter ants were removed with forceps from the colony, frozen in liquid nitrogen, and stored at -80C until dissection. Dissections were performed in November 2020. The antennae and the brain were dissected of each individual. Both tissues were separately placed into 75l of Trizol and stored at -80C. mRNA was extracted using the Qiagen MiniKit following instructions and samples were sent to Novogene (Cambridge, UK). Here, mRNA was enriched using oligo(dT) beads. Next, the mRNA was fragmented randomly by adding fragmentation buffer, then cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment.