Samples preparation
All H. horticola wasps emerging from the field-collected
caterpillars were individually preserved in ethanol in the freezer
(-20°C) until use. The DNA was extracted from the abdomen of each wasp
using a Qiagen DNeasy blood and tissues extraction kit, following the
manufacturer’s protocol (Qiagen®, USA). We amplified the mitochondrialCOI gene by PCR using the primer pair LCO/HCO developed by Folmer
et al. (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994).
Mitotype and Wolbachia-infection status
Cytoplasmic entities such as the mitochondria and Wolbachiasymbionts are passed on only from mothers. These maternally inherited
entities can thus change frequency in a population at different rates
than genotypes determined using nuclear microsatellite markers. To
evaluate maternal inheritance, we sequenced a mitochondrial gene and
screened for Wolbachia in the majority of our samples.
Two common H. horticola mitotypes were previously characterised
from the Åland islands (Duplouy et al., 2015). We determined the
mitotype of 222 wasps picked at random from the 324 samples genotyped
above (37 from Finström, 41 from Föglö, 43 from Seglinge-Kumlinge, 63
from Saltvik and 38 from Sottunga) by direct Sanger sequencing theCOI gene described above using an ABI 3730 DNA Sequencer (Applied
BiosystemsTM, USA). These samples included males and
females from the five localities and each collection year.
Duplouy et al. (Duplouy et al., 2015) showed earlier that about 50% of
the H. horticola population on the Åland islands is infected by
the Wolbachia strain w Hho. We screened forw Hho in 296 samples (90 from Finström, 41 from Föglö, 40 from
Seglinge-Kumlinge, 86 from Saltvik and 39 from Sottunga), picked at
random from the 324 samples genotyped above, using the primer pair
81F/691R to amplify the conserved Wolbachia wsp gene
(Zhou, Rousset, & O’Neil, 1998).