Computational protocol: Wolbachia Infection in a Natural Parasitoid Wasp Population

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Protocol publication

[…] DNA was extracted from the wasp abdomen using a Qiagen DNeasy blood and tissues extraction kit, following the manufacturer’s protocol (Qiagen, USA). The DNA quality was tested by PCR amplifying the mitochondrial COI gene (primer pair LCO/HCO []). Any sample with no amplification for COI was removed. The COI locus was sequenced from 514 samples (422, 45, 6, 6 and 35 from Finland, Estonia, France, Spain and Sweden, respectively, ). Wolbachia infection was assessed through the amplification of two to seven Wolbachia loci, including the wsp gene (primer pair 81F/691R []), the 16S-RNA gene (553F_W/1334R_W []) and the five MLST loci (MultiLocus Sequence Typing genes: CoxA, FbpA, HCpA, GatB and FtSZ using respective primers from []). We also sequenced two to seven Wolbachia loci (MLST, wsp and 16s loci) from 249 (218, 5 and 26 from Finland, France and Sweden, respectively) of the 340 wasps infected with Wolbachia. The Fst values, based on the COI sequences, were calculated using Arlequin 3 [] (). Each consensus sequence was submitted to GenBank (KF722987–94, KJ150624, KM598264–68 & KR185318–21). [...] Phylogenies (, and ) were built using the online tree-building program Phylogeny.fr [, ] in the One-click mode with default settings. For Wolbachia, we used the concatenated wsp, 16S and MLST consensus sequences from our three strains and eight additional Wolbachia strains (GenBank AM999887, CP001391, CP003883, AE017321, AE017196, EF025179 to -183, EF078895, AB474245 to -249, AB094382, AB052745). We rooted the tree using wBm as an outgroup. For H. horticola mtDNA, we used the seven mitotypes obtained from our samples together with sequences from five other species of Hyposoter (GenBank JF963449, JQ576453, JQ576979 & DQ538855 to -58) and two Campoletis sonorensis (Ichneumonidae, GenBank DQ538849 & -50). We rooted the tree using two Cotesia melitaearum (Braconidae, GenBank KM598269 & -70) as outgroups. […]

Pipeline specifications

Software tools Arlequin, Phylogeny.fr
Applications Phylogenetics, Population genetic analysis
Organisms Melitaea cinxia
Diseases Infection