Computational protocol: The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone

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Protocol publication

[…] Arthropod DNA was extracted using the PROMEGA Wizard® SV 96 Genomic DNA Purification System following the instructions of the manufacturer. DNA extraction was performed on the entire body or abdominal tissue rather than legs, to reduce the risk of missing infection with reproductive parasites when they are present (false negatives). The DNA quality was systematically tested using polymerase chain reaction (PCR) amplification of a conserved region of the arthropod 18S rDNA using primers listed in Table . Infections have been investigated in each individual host for seven reproductive parasites: spotted fever group Rickettsia, Wolbachia, Arsenophonus, Cardinium, male killers (MKs) within the flavobacteria, S. ixodetis and S. poulsonii. Independent assays for infection by each reproductive parasite were performed using PCR amplification of a fragment of either the 16S rDNA gene or the 17-kDa ompA gene using specific primers (Table ). Infected-positive individuals were used as positive controls in each PCR assay (Table ). Additional PCR amplifications of the Wolbachia surface protein gene (wsp) were completed in some cases to differentiate the diversity of strains present (primers listed in Table ). PCRs were performed under the following conditions: initial denaturation at 95°C for 2 minutes, 35 cycles of denaturation (94°C, 30 seconds), annealing (50 to 55°C, depending on primers, 30 seconds), extension (72°C, 1 minute to 1 minute 30 seconds) and a final extension at 72°C for 5 minutes. The PCR products were electrophoresed in a 1.5% agarose gel. Where a PCR product was obtained, this was sequenced from two randomly sampled individuals per infected species to ensure that the record represented a true positive and not a PCR artefact or related bacterium. PCR products were sequenced directly (through both strands) and analysed using the Basic Local Alignment Search Tool (BLAST) to establish that they were within the target clade. The relationship between strains was then estimated. Sequences were first aligned and modified visually using MEGA version 3.1 []. Phylogenetic analyses were conducted by the neighbour-joining (NJ) method and the maximum-parsimony (MP) method using MEGA version 3.1 []. NJ phylogenies were constructed based upon unambiguously aligned sites using the Tajima-Nei model of nucleotide substitution []. MP phylogenies were constructed using the close-neighbour-interchange method []. Bootstrap probabilities were calculated by generating 1000 bootstrap replicates. The sequences are deposited in GenBank (accession numbers EU333926–EU333941 and EU727094–EU727140). […]

Pipeline specifications

Software tools BLASTN, MEGA
Application Phylogenetics
Diseases Infection, Genetic Diseases, Inborn