Torix Rickettsia is the most common bacterial contaminant produced
during barcoding projects
Out of 3,817 sequences considered contaminants, 1,126 of these were
deemed by BOLD to be bacterial in origin (Figure 1, Table S4).
Phylogenetic placement supported the correct designation of these
sequences as of microbial origin (Figure 2). The dominant genus wasRickettsia with 753 (66.9%) amplifications, compared toWolbachia with 306 (27.2%). Of the remaining 67 non-target
sequences, 16 formed a monophyletic group with other Anaplasmataceae and
51 were undesignated proteobacteria. When considering the 184,585
specimens in the total project, this analysis gave an overallRickettsia and Wolbachia prevalence of 0.41% and 0.17%
respectively within the dataset. Through later access to the 55,366
representative data subset from where the contaminants originated,
further unique bacteria contaminants were also detected (possibly missed
by BOLD’s automated contaminant filtering system). This suggests these
prevalences are conservative estimates.
BOLD Rickettsia contaminants were dominated by amplicons from the
Torix group of Rickettsia (716/753; 95.1%) (Figure 3). The
remaining 37 Rickettsia clustered with Transitional/Spotted Fever
(n=15), Belli (n=9), Rhyzobius (n=1) groups, while 12 sequences formed
two unique clades (Table S4). Across arthropod hosts: 292 (38.8%) were
derived from Hymenoptera; 189 (25.1%) from Diptera; 177 from Hemiptera
(23.5%); 41 from Psocoptera (5.4%); 40 from Coleoptera (5.3%); 7 from
Arachnida (0.9%); 4 from Trichoptera (0.5%); and single cases of
Thysanoptera, Diplopoda and Dermaptera (0.1% each). Mapping the 753Rickettsia to collection site (Figure S1) revealed arthropod
infections predominantly from Canada with other locations in
South/Central America, Europe, Africa and Asia.
We observed that two sets of COI primers were responsible for
99% of Rickettsia amplifications (Table S5) with a majority
(89%) amplifying with the primer combination C_LepFolF/C_LepFolR
(Hernández-Triana
et al., 2014). Torix Rickettsia COI showed a stronger
match to these primers at the 3’ end (the site responsible for efficient
primer annealing) compared to Wolbachia and otherRickettsia groups. Whilst all contained a SNP at the 3’
priming end of C_LepFolR, Torix Rickettsia (Rickettsiaendosymbiont of Culicoides newsteadi ; MWZE00000000) was the only
sequence to not contain a similar SNP at the 3’ priming site of
C_LepFolF (Tables S6.1 and S6.2).