Torix Rickettsia are widespread in arthropods and reflect a neglected symbiosis

Abstract

Background: Rickettsia are intracellular bacteria best known as the causative agents of human and animal diseases. Although these medically important Rickettsia are often transmitted via haematophagous arthropods, other Rickettsia, such as those in the Torix group, appear to reside exclusively in invertebrates and protists with no secondary vertebrate host. Importantly, little is known about the diversity or host range of Torix group Rickettsia.

Results: This study describes the serendipitous discovery of Rickettsia amplicons in the Barcode of Life Data System (BOLD), a sequence database specifically designed for the curation of mitochondrial DNA barcodes. Of 184,585 barcode sequences analysed, Rickettsia is observed in ∼0.41% of barcode submissions and is more likely to be found than Wolbachia (0.17%). The Torix group of Rickettsia are shown to account for 95% of all unintended amplifications from the genus. A further targeted PCR screen of 1,612 individuals from 169 terrestrial and aquatic invertebrate species identified mostly Torix strains and supports the "aquatic hot spot" hypothesis for Torix infection. Furthermore, the analysis of 1,341 SRA deposits indicates that Torix infections represent a significant proportion of all Rickettsia symbioses found in arthropod genome projects.

Conclusions: This study supports a previous hypothesis that suggests that Torix Rickettsia are overrepresented in aquatic insects. In addition, multiple methods reveal further putative hot spots of Torix Rickettsia infection, including in phloem-feeding bugs, parasitoid wasps, spiders, and vectors of disease. The unknown host effects and transmission strategies of these endosymbionts make these newly discovered associations important to inform future directions of investigation involving the understudied Torix Rickettsia.

Keywords: DNA barcoding; Rickettsia; endosymbiont; symbiosis: arthropods.

Figure 1:

Workflow of the BOLD project demonstrating the acquisition and fates of contaminant and non-contaminant COI barcoding sequences.

Figure 2:

Cladogram of the maximum likelihood (ML) tree of 1,126 proteobacteria COI contaminants retrieved from a BOLD project incorporating 184,585 arthropod specimens. The tree is based on 561 bp and is rooted with the free-living alphaproteobacteria Pelagibacter ubique. Parentheses indicate the number of BOLD contaminants present in each group. Tips are labelled by BOLD processing ID and host arthropod taxonomy. The Rickettsiales genera of Anaplasma, Rickettsia (collapsed node), Orientia, and Wolbachia supergroups (A, B, E, and F), as well as the Legionellales genera Legionella and Rickettsiella, are included as reference sequences (Accession numbers: Additional file 10).

Figure 3:

Cladogram of a maximum likelihood (ML) tree of 753 COI Rickettsia contaminants retrieved from a BOLD project incorporating 184,585 arthropod specimens. The tree is based on 561 bp and is rooted by the Rickettsia endosymbiont of Ichthyophthirius multifiliis (Candidatus Megaira) using the TVM+F+I+G4 model. Parentheses indicate the number of BOLD contaminants present in Torix and non-Torix Rickettsia groups. Tips are labelled by BOLD processing ID and host arthropod taxonomy. The Rickettsia groups Spotted Fever, Transitional, Belli, Typhus, Rhyzobius, and Torix are included as references (Accession numbers: Additional file 10).

Figure 4:

Phylogram of the maximum likelihood (ML) tree of 99 COI Rickettsia contaminants (prefix “BIOUG”) used for further phylogenetic analysis and 53 non-BOLD reference profiles (Accession numbers: Additional file 10). The tree is based on the concatenation of 4 loci, 16S rRNA, 17KDa,  gltA, and COI under a partition model, with profiles containing ≥3 of 4 sites included in the tree (2,834 bp total) and is rooted by Rickettsia endosymbiont of Ichthyophthirius multifiliis (Candidatus Megaira). Tips are labelled by host arthropod taxonomy.

Figure 5:

16S rRNA and gltA concatenated maximum likelihood (ML) phylogram (1,834 bp total) including Rickettsia hosts from SRA (triangles) and targeted screens (stars). The TIM3+F+R2 (16S) and K3Pu+F+G4 (gltA) models were chosen as best-fitting models. Rooting is with Orientia tsutsugamushi. Accession numbers found in Additional file 10.

Figure 6:

Phylogram of a maximum likelihood (ML) tree of COI Rickettsia contaminants (prefix “BIOUG”) giving a host barcode and 43 non-BOLD reference profiles. The tree is based on 4 loci, 16S rRNA, 17KDa,  gltA, and COI, under a partition model with profiles containing ≥2 out of 4 sites included in the tree (2,781 bp total) and is rooted by the Rickettsia endosymbiont of Ichthyophthirius multifiliis (Candidatus Megaira). The habitats and lifestyles of the host are given to the right of the phylogeny. Accession numbers found in Additional file 10.