Widespread Torix Rickettsia in New Zealand amphipods and the use of blocking primers to rescue host COI sequences

Abstract

Endosymbionts and intracellular parasites are common in arthropod hosts. As a consequence, (co)amplification of untargeted bacterial sequences has been occasionally reported as a common problem in DNA barcoding. While identifying amphipod species with universal COI primers, we unexpectedly detected rickettsial endosymbionts belonging to the Torix group. To map the distribution and diversity of Rickettsia species among amphipod hosts, we conducted a nationwide molecular screening of seven families of New Zealand freshwater amphipods. In addition to uncovering a diversity of Torix Rickettsia species across multiple amphipod populations from three different families, our research indicates that: (1) detecting Torix Rickettsia with universal primers is not uncommon, (2) obtaining ‘Rickettsia COI sequences’ from many host individuals is highly likely when a population is infected, and (3) obtaining ‘host COI’ may not be possible with a conventional PCR if an individual is infected. Because Rickettsia COI is highly conserved across diverse host taxa, we were able to design blocking primers that can be used in a wide range of host species infected with Torix Rickettsia. We propose the use of blocking primers to circumvent problems caused by unwanted amplification of Rickettsia and to obtain targeted host COI sequences for DNA barcoding, population genetics, and phylogeographic studies.

Genomic evolution and adaptation of arthropod-associated Rickettsia

“…(Order Rickettsiales, Family Rickettsiaceae ) are obligate intracellular α-proteobacteria of both ecological and medical interest. These bacteria live in close association with a diverse range of hosts including arthropods, mostly, and vertebrates, plants, algae, annelids, amoebae, ciliate and medusae (as primary hosts and vectors) 1 – 6 . Rickettsia spp.…”

Torix Rickettsia are widespread in arthropods and reflect a neglected symbiosis

“…These deposits derived from studies using mitochondrial DNA (mtDNA) barcoding for phylogeographic inference [ 30 ], or in barcoding-based species identification approaches [ 31 , 32 ]. Non-target amplification of Rickettsia COI using mitochondrial COI barcoding primers has been reported in spiders [ 31 , 32 ] and freshwater amphipods [ 30 , 33 ]. Furthermore, we have noted 2 cases in our laboratory where amplicons obtained for mtDNA barcoding of an arthropod have, on sequence analysis, revealed Rickettsia COI amplification (Belli group Rickettsia from Collembola, and Torix group Rickettsia from Cimex lectularius bedbugs).…”

Transmission, Tropism, and Biological Impacts of Torix Rickettsia in the Common Bed Bug Cimex lectularius (Hemiptera: Cimicidae)

“…The Rickettsia was a sister group to all other Rickettsia described previously, and the clade were named “torix Rickettsia” (Kikuchi et al, 2002 ). Torix Rickettsia have since been found across multiple arthropod taxa and seem to be widespread and especially common in species associated with freshwater [e.g., Culicoides midges (Pilgrim et al, 2017 ), dytiscid water beetles (Küchler et al, 2009 ), Odonata (Thongprem et al, 2020a ), and Amphipoda (Park and Poulin, 2020 )], but have also been detected in some terrestrial arthropods, e.g., Araneidae (Goodacre et al, 2006 ), Siphonaptera (Song et al, 2018 ), and Hemiptera (Wang et al, 2020 ). Whilst we now understand symbioses between invertebrates and torix Rickettsia are common, much less is known of their biological significance.…”