bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Eunji Park, 0000-0002-5087-2398 2 Robert Poulin, 0000-0003-1390-1206 3 4 5 Article type: Original article 6 7 8 Torix group Rickettsia are widespread in New Zealand freshwater 9 amphipods: using blocking primers to rescue host COI sequences 10 11 12 Eunji Park1* and Robert Poulin1 13 14 1 Department of Zoology, University of Otago, 340 Great King Street, Dunedin 9016, New Zealand 15 * Corresponding author: [email protected] 16 17 18 19 20 21 22 23 24 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 25 Abstract 26 Endosymbionts and intracellular parasites are common in arthropods and other invertebrate 27 hosts. As a consequence, (co)amplification of untargeted bacterial sequences has been 28 occasionally reported as a common problem in DNA barcoding. The bacterial genus 29 Rickettsia belongs to the order Rickettsiales and consists of two lineages: one including 30 diverse pathogens infecting arthropod hosts, the other consisting of non-pathogenic species 31 with a broaDer host taxonomic range. While discriminating among amphipod species with 32 universal primers for the COI region, we unexpectedly detected rickettsial endosymbionts 33 belonging to the Torix group. To map the distribution and diversity of Rickettsia among 34 amphipods hosts, we conducted a nationwide molecular screening of seven families of 35 freshwater amphipods collected throughout New Zealand. In addition to uncovering a 36 diversity of Torix group Rickettsia across multiple amphipod populations from three different 37 families, our research indicates that 1) detecting Torix Rickettsia with universal primers is not 38 uncommon, 2) obtaining ‘Rickettsia COI sequences’ from many host individuals is highly 39 likely when a population is infected, and 3) obtaining ‘host COI’ may not be possible with a 40 conventional PCR if an individual is infected. Because Rickettsia COI is highly conserved 41 across diverse host taxa, we were able to design blocking primers that can be used in a wide 42 range of host species infected with Torix Rickettsia. We propose the use of blocking primers 43 to circumvent problems caused by unwanted amplification of Rickettsia and to obtain targeted 44 host COI sequences for DNA barcoding, population genetics, and phylogeographic studies. 45 46 Keywords 47 Rickettsiales, bacterial COI, Amphipoda, DNA contamination, blocking oligos, universal 48 primers 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 49 Introduction 50 The cytochrome c oxidase subunit 1 gene (COI), a partial fragment of mitochondrial DNA, is 51 the marker of choice for DNA barcoding, and is also widely used for population genetics and 52 phylogeographic studies (Bucklin et al., 2011; Hajibabaei et al., 2007; Hebert et al., 2004). A 53 variable region is flankeD by highly conserved regions; this allowed for the design of a pair of 54 universal primers and their application to various organisms (Folmer et al., 1994; Hebert et 55 al., 2003). With the advancement of fast and cost-effective next-generation sequencing 56 technologies, which enables metabarcoding (Elbrecht and Leese, 2015; Taberlet et al., 2012), 57 the number of COI sequences is increasing rapidly in public databases such as GenBank and 58 The Barcode of Life DataSystems (BOLD) (Porter and Hajibabaei, 2018). However, quality 59 control is often an issue due to the presence of questionable “COI-like” sequences (Buhay, 60 2009) or nuclear mitochondrial pseudogenes (numts) that are often coamplified with 61 orthologous mtDNA (Song et al., 2008). Bacterial sequences are also often coamplified with 62 universal primers. Indeed, there have been reports of the amplification of untargeted 63 sequences of endosymbiotic bacteria such as Wolbachia and Aeromonas during DNA 64 barcoding with universal primers and their misidentification as those of invertebrate hosts 65 during deposition in databases (Mioduchowska et al., 2018; Smith et al., 2012). 66 67 The bacterial genus Rickettsia is another of these endosymbiotic taxa. This genus belongs to 68 the order Rickettsiales along with Wolbachia, and comprises diverse pathogenic species that 69 cause vector-borne diseases in birds and mammals including humans. Some rickettsioses 70 (diseases that are caused by Rickettsia) with severe symptoms are well known, and include 71 Rocky Mountain spotted fever, Queensland tick typhus, Rickettsial pox, and murine or 72 epidemic typhus (Parola et al., 2005; Perlman et al., 2006; Weinert, 2015). To date, at least 13 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 73 groups are known within the genus Rickettsia: Adalia, Bellii, Canadensis, Guiana, Helvetica, 74 Meloidae, Mendelii, Rhyzobious, Spotted fever, Scapularis, Torix, Transitional, and Typhus 75 (Binetruy et al., 2020; Hajduskova et al., 2016; Weinert et al., 2009). All these groups except 76 the Torix group are exclusively associateD with arthropod hosts, such as mites, fleas, ticks, 77 and spiders. The Torix group, which is sister to all other groups, is the only group that 78 includes non-arthropod hosts such as amoeba and leeches (Galindo et al., 2019; Kikuchi et al., 79 2002; Kikuchi and Fukatsu, 2005). In addition to these freshwater hosts, the Torix group 80 occurs in diverse arthropod groups that spend part of their life cycle in the aquatic 81 environment (e.g. Coleoptera and Diptera) (Küchler et al., 2009; Pilgrim et al., 2017; Weinert 82 et al., 2009). 83 84 Although Rickettsia are known as common pathogens or endosymbionts in arthropod hosts, 85 this group has never been reported in crustaceans. Rickettsia-like organisms (RLO) have been 86 reported in some groups of crustaceans including crabs, crayfish, lobsters, shrimps, and 87 amphipods (Gollas-Galván et al., 2014). However, most reports of these RLOs were based on 88 morphological similarity with Rickettsia and were rarely confirmeD by molecular data. In 89 amphipods, RLOs were reported in several species of gammarids, as well as other taxa (e.g., 90 Crangonyx floridanus and Diporeia sp.) (Graf, 1984; Larsson, 1982; Messick et al., 2004; 91 Winters et al., 2015). 16S rRNA sequences of RLOs are available for Diporeia sp. and some 92 gammarids, but none of them belong to the genus Rickettsia (Bojko et al., 2017; Winters et 93 al., 2015). 94 95 In the course of an investigation of the diversity of New Zealand freshwater amphipods, we 96 obtained a COI sequence that was highly divergent from that obtained from other populations 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 97 (~57%) of the same host group (Paracalliope species complex). DNA from the individual 98 amphipod was extracteD from its legs (i.e., low chance of contamination due to gut contents). 99 We obtained a clear chromatogram with no ambiguous peaks. Furthermore, this sequence was 100 similar to other COI sequences obtained from diverse insects (Coleoptera, Diptera, 101 Hemiptera, Hymenoptera, Odonata) in GenBank with sequence similarity ranging from 80 to 102 99 %. However, these sequences were also similar (~92%) to sequences of rickettsial 103 endosymbionts of insects and spiders that have been recently registered in GenBank. Because 104 such highly conserved COI sequences among distantly related arthropod groups are unlikely, 105 we assumed that these sequences were actually obtained from their rickettsial endosymbionts. 106 We independently confirmed the presence of Rickettsia in our amphipod hosts using three 107 genetic markers that were designed to be specific to Rickettsia. 108 109 Because Rickettsia is an endosymbiont within host cells, DNA extracts from infected host 110 tissue will inevitably include DNA of endosymbionts as well. If binding sites for ‘universal 111 primers’ are conserved in both hosts and their endosymbionts, PCR products obtained from 112 these mixed templates may result in mixed signals in chromatograms, or in the amplification 113 of endosymbiont instead of host sequences. Using primers that are designed to bind uniquely 114 and specifically to host templates woulD reduce this problem. However, designing group- 115 specific primers is not always possible, especially when reference sequences are scarce or not 116 available. Also, finding conserved regions across a given taxonomic group may not be 117 achievable. Alternatively, blocking primers can be useD to prevent the amplification of 118 unwanted or dominant sequences among DNA templates (Vestheim and Jarman, 2008). For 119 example, this method has been successfully applied to identify prey items (by suppressing the 120 amplification of predator DNA in gut contents), or to obtain rare mammal sequences from 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.120196; this version posted May 30, 2020.