A Covr Myrmica

UVicSPACE: Research & Learning Repository Faculty of Science Faculty Publications This is a post-review version of the following article: Evolution and diversity of inherited Spiroplasma in Myrmica ants Matthew J. Ballinger, Logan D. Moore and Steve J. Perlman February 2018 The final publication is available at: https://doi.org/10.1128/AEM.02299-17 Citation for this paper: Ballinger, M.J., Moore, L.D., Perlman, S.J. (2018). Evolution and diversity of inherited Spiroplasma symbionts in Myrmica ants. Applied and Environmental Microbiology, 84(4):e02299-17. https://doi.org/10.1128/AEM.02299-17 Accepted Manuscript: Ballinger MJ, Moore LD, Perlman SJ. 2018. Evolution and diversity of inherited Spiroplasma in Myrmica ants. Applied and Environmental Microbiology. 84 (4): e02299- 17. 1 2 3 4 5 6 7 8 Evolution and diversity of inherited Spiroplasma in Myrmica ants 9 10 Matthew J. Ballingera,1, Logan D. Moorea, and Steve J. Perlmana, 11 12 aDepartment of Biology, University of Victoria, Victoria, BC, Canada V8W 2Y2 13 14 15 1To whom correspondence should be addressed: [email protected] 16 Abstract (204 words) 17 Microbial partners play an important role in the biology and ecology of animals. In 18 insects, maternally-transmitted symbionts are especially common and can have host 19 effects ranging from reproductive manipulation to nutrient provisioning and defense 20 against natural enemies. In this study, we report a genus-wide association of Myrmica 21 ants with the inherited bacterial symbiont, Spiroplasma. We screen Myrmica ants 22 collected from the wild, including the invasive European fire ant, Myrmica rubra, and find 23 an extraordinarily high prevalence of this symbiont – 8 of 9 species, 42 of 43 colonies, 24 and 250 of 276 individual workers were harboring Spiroplasma – only one host species 25 was uninfected. In our screens, each host species carried a distinct Spiroplasma strain, 26 and none were infected with more than one strain. All symbionts belong to the citri 27 clade, allied most closely with pathogenic Spiroplasma of corn crops and honeybees, 28 and there is strong evidence of host-symbiont persistence across evolutionary 29 timescales. Genome sequencing of two Spiroplasma symbionts revealed candidate 30 genes that may play a part in the symbiosis, a nutrient transporter absent from other 31 Spiroplasma, and a ribosome-inactivating protein previously implicated in parasite 32 defense. These results together suggest long-term, likely mutualistic relationships 33 atypical of Spiroplasma-insect associations with potential significance for broad 34 ecological interactions of Myrmica. 35 2 36 Importance (129 words) 37 Animal-associated microbial symbionts can dramatically affect the biology of their hosts. 38 Identification and characterization of these intimate partnerships remains an essential 39 component of describing and predicting species interactions, especially for invasive host 40 species. Ants perform crucial ecological functions as ecosystem engineers, scavengers, 41 and predators, and ants in the genus Myrmica can be aggressive resource competitors 42 and reach high densities in their native and invaded habitats. In this study, a novel 43 symbiosis is identified between Myrmica ants and the facultative bacterial symbiont, 44 Spiroplasma. Broad host distribution, high frequencies of infection, and host-symbiont 45 codivergence over evolutionary timescales, an uncommon feature of Spiroplasma 46 associations, suggest an important, likely mutualistic interaction. Genome sequencing 47 identified highly divergent gene candidates that may contribute to Spiroplasma’s role as 48 a possible defensive or nutritional partner in Myrmica. 49 3 50 Introduction 51 It is now well established that most insects harbor maternally inherited bacterial 52 endosymbionts that play critical roles in the ecology and evolution of their hosts (1). 53 Insect lineages that feed exclusively on nutrient-poor diets, such as plant sap or animal 54 blood, typically host obligate nutritional endosymbionts that provide essential vitamins 55 and amino acids. These obligate endosymbionts are often housed in specialized 56 symbiont organs and show patterns of strict and ancient co-diversification with their 57 hosts. 58 More common still are facultative inherited symbionts, of which the best known is 59 Wolbachia (2). While these symbionts are transmitted almost exclusively through 60 females over ecological timescales, they very rarely cospeciate with their hosts, and 61 instead, repeatedly colonize new host lineages over evolutionary timescales via 62 horizontal transmission. Although not essential for host survival or reproduction, many 63 facultative inherited symbionts increase host fitness under certain conditions, for 64 example by protecting their hosts against natural enemies or environmental stresses 65 (3–8). Others manipulate their host's reproduction in order to increase the frequency of 66 symbiont-infected females (9–13). Five bacterial lineages are particularly widespread as 67 facultative symbionts of insects. In addition to Wolbachia, these are Arsenophonus, 68 Cardinium, Rickettsia, and Spiroplasma (14, 15). Initial surveys have found that these 69 bacteria infect ~5-30% of insect species – this represents millions of infected species. 70 However, most insect lineages have been poorly sampled, and even when infections 71 have been reported, it is often not understood how these facultative symbionts affect 72 host fitness or persist in host populations. 4 73 Spiroplasma is an incredibly diverse genus of bacteria that infect arthropods, with 74 a wide range of fitness effects and transmission strategies (16). Many Spiroplasma are 75 pathogenic, including pathogens of bees, crayfish, and plants (17–20). Even more 76 prevalent are horizontally transmitted gut commensals that have been isolated from a 77 wide range of insects, including beetles and flies (21, 22). Finally, maternal transmission 78 has evolved independently in a number of Spiroplasma lineages. Vertically transmitted 79 Spiroplasma can be found both inside and outside cells, often at high densities in insect 80 hemolymph, as well as in ovarian tissues (23). While the effects of most vertically 81 transmitted Spiroplasma are not known, a number of strains manipulate their host's 82 reproduction by killing male embryos; male-killing Spiroplasma strains have been 83 documented in butterflies, planthoppers, beetles, flies, and lacewings (10, 11, 24–26). 84 Some Spiroplasma protect their hosts against natural enemies, with strains that infect 85 aphids providing protection against pathogenic fungi (27), and strains that infect 86 Drosophila flies protecting against parasitic wasps and nematodes (4, 8). Recent 87 studies have implicated a diverse arsenal of toxins called ribosome-inactivating proteins 88 (RIPs) in Drosophila defense (28, 29). Identification of a Spiroplasma-encoded RIP 89 transcript in the publically-available transcriptome of the European invasive fire ant, 90 Myrmica rubra, motivated closer examination of the relationship between Spiroplasma 91 and this ant genus in the present study. 92 Although Spiroplasma infects a wide range of arthropods, few studies have 93 examined a specific group of hosts in detail. The best studied inherited Spiroplasma are 94 those that infect Drosophila. At least 18 species have been found to harbor Spiroplasma 95 (8, 30, 31), with infection frequencies ranging from less than 5% to greater than 85% 5 96 (32, 33). Drosophila flies have been independently colonized by five different lineages of 97 inherited Spiroplasma, from the citri, poulsonii, ixodetis, and tenebrosa clades (30). In 98 this study, ant species in the genus Myrmica were surveyed for Spiroplasma. This 99 genus also appears to be a hotspot for Spiroplasma infection, with all species except 100 one infected at high frequency. Unlike Drosophila, however, Spiroplasma infecting 101 Myrmica are all members of the citri clade, and there is a strong phylogenetic signal 102 suggesting persistent host-symbiont associations across evolutionary timescales. 103 Results 104 Spiroplasma symbionts are widespread in Myrmica 105 Nine species of Myrmica, broadly distributed across the genus (Fig 1), were screened 106 for Spiroplasma by PCR amplification of the ftsZ gene; all but one were positive (Table 107 1). Spiroplasma genes were also detected in all three publicly available Myrmica 108 transcriptomes (M. rubra, M. ruginodis, and M. sulcinodis; NCBI BioProject 109 PRJDB4088). Among screened species, the prevalence of infection was high, with 250 110 out of 276 individuals, and 42 out of 43 colonies, testing positive. COI failed to amplify 111 or yield quality sequence from nine of the 284 DNA extractions and these samples were 112 excluded from prevalence calculations. In one case, ftsZ amplified from a Myrmica sp. 113 sample in which COI failed and this sample was conservatively excluded from 114 subsequent analysis. For the two best sampled species, M. rubra and M. scabrinodis 115 (two mtDNA haplotypes), infection frequencies were 86% and 96%, respectively, and all 116 colonies were infected. Infection frequencies were similarly high in juvenile stages, with 117 9 of 10 larvae and 8 of 9 pupae from one M. scabrinodis colony testing positive. Many of 118 the species in our data set are represented by a single colony or individual, yet in most 6 119 cases Spiroplasma was consistently detected despite limited sampling; however, high 120 prevalence for these species should not

Load more