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bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 Meta-transcriptomic comparison of the RNA viromes of the

2 vectors pipiens and Culex torrentium in northern Europe 3 4 5 John H.-O. Pettersson1,2,3,*, Mang Shi2, John-Sebastian Eden2,4, Edward C. Holmes2 6 and Jenny C. Hesson1 7 8 9 1Department of Medical Biochemistry and Microbiology/ Science Center, Uppsala 10 University, Sweden. 11 2Marie Bashir Institute for Infectious and Biosecurity, Charles Perkins Centre, 12 School of Life and Environmental Sciences and Sydney Medical School, the University of 13 Sydney, Sydney, New South Wales 2006, Australia. 14 3Public Health Agency of Sweden, Nobels väg 18, SE-171 82 Solna, Sweden. 15 4Centre for Research, The Westmead Institute for Medical Research, Sydney, Australia. 16 17 18 *Corresponding author: [email protected] 19 20 Word count abstract: 247 21 22 Word count importance: 132 23 24 Word count main text: 4113 25 26

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27 Abstract 28 There is mounting evidence that mosquitoes harbour an extensive diversity of 'insect-specific' 29 RNA in addition to those important to human and health. However, because 30 most studies of the mosquito virome have been conducted at lower latitudes there is a major 31 knowledge gap on the genetic diversity, evolutionary history, and spread of RNA viruses 32 sampled from mosquitoes in northern latitudes. Here, we determined and compared the RNA 33 virome of two common northern Culex mosquito species, Cx. pipiens and Cx. torrentium, 34 known vectors of and , respectively, collected in south-central 35 Sweden. Following bulk RNA-sequencing (meta-transcriptomics) of 12 libraries, comprising 36 120 specimens of Cx. pipiens and 150 specimens of Cx. torrentium, we identified 40 viruses 37 (representing 14 virus families) of which 28 were novel based on phylogenetic analysis of the 38 RNA-dependent RNA polymerase (RdRp) . Hence, we found similar levels of virome 39 diversity as in mosquitoes sampled from the more biodiverse lower latitudes. Four libraries, 40 all from Cx. torrentium, had a significantly higher abundance of viral reads, spanning ~7– 41 36% of the total amount of reads. Many of these viruses were also related to those sampled on 42 other continents, indicative of widespread global movement and/or long -virus co- 43 evolution. Importantly, although the two mosquito species investigated have overlapping 44 geographical distributions and share many viruses, approximately one quarter of the viruses 45 were only found at a specific location, such that geography must play an important role in 46 shaping the diversity of RNA viruses in Culex mosquitoes. 47 48 Importance 49 RNA viruses are found in all domains of life and all global habitats. However, the factors that 50 determine virome composition and structure within and between organisms are largely 51 unknown. Herein, we characterised RNA virus diversity in two common mosquito 52 species, and Culex torrentium, sampled from northern Europe. Our analysis 53 revealed extensive viral diversity, including 28 novel viruses, and was comparable to the 54 levels of diversity found in other temperate and tropical regions globally. Importantly, as well 55 as harbouring RNA viruses that are closely related to other mosquito-derived viruses sampled 56 in diverse global locations, we also described a number of viruses that are unique to specific 57 sampling locations in Sweden. Hence, these data showed that geographical factors can play an 58 important role in shaping virome structure even at local scales. 59

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60 Introduction 61 The mosquito (Diptera; Culicidae) Culex comprises more than a thousand species, with 62 representatives found globally (1). Culex species are vectors of a number of important 63 pathogens including West Nile virus (WNV) (), Japanese virus (JEV) 64 (Flaviviridae) and Sindbis virus (SINV) (Togaviridae), as well as a variety of nematodes (1– 65 3). One of the most widespread Culex species is the Northern House mosquito, Cx. pipiens, 66 that is distributed across the northern hemisphere. In Europe and the Middle East it occurs 67 together with Cx. torrentium, another Culex species with females and larvae that are 68 morphologically identical to Cx. pipiens. These two species have overlapping distributions 69 and share larval habitats. However, Cx. torrentium dominates in northern Europe while Cx. 70 pipiens is more abundant in the south (4). Both species are vectors for a number of - 71 associated viruses that can cause in Europe; for example, WNV, that may cause a 72 febrile disease with encephalitis, and SINV that may result in long lasting arthritis (2, 5). Cx. 73 pipiens is one of the most common WNV vectors in both southern Europe and North 74 America, and Cx. torrentium is the main vector of SINV in northern Europe (2, 6). Infections 75 with these pathogenic viruses occur in late summer when the viral prevalence accumulates in 76 passerine , the hosts of both of these viruses (7, 8). Despite their importance as 77 vectors, little is known about the detailed biology of Cx. pipiens and Cx. torrentium due to the 78 difficulties in species identification, which can only be reliably achieved through molecular 79 means. Much of the biology of these species, such as their larval habitat and feeding 80 preferences, is considered similar. However, one significant difference between the two 81 species is that while Cx. pipiens harbours a high prevalence of the intracellular bacteria 82 pipientis, it is seemingly absent in Cx. torrentium (9). 83 84 In recent years, studies utilizing RNA-sequencing (RNA-Seq, or 'meta-transcriptomics') have 85 revealed an enormous RNA virus diversity in both and (10, 11). 86 Mosquitoes are of particular interest as many are well-known vectors of pathogenic viruses. 87 Importantly, recent studies have shown that these pathogenic viruses represent only a fraction 88 of the total virome in the mosquito species investigated. Indeed, mosquitoes clearly carry a 89 large number of newly described and divergent -specific viruses, with 90 representatives from many genetically diverse virus families and orders, such as the 91 Flaviviridae, Togaviridae and the (12–16). However, most studies have been 92 conducted on latitudes below 55°, such that there is a marked lack of data of the mosquito 93 viral diversity present in northern temperate regions where the composition of mosquito

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94 species as well as environmental parameters differ significantly from lower latitudes. Indeed, 95 for many forms of life, biodiversity increases towards the equator (17), and the species 96 richness of mosquitoes is greater in tropical regions than temperate regions (18). A central 97 aim of the current study was therefore to investigate whether viral diversity co-varies in the 98 same manner. Given that Cx. pipiens and Cx. torrentium are two common Culex species in 99 northern and central Europe, and known vectors of SINV and WNV, they were chosen for 100 RNA virome investigation and comparison by RNA-Seq. 101 102 Results 103 RNA virome characterisation 104 We characterised the RNA viral transcriptome of two mosquito species, Cx. pipiens and Cx. 105 torrentium, collected from central and southern Sweden (Suppl. table 1). After high- 106 throughput sequencing, a total of 569,518,520 (range 34,150,856–62,936,342) 150bp reads 107 were produced from 12 ribosomal RNA-depleted sequence libraries that were assembled into 108 153,583 (4,333–33,893) contigs. From all the contigs assembled, we identified 40 that 109 contained RdRp sequence motifs and hence indicative of viruses, belonging to 14 different 110 viral families/orders: Alphaviridae, Bunyavirales, , Luteoviridae, 111 , , , , Picornaviridae, , 112 , and representatives from the divergent , Negeviridae- 113 and Qin-viruses. For each viral family/, between one and five virus species were 114 identified and in total 28 novel RNA virus species were discovered here, which were named 115 based on geographical location. 116 117 The relative number of all virus reads, as mapped to contigs with RdRp-motifs, compared to 118 the total amount of non-ribosomal RNA reads per library varied between 0.1–36.6% (Table 119 1). Notably, libraries 2, 10, 11 and 12 from Cx. torrentium were characterised by a 120 significantly higher number of viral reads compared to non-viral reads (Figure 1, Table 1). 121 The individual abundance of each viral species, measured as the number of reads mapped to 122 each RdRp contig divided by the total amount of reads in the library x 1000,000 (i.e. reads per 123 million, RPM), varied between 1.09–10,006.67 RPM for Cx. pipiens and 1.08–303,145.83 124 RPM for Cx. torrentium. In comparison, the abundance of host reads, as measured by the 125 presence of the host mitochondrial protein COX1, was more stable and varied only between 126 4.22–66.99 RPM across all libraries (Table 2). 127

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128 Virome comparison between mosquito species and geographical regions 129 Both the composition and abundance of the virus species and families observed differs 130 between the two mosquito species (Figure 2, Table 2). Of the 40 virus species discovered, 131 most were found in Cx. pipiens which harboured 34 species: 23 of these are newly described 132 in Cx. pipiens and 11 have been described previously. Sixteen of these 34 virus species were 133 unique to Cx. pipiens and hence not present in Cx. torrentium. Similarly, 24 of the 40 virus 134 species were discovered in Cx. torrentium: 18 of these are newly described in Cx. torrentium 135 and 6 have been described previously. Six viruses found Cx. torrentium were not present in 136 Cx. pipiens (Figure 3, Table 2). 137 138 We next analysed potential host-relationships by comparing abundance, presence across 139 libraries and phylogenetic relationship with other viruses (Table 3). These data suggest that 16 140 of the 40 viruses were likely mosquito associated, of which one and two were unique to Cx. 141 pipiens and Cx. torrentium, respectively (Figure 4–6). The host association was unclear in the 142 remaining viruses (for example, they could be associated with micro-organisms co-infecting 143 the mosquitoes) and could not be safely assumed to infect mosquitoes. For example, Ahus 144 virus (Totiviridae) was at low abundance, was not present in several libraries, and clustered 145 with viruses derived from various environmental samples, suggesting that it is less likely to be 146 mosquito-associated. Similarly, although Gysinge virus (Mononegavirales) was abundant and 147 present in several libraries (Table 3), that its closest relative (Figure 5) was a soy bean leaf 148 associated virus (19) means that it cannot be safely assigned to mosquitoes. Conversely, 149 Culex mononega like virus 2 (Figure 5) was found to be abundant, present in several libraries 150 and clustered with other mosquito viruses, suggesting that it is likely to be mosquito 151 associated. All potential mosquito host-association data is summarised in Table 3. 152 153 Notably, Cx. torrentium harboured four viruses of significantly higher abundance compared 154 to Cx. pipiens: (i) Nam Dinh virus (303,145 RPM, or 42% of all viral reads and more than 155 30% of all [non rRNA] reads, respectively, in library L2), (ii) Biggie virus (35,063 RPM, or 156 4.6% of all viral reads and 3.5% of all reads, respectively, in library L12), as well as two 157 newly identified viruses, (iii) Valmbacken virus (52,264 RPM, or 27% of all viral reads and 158 3.5% of all reads, respectively, in library L12), and (iv) Jotan virus (41,738 RPM, or 56% of 159 all viral reads and 4.2% of all reads, respectively, in library L11) (Table 2, Suppl. table 2). Cx. 160 pipiens had a more even composition of viral families across libraries (Figure 2), and the most

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161 abundant virus, Nam Dinh virus, reached 10,006 RPM (or 73% of all viral reads and 1% of all 162 reads, respectively) in library L5. 163 164 We next compared the virome composition between Kristianstad in the south and the 165 floodplains of the Dalälven river situated roughly 600 km further north. In the case of Cx. 166 pipiens this analysis revealed a total of 20 virus species from Kristianstad, 12 of which were 167 unique to Cx. pipiens and five detected in Kristianstad only, all of which were unique to Cx. 168 pipiens: Asum virus (Bunyaviridae), Eskilstorp virus (), Kristianstad virus 169 (Bunyaviridae), Rinkaby virus (Virga–Negev virus), and Vittskovle virus (Qinvirus). A total 170 of 28 viruses were found in Cx. pipiens from Dalälven. Eleven of these were unique to Cx. 171 pipiens and four were unique to Cx. pipiens from Dalälven: Salari virus (Bunyavirales), 172 Sonnbo virus (Partitiviridae), Culex mononega-like virus 1 (Mononegavirales), and Berrek 173 virus (Luteoviridae) (Table 2, Figure 2, Figure 4–6). A similar relationship was found for Cx. 174 torrentium. In the case of Dalälven, 24 viruses were found in Cx. torrentium, of which 18 175 were shared with Cx. pipiens and six of which were unique to Cx. torrentium (Table 2, Figure 176 2, Figure 4–6). Hence, the majority of the mosquito viruses identified here were shared both 177 between species and geographical regions, even though only 30 specimens of Cx. pipiens 178 were available from Kristianstad. In contrast, approximately one quarter of the total number 179 of virus species found at each location were unique to that location, indicative of some virome 180 differentiation at a local geographic scale. 181 182 Evolutionary history and host-associations of the discovered RNA viruses 183 Our phylogenetic analyses of the viruses discovered showed that several are closely related to 184 previously identified viruses, and that many form clusters with mosquito-associated and/or 185 Culex associated viruses within particular viral families, such as Merida virus and Gysinge 186 virus (Mononegavirales), and Tarnsjo virus () (Figure 4–6). In contrast, other 187 novel viruses clustered with viruses neither associated with mosquitoes nor other : 188 that they are distinguished by long branches suggests that they might infect diverse host taxa. 189 190 Positive-sense RNA viruses 191 We identified 16 positive-sense RNA viruses, of which 12 were likely novel. The majority of 192 the positive-sense RNA viruses fell within the Hepe-Virga-Endorna-Tymo-like virus complex 193 (N = 8), whereas the others fell within Nidovirales (N = 1), Luteoviridae (N = 4), 194 (N = 2) and Togaviridae (N = 1), respectively (Figure 4). The viruses

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195 discovered contain both those that are closely related to other mosquito-associated viruses, 196 such as the highly abundant Nam Dinh virus (Nidovirales), as well as those without clear host 197 associations. For example, Biggie virus clusters in a distinct group of Biggie viruses 198 (Virga/Endorna-viridae) sampled from other Culex mosquitoes (15). We also identified 199 several novel and divergent viruses in the family Endornaviridae - specifically Kerstinbo 200 virus and Hallsjon virus - that do not cluster with other arthropod-associated viruses (Figure 201 4). Similarly, within the Tymoviridae we detected a two variants of a Culex-associated virus, 202 Tarnsjo virus, that are closely related to a Culex associated Tymoviridae-like virus (15). 203 204 We identified four viruses within the Luteoviridae: Culex associated luteo-like virus, as well 205 as the novel Berrek, Fagle and Marma viruses. Culex associated luteo like-virus has 206 previously been found in a pool of Culex sp. mosquitoes from North America (15). Both of 207 the newly discovered Berrek virus and Marma virus grouped with other found in 208 mosquitoes (Figure 4), but only Marma virus was abundant, suggesting that it is Culex 209 associated (Table 2, Table 3). 210 211 Two novel were also identified. The abundant Rinkaby virus clusters with 212 Yongsan iflavirus 1 virus, sampled from Cx. pipiens mosquitoes from South Korea, and was 213 therefore considered a bona fide Culex associated . Although Ista virus did not 214 cluster with viruses derived from mosquitoes, its high abundance and the fact that it was 215 present in all libraries (Figure 4, Table 3) suggest that it is also Culex associated. 216 217 Finally, four of our libraries - L1 and L2 for Cx. torrentium and L3 and L6 for Cx. pipiens - 218 contained reads for SINV. Importantly, whereas as the presence of SINV could be confirmed 219 with PCR in library L1, L2 and L3, it was not PCR confirmed in L6 such that contamination 220 cannot be excluded in this case. 221 222 Negative-sense RNA viruses 223 In total, we identified 16 negative-sense RNA viruses, including 9 novel viruses. These were 224 distributed as follows: Bunyavirales (N = 5), Mononegavirales (N = 5), Qin-like viruses (N = 225 4) and Orthomyxoviridae (N = 2) (Figure 5). As was the case for the positive-sense RNA 226 viruses, some of these viruses that have been identified previously and cluster with viruses 227 found in mosquitoes of the same genera, including Salari virus (Bunyavirales) and a number 228 of novel viruses such as Anjon virus (Bunyavirales).

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229 230 Within the order Mononegavirales, Merida virus, Culex mononega-like virus 2, Culex 231 mosquito virus 4, and Culex mononega-like virus 1 have previously described been in 232 mosquitoes (12, 15, 20). Although abundant, the novel Gysinge virus was not found to cluster 233 with any mosquito sequences (Figure 5), so that its true host is uncertain. 234 235 The Qinviruses are a newly described and highly divergent group of RNA viruses (10). We 236 identified four novel Qin-like viruses: Nackenback virus, Gran virus, Vinslov virus and 237 Vittskovle virus. The latter three are more closely related to the Hubei qinvirus like virus 2 238 previously found in a pool of different arthropods (10). Nackenback virus was found to share 239 a more recent common ancestor with Wilkie Qin-like virus previously found in and 240 Culex mosquitoes in Australia (12). Although Qin-like was most closely related to fungal 241 viruses (12), it is notable that Nackenback virus was found in both Cx. pipiens and Cx. 242 torrentium libraries and was also more abundant than host non-RNA in the Cx. torrentium 243 libraries (Table 2, Table 3). Hence, this virus may be truly mosquito-associated. We also 244 detected two orthomyxoviruses, Wuhan Mosquito Virus 6 and Wuhan Mosquito Virus 4, both 245 of which have previously been found in pools of Culex mosquitoes and are known to be 246 mosquito associated (12). 247 248 Double-stranded RNA viruses 249 We identified a total of eight double-stranded RNA viruses in our Swedish mosquitoes, all of 250 which were novel. These belong to the Partitiviridae (N = 2), Reoviridae (N = 1) and 251 Toti/Chrysoviridae (N = 5). For the family Reoviridae, Valmbacken virus clustered with 252 reo-like virus, previously discovered in mosquitoes (12). Valmbacken 253 virus was also abundant and found in all libraries, and is therefore most likely a Culex 254 associated reovirus (Table 2, Table 3). 255 256 In comparison, four of the five novel clustered with other mosquito associated 257 totiviruses (Figure 6), but only two (Lindangsbacken virus and Eskilstorp virus) were also 258 found to be abundant. The fifth , Ahus virus, was highly divergent, had low 259 abundance and clustered distantly with potentially protist originating viruses (Figure 6). Thus, 260 the host association of Ahus virus remains uncertain. 261

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262 The two novel partiti-like viruses, Vivastbo virus and Sonnbo virus, did not cluster with any 263 viruses sequenced from mosquitoes, but rather grouped with viruses originating from various 264 arthropod hosts (Figure 6). However, the relatively high abundance levels of Vivastbo virus 265 suggest that it may be associated with mosquitoes (Table 2, Table 3). 266 267 Finally, all sequencing libraries generated here were negative for Wolbachia as assessed by 268 mapping against the COX1 and WSP of Wolbachia pipientis. Although it is not 269 possible to completely exclude the presence of other Wolbachia variants, our results suggest 270 that differential presence/absence of Wolbachia has not affected the viral load or diversity 271 observed. 272 273 Discussion 274 Through total RNA-sequencing of 270 Culex mosquitoes collected in Sweden we identified 275 40 viruses, including 28 that are novel. A virome comparison between the two vector species 276 Cx. pipiens and Cx. torrentium revealed that although these mosquitoes are from the same 277 genus and have overlapping geographical distribution, the virome family and species 278 composition and abundance differed to some extent between the two species, and also by the 279 geographic location of sampling (Figure 1, Figure 2, Table 2). 280 281 Viewed at the family/order level, the relative virome abundance of Cx. pipiens was dominated 282 by nido-, luteo-, and orthomyxo-viruses. In comparison, Cx. torrentium had a greater 283 proportion of picorna-, nido-, mononega-, and reo-viruses. It should be noted, however, that 284 family-wide comparisons could be significantly skewed in the presence of single highly 285 abundant viruses, as was the case here (e.g. the Nam Dinh virus in library 2 that reached 30% 286 of all reads in the library), such that analyses of relative abundance and diversity are better 287 conducted at the species level. Viewed at the level of species per region, about one quarter of 288 the viruses were unique to their respective sampling location (Figure 3). This suggests that 289 local acquisition, as well as local ecosystem and habitat composition, may be important in 290 shaping virome compositions. 291 292 Direct comparisons between virome studies are greatly complicated by such factors as 293 differences in sequencing technologies, bioinformatic analyses, criteria for species 294 demarcation, and study focus. Despite these important caveats, it is noteworthy that the 295 number of viruses found in here is of a similar magnitude and diversity to those found at

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296 lower latitudes (12, 15). Hence, the virome composition appears not to follow the same trend 297 as mosquito biodiversity, with fewer species in temperate regions (17, 18). Specifically, 24 298 different viruses were found in Cx. torrentium, of which six were unique to that species, and 299 34 viruses were found in Cx. pipiens, of which 16 were unique. Hence, 18 viruses were shared 300 between both mosquito species, 16 of which we tentatively consider to be mosquito 301 associated based on their abundance and phylogenetic position (Figure 3, Figure 4–6, Table 302 3). 303 304 Given their relatively close phylogenetic relationship (Figure 7) and the fact that both 305 mosquito species inhabit the same region, share larval habitat (4), and blood-meal hosts 306 (Hesson, J. unpublished), the difference in their viromes is striking. By considering virus 307 abundance and phylogenetic position we suggest that 26 of the viruses discovered were likely 308 mosquito-associated (Figure 4–6, Table 3), although we cannot exclude either false-negative 309 or false-positive associations. For example, the divergent Ista virus (Picornaviridae) was 310 found in high abundance and in multiple libraries, but did not cluster with any viruses that 311 originated from mosquitoes, although it did group with other arthropods (Figure 4–6). The 312 fact that it did not cluster with other mosquito viruses is, however, perhaps unsurprising as 313 studies from temperate regions are few, and this is the first study investigating the virome of 314 Cx. torrentium. It is clear that many viruses are seemingly ubiquitous in mosquitoes, covering 315 a wide variety of climates and habitats (12, 15, 21), but whether Ista virus and many other 316 viruses are truly mosquito-associated will need to be considered in additional studies. It was 317 also noteworthy that no insect-specific were discovered in this study, even though 318 these are relatively commonplace (22) and have previously been found in mosquitoes in 319 northern Europe (23, 24). 320 321 Notably, our study suggests that pathogenic viruses, such as SINV, can sometimes have 322 similar abundance to viruses not associated with human disease (Table 2), suggesting that 323 presence of pathogenic viruses does not necessarily impact overall virome composition. The 324 difference in viral load of certain viruses between Cx. torrentium and Cx. pipiens is 325 interesting. One potential explanation is that the virome composition has been impacted 326 through differential associations with the intracellular bacteria Wolbachia pipientis. Indeed, it 327 has previously been shown that Cx. pipiens is commonly infected with Wolbachia, while this 328 bacterium is absent from Cx. torrentium (9). Wolbachia is well-known for its ability to 329 influence virus infection in mosquitoes, but has mostly been studied in systems with

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330 pathogenic viruses such as dengue (25). However, we found no compelling evidence for 331 Wolbachia in any of the Culex samples studied here. 332 333 The species separation between Cx. pipiens and Cx. torrentium has long been ignored, largely 334 because of the need for molecular demarcation, so that it has been assumed that most of the 335 biology of the two species is comparable. Our study reveals that Culex mosquitoes in northern 336 temperate regions harbour as much, and sometimes more, viral diversity as mosquitoes in 337 tropical and sub-tropical regions. We also show that the two vector species share many 338 viruses, but also differ noticeably in which RNA viruses they harbour, and at significantly 339 different abundance levels. Further studies should aim to study how mosquito host structure 340 and feeding preference, evolutionary history, ecological relationships, geography and 341 environment determines virome composition and the potential interaction with pathogenic and 342 non-pathogenic viruses, as well as the impact of virome composition on mosquito health. 343 344 Materials and Methods 345 Mosquito collection 346 Mosquitoes were collected from two regions in Sweden: (i) from floodplains of the Dalälven 347 river in central Sweden (60.2888; 16.8938) in 2006, 2009, and 2011, and (ii) around the city 348 of Kristianstad, in southern Sweden (56.0387; 14.1438) in 2006 and 2007. Collections were 349 performed using CDC-light traps baited with carbon dioxide, and catches were sorted and 350 identified to species on a chilled table, using keys by Becker et al. (26). In total, legs from 270 351 Cx. pipiens/torrentium mosquitoes were removed to enable molecular identification to species 352 (27). Bodies were homogenized in PBS buffer supplemented with 20% FCS and antibiotics 353 and stored at -80C degrees until further processing. 354 355 Sample processing and sequencing 356 Total RNA was extracted from 12 pools from the homogenate of individual Cx. torrentium (n 357 = 150) and Cx. pipiens mosquitoes (n = 120) (Supplementary table 1), using the RNeasy® 358 Plus Universal kit (Qiagen) following the manufacturer’s instructions. The extracted RNA 359 was subsequently DNased and purified using the NucleoSpin RNA Clean-up XS kit 360 (Macherey-Nagel). Prior to library construction, ribosomal RNA (rRNA) was depleted from 361 the purified total RNA using the Ribo-Zero Gold (human-mouse-rat) kit (Illumina) following 362 the manufacturer’s instructions. Sequencing libraries were then constructed for all rRNA- 363 depleted RNA-samples using the TruSeq total RNA library reparation protocol (Illumina). All

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364 libraries were sequenced on a single lane (paired-end, 150 bp read-length) on an Illumina 365 HiSeq X10 platform. Library preparation and sequencing was carried out by the Beijing 366 Genomics Institute (www.bgi.com/global/). All 12 libraries were quality trimmed with 367 trimmomatic v.0.36 (28) and then assembled de novo using Trinity v.2.5.4 (29). 368 369 Discovery of viruses and Wolbachia bacteria 370 Trinity assemblies were screened against the complete non-redundant NCBI GenBank 371 (nt) and protein (nr) databases using blastn and diamond (30) blastx with a cut-off 372 e-value of 1×10−5. Assemblies identified as RNA viruses were screened against the 373 Conserved Doman Database (www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi) with an 374 expected value threshold of 1×10−3 to identify viral sequence motifs. The mitochondrial 375 COX1 , mined from the sequence data, and all contigs with RdRp-motifs was mapped 376 back, using Bowtie2 (31), against all quality trimmed libraries to estimate abundance. A virus 377 was considered to be in high abundance if: (i) it represented >0.1% of total non-ribosomal 378 RNA in the library, and (ii) if the abundance was higher to that of abundant host COX1 gene 379 (12, 32), and hence likely to be mosquito associated. Hits that were below the level of cross- 380 library contamination due to index-hopping, measured as 0.1% of the most abundant library 381 for the respective virus species or less than 1 read per million mapped to a specific virus 382 contig, was considered negative (coloured grey in Table 1 and Table 2, respectively). To 383 investigate the presence of Wolbachia bacteria in the libraries, published sequences of the 384 Wolbachia Cx. pipiens wsp surface protein gene (DQ900650.1) and the mitochondrial COX1 385 gene (AM999887.1) were mapped backed against all libraries using the above criteria for 386 abundance and presence/absence. 387 388 Inference of virus evolutionary history and host associations 389 The evolutionary (i.e. phylogenetic) history of the viruses discovered was inferred by aligning 390 protein translated open reading frames with representative sequences from the Alphaviridae, 391 (Order) Bunyavirales, Endornaviridae, Luteoviridae, (Order) Mononegavirales, Nido-like 392 viruses, (Order) Orthomyxovirales, Partitiviridae, Picornaviridae, Qin-like viruses, 393 Reoviridae, Totiviridae, Tymoviridae and Virgaviridae and Negev-like viruses. All RdRp 394 amino acid sequence alignments were performed using the E-INS-i algorithm in Mafft (33). 395 Poorly aligned regions, in which amino acid positional homology could not be confirmed, 396 were then removed from the alignments using TrimAl utilizing the ‘strict’ settings. Finally, 397 phylogenetic trees were computed with a maximum likelihood approach as implemented in

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398 PhyML (34) employing the LG +Γ amino acid model, SPR branch-swapping and the 399 approximate likelihood ratio test (aLRT) with the Shimodaira-Hasegawa-like procedure used 400 to assess branch support. The resultant phylogenetic trees were edited and visualised with 401 FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/so ware/figtree). 402 403 To help assess whether the novel viruses discovered are mosquito-associated - that is, to 404 distinguish those that actively replicate in the host from those present in diet mosquito or a co- 405 infecting micro-organism - we considered four factors: (i) the abundance of viral contigs per 406 total number of reads in a library, (ii) the abundance in relation to the COX1 host gene, (iii) 407 the presence in the libraries, and (iv) phylogenetic clustering with other mosquito derived 408 viruses. 409 410 The raw sequence data generated here has been deposited in the NCBI short read archive 411 (BioProject: PRJNA516782) and all viral contigs has been deposited in NCBI GenBank 412 (accession numbers: MK440619–MK440659). 413 414 Acknowledgments 415 The authors are grateful to Dr. JO Lundström for access to mosquito material. JHOP is 416 supported by the Swedish research council FORMAS (grant nr: 2015-710). ECH is supported 417 by an ARC Australian Laureate Fellowship (FL170100022). JCH is supported by E and R 418 Börjeson's Foundation and the Swedish Society for Medical Research. 419 420 References 421 1. Mullen GR, Durden L. 2009. Medical and veterinary entomology2. ed. Elsevier, 422 Amsterdam. 423 2. Gould E, Pettersson J, Higgs S, Charrel R, de Lamballerie X. 2017. Emerging 424 : Why today? One Health 4:1–13. 425 3. Weaver SC, Lecuit M. 2015. virus and the global spread of a 426 mosquito-borne disease. N Engl J Med 372:1231–1239. 427 4. Hesson JC, Rettich F, Merdić E, Vignjević G, Ostman O, Schäfer M, Schaffner 428 F, Foussadier R, Besnard G, Medlock J, Scholte E-J, Lundström JO. 2014. The 429 vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe. 430 Med Vet Entomol 28:179–186. 431 5. Kurkela S, Helve T, Vaheri A, Vapalahti O. 2008. Arthritis and arthralgia three 432 years after Sindbis virus infection: clinical follow-up of a cohort of 49 patients. Scand J Infect 433 Dis 40:167–173. 434 6. Hesson JC, Verner-Carlsson J, Larsson A, Ahmed R, Lundkvist Å, Lundström 435 JO. 2015. Culex torrentium Mosquito Role as Major Enzootic Vector Defined by Rate of 436 Sindbis Virus Infection, Sweden, 2009. Emerging Infect Dis 21:875–878.

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515 Tables 516 Table 1. Overview of viral RdRp-motif library content compared to the total number of non- 517 ribosomal RNA reads per library. 518 519 Table 2. Individual abundance, measured as reads per million, of each virus, as well as 520 Wolbachia bacteria, in comparison with the abundance of host COX1 gene. 521 522 Table 3. Indication of host associations for the discovered viruses. Host-association was 523 assessed using (i) the abundance of viral contig per total amount of reads in a library, (ii) virus 524 abundance in relation to the COX1 host gene, (iii) presence amongst the libraries, and (iv) 525 phylogenetic clustering with other mosquito derived viruses. 526 527 Figures 528 Figure 1. Estimation of virome composition compared to host (non-viral) content in each 529 library. 530 531 Figure 2. Comparison of the virome family composition and abundance between the Cx. 532 pipiens and Cx. torrentium. For ease of presentation, abbreviations are used to indicate virus 533 in each case. 534 535 Figure 3. Venn diagram showing the number of unique and shared viruses per location per 536 mosquito species. 537 538 Figure 4. Phylogenetic analysis of all the positive-sense RNA viruses identified here (marked 539 by coloured circles) along with representative publicly available viruses. Those viruses most 540 likely associated with mosquitoes are marked by an *. Numbers on branches indicate SH 541 support, and only branches with SH support ≥80% are indicated. Branch lengths are scaled 542 according to the number of amino acid substitutions per site. All phylogenetic trees were 543 midpoint-rooted for clarity only. 544 545 Figure 5. Phylogenetic analysis of all the negative-sense RNA viruses identified here 546 (marked by coloured circles) along with representative publicly available viruses. Those 547 viruses most likely associated with mosquitoes are marked by an *. Numbers on branches 548 indicate SH support, and only branches with SH support ≥80% are indicated. Branch lengths

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549 are scaled according to the number of amino acid substitutions per site. All phylogenetic trees 550 were midpoint-rooted for clarity only. 551 552 Figure 6. Phylogenetic analysis of all the double-stranded RNA viruses identified here 553 (marked by coloured circles) along with representative publicly available viruses. Those 554 viruses most likely associated with mosquitoes are marked by an *. Numbers on branches 555 indicate SH support, and only branches with SH support ≥80% are indicated. Branch lengths 556 are scaled according to the number of amino acid substitutions per site. All phylogenetic trees 557 were midpoint-rooted for clarity only. 558 559 Figure 7. Phylogenetic relationships, based on partial COX1-gene, of Cx. pipiens and Cx 560 torrentium for all libraries (L1–L12) together with representative publicly available reference 561 sequences (with their associated GenBank accession numbers). Numbers on branches indicate 562 SH support, and only branches with SH support ≥80% are indicated. Branch lengths are 563 scaled according to the number of nucleotide substitutions per site. The tree is midpoint 564 rooted for clarity only. 565 566 Supplementary Material 567 Supplementary Table 1. Information on collection site, year of collection and pool size for 568 all Cx. pipiens and Cx. torrentium libraries. 569 570 Supplementary Table 2. Number of reads mapped to each virus, Wolbachia and host genes 571 per library per mosquito species.

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100% Virga–Negev

90% Tymo Toti 80% Reo

Qin 70% Picorna

60% Phasma

Partiti 50% Orthomyxo

40% Nido Mononega

30% Luteo

Endorna 20% Chryso

10% Bunya Alpha 0% L1 L2 L9 L10 L11 L12 L3 L4 L5 L6 L7 L8 Cx. torrentium Cx. pipiens bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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 100% aCC-BY-NC-ND 4.0 International license. 40%

30%

Non-viral reads 20% Virus reads

10%

0% L1 L2 L9 L10 L11 L12 L3 L4 L5 L6 L7 L8 Cx. torrenium Cx. pipiens bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Cx. torrentium – Dalälven # unique viruses = 6

Shared Cx. torrentium and Shared Cx. torrentium (Dalälven) Cx. pipiens – Dalälven and Cx. pipiens (Kristianstad) # viruses = 10 # viruses = 1

Shared between both species and all locations # viruses = 7

Cx. pipiens – Dalälven Cx. pipiens – Kristianstad # unique viruses = 4 # unique viruses = 5

Shared Cx. pipiens (Dalälven) and Cx pipiens (Kristianstad) # viruses = 7 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Hepe-Virga-Endorna-Tymo-like Luteoviridae Picornaviridae Negev virus 1 1 Wuhan insect virus 13 0.93 Yongsan iflavirus 1 Negev virus Hubei mosquito virus 2 1 1 Negev virus Hubei mosquito virus 2 Bee iflavirus 1 Yongsan iflavirus 1 1 1 0.99 Negev virus Marma virus Ista virus 1 1 Jotan virus * * 1 * Negev virus Wenzhou sobemo like virus 4 * 1 Hubei picorna-like virus 30 Armigeres iflavirus Brejeira virus 1 1 Hubei sobemo like virus 8 len2721 Hubei negev like virus 2 Venturia canescens picorna-like virus 1 Slow bee paralysis virus 1 Hubei sobemo like virus 10 1 1 Loreto virus 0.98 Dinocampus coccinellae paralysis virus Hubei odonate virus 4 1 Hubei sobemo like virus 9 1 Wuhan house centipede virus 1 1 1 1 1 Wuhan coneheads virus 1 Hubei tetragnatha maxillosa virus 2 Wuhan insect virus 8 Wuhan arthropod virus 4 1 1 Hubei virga like virus 4 Shuangao insect virus 9 Nasonia vitripennis virus Hubei picorna-like virus 28 0.93 Hubei negev like virus 1 Baird Spence virus Hubei myriapoda virus 1 Biggie virus Sanxia water strider virus 10 Biggie virus * 1 1 0.81 Culex associated luteo like virus Culex Biggie like virus * 0.5 0.5 1 1 Culex associated Luteo like virus Culex negev like virus 3 0.93 1 Tanay virus 0.99 Wenzhou sobemo like virus 3 Sanxia sobemo like virus 2 1 Hubei virga like virus 7 1 Wuhan insect virus 9 0.99 Hubei sobemo like virus 13 Togaviridae Nido virus-like 1 Hubei virga like viurs 8 0.91 Sanxia sobemo like virus 1 1 1 Alphamesonivirus 1 1 1 0.99 1 Bebaru virus 1 1 1 Nam Dinh virus 1 Hubei sobemo like virus 41 Mayaro virus * 1 1 Ngewotan virus Hubei sobemo like virus 41 1 Getah virus 1 1 0.99 1 Hubei virga like virus 23 virus 1 Culex luteo like virus Nam Dinh virus Hubei virga like virus 23 1 1 1 1 Berrek virus Chikungunya virus Rinkaby virus 1 Cavally virus 1 1 Onyong nyong virus Hubei virga* like virus 22 Humaita Tubiacanga virus 0.9 1 Dianke virus Hubei virga like virus 21 Wuchan romanomermis nematode virus 3 Ndumu virus 1 1 1 Shuangao sobemo like virus 2 Bontang Baru virus 1 Hubei sobemo like virus 40 1 Bontang virus 1 1 1 1 Beihai sobemo like virus 27 1 Western virus Karang Sari virus 1 1 1 1 Wenzhou shrimp virus 9 Fort Morgan virus 1 Shahe endorna like virus 1 Kamphang Phet virus 1 1 Shahe endorna like virus 1 Barns Ness beadlet anemone sobemo like 1 Madariaga virus Casuarina virus Osterfarnebo virus Eastern equine encephalitis virus 0.89 Venezuelan equine encephalitis virus Nse virus strain 1 1 1 0.85 Southern elephant seal virus 0.94 Helicobasidium mompa alphaendornavirus 1 Nse virus Norway luteo like virus 1 0.85 0.99 Hallsjon virus 0.92 Sindbis virus Helianthus annuus alphaendornavirus Hubei sobemo like virus 29 Sindbis virus 1 1 Meno virus strain 1 1 0.81 Soybean leaf associated endornavirus 1 Hubei sobemo like virus 30 Ockelbo virus Meno virus 0.98 0.88 Rhizoctonia cerealis alphaendornavirus 1 0.95 Fagle virus Sindbis virus 1 1 Beihai Nido like virus 1 Ceratobasidium endornavirus D 1 Sanxia sobemo like virus 4 Sindbis virus 1 Rhizoctonia solani endornavirus Beihai Nido like virus 1 0.92 Tama virus Sindbis virus * 1 Phytophthora alphaendornavirus 1 Babanki virus 0.95 1 Sanxia sobemo like virus 5 1 1 Kerstinbo virus Sindbis like virus * Hubei sobemo like virus 25 1 0.7 Grapevine endophyte endornavirus Sindbis virus 1 0.97 Vicia faba alphaendornavirus Hubei sobemo like virus 36 0.99 Whataroa virus Wuchan romanomermis nematode virus 1 1 1 Aura virus 1 Tai Forest Forneby virus 0.6 1 Endornavirus like virus Sclerotinia sclerotiorum endornavirus 4 1 0.2 1

0.94 Tarnsjo virus 1 1 * Tarnsjo virus = Associated with moquitoes (Tab. 3) 0.95 Culex originated *Tymoviridae like virus * 1 = Found both in Cx. pipiens and Cx. torrentium 1 Hubei macula like virus 1 1 Hubei macula like virus 2 = Found only in Cx. pipiens Hubei macula like virus 3 1 = Found only in Cx. torrentium 0.5 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Mononegavirales Bunyavirales Bunyavirales Orthomyxoviridae 0.94 Bunyavirus sp 1 Simbu orthobunyavirus 1 Wuhan Louse Fly Virus 4 1 Culex mononega like virus 1 * 1 0.98 1 Hubei bunya like virus 13 Aino virus Wuhan Louse Fly Virus 3 1 Culex mononega like virus 1 1 1 Hubei insect virus 1 Shamonda virus Jingshan Fly Virus 1 1 Xincheng Mosquito Virus 1 1 Rhinolophus pearsoni bunyavirus 1 Akabane virus Shuangao Insect Virus 4 0.99 Xincheng Mosquito Virus 1 1 Cat Que virus Ostrinia furnacalis 1 Sanxia Water Strider Virus 3 Aedes anphevirus 1 1 1 Pararge aegeria Buttonwillow virus 1 Wuhan Mosquito Virus 5 Aedes aegypti anphevirus 1 1 1 Oropouche virus 1 Hubei bunya like virus 2 0.95 Aedes alboannulatus orthomyxo-like virus Aedes anphevirus 1 Hubei bunya like virus 3 Leanyer virus 1 Wuhan Mosquito Virus 3 1 Bolahun virus variant 2 Wuhan insect virus 16 Mburo virus 1 1 1 Wuhan Mosquito Virus 6 Bolahun virus variant 1 1 Salari virus 1 Bwamba orthobunyavirus 1 * 1 Wuhan Mosquito Virus 6 Gambie virus Pongola virus SAAr1 1 Salarivirus Mos8CM0 1 1 1 Wuhan Mosquito Virus 6 1 Culex mononega like virus 2 1 uncultured virus La Crosse virus 1 1 Wuhan Mosquito Virus 4 Culex mononega like virus 2 1 Xinzhou Mosquito Virus Kowanyama virus * * 1 Wuhan Mosquito Virus 4 Zhee mosquito virus Bunyamwera virus 1 Shuangao Insect Virus 3 1 1 Wuhan Louse Fly Virus 1 Culex tritaeniorhynchus rhabdovirus Wolkberg virus 0.7 1 Crithidia G15 virus 1 Culex tritaeniorhynchus rhabdovirus 1 Murrumbidgee virus Apis bunyavirus 1 0.98 1 0.93 Orthobunyavirus sp 1 Culex tritaeniorhynchus rhabdovirus Crithidia ZM virus 1 1 1 Caraparu virus 0.99 Merida like virus Hubei bunya like virus 5 1 1 Caraparu virus Qin-like viruses 1 Merida virus Hubei bunya like virus 6 1 1 Itaqui virus 1 Hubei qinvirus like virus 2 Merida virus Humpbacked Fly Virus 1 * 0.87 1 Nepuyo virus Hubei dimarhabdovirus virus 3 Rasbo virus Hubei qinvirus like virus 2 1 Gumbo Limbo virus 1 1 1 1 Wuhan Virus Vinslov virus Ohlsdorf virus 1 1 Moju virus 1 Procotyla fluviatilis Ohlsdorf virus 1 Vittskovle virus Bimiti virus Hubei bunya like virus 4 Gran virus 1 Culex mosquito virus 4 1 1 Ananindeua virus * 1 1 Jiangxia Mosquito Virus 1 1 Culex mosquito virus 4 1 Capim virus Wilkie qin like virus 0.88 Jiangxia Mosquito Virus 1 1 1 Culex mosquito virus 5 0.96 Guajara orthobunyavirus Wilkie qin like virus Kristianstad virus 1 1 1 Wuhan Mosquito Virus 8 1 Brazoran virus 1 Wilkie qin like virus 1 Beihai barnacle virus 6 Asum virus Wuhan Mosquito Virus 8 Nackenback virus 1 0.95 Beihai blue swimmer crab virus 2 0.97 Kibale virus * Imjin River virus 1 Shahe bunya like virus 1 1 1 Wenzhou qinvirus like virus 2 Herbert virus Shuangao Fly Virus 1 0.98 Ixodes scapularis associated virus 6 0.98 Wenzhou qinvirus like virus 2 Tai virus 1 1 Sclerotinia sclerotiorum negative RNA virus 1 Shuangao Insect Virus 1 0.4 0.9 Fusarium graminearum negative RNA virus 1 0.5 Khurdun virus 1 Gysinge virus * 1 Soybean leaf associated negative RNA virus 0.97 Apis bunyavirus 2 0.5 1 Hubei rhabdo like virus 4 1 Hubei diptera virus 6 1 0.9 Hubei diptera virus 7 Hubei rhabdo like virus 4 Wuhan mosquito virus 1 Kiln Barn virus 1 Anjon virus 1 * Culex phasma like virus 1 1 0.6 Wuhan Mosquito Virus 2 Kigluaik phantom orthophasmavirus Nome phantom orthophasmavirus

0.4 * = Associated with moquitoes (Tab. 3) = Found both in Cx. pipiens and Cx. torrentium = Found only in Cx. pipiens = Found only in Cx. torrentium bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Partitiviridae Reoviridae Toti- and Chryso-viridae

0.99 0.97 1 0.81 0.98 Eskiltorp virus 1 0.99 1 Hubei partiti like virus 14 1 Hubei chryso like *virus 1 0.82 1 Hubei partiti like virus 14 1 Shuangao chryso like virus 1 1 1 Hubei partiti like virus 13 1 Hubei chryso like virus 2 1 0.93 Partitivirus-like 2 Phlebiopsis gigantea mycovirus dsRNA 1 Micromonas pusilla reovirus 1 Ustilago maydis virus H1 Sonnbo virus 0.97 1 1 Homalodisca vitripennis reovirus Botrytis porri RNA virus 1 1 Hubei partiti like virus 15 1 Rice dwarf virus 1 Beihai barnacle virus 15 Hubei partiti like virus 16 0.83 0.99 1 Rice gall dwarf virus Beihai barnacle virus 15 Beihai partiti like virus 2 1 1 Ahus virus Hubei reo-like virus 10 1 1 1 Diatom colony associated dsRNA virus 17 type B 1 Aedes camptorhynchus reo-like virus 0.94 0.86 Diatom colony associated dsRNA virus 17 genome type A 0.99 Hubei partiti like virus 12 1 1 Valmbacken virus 1 Hubei toti like virus 5 * 1 1 1 Hubei reo-like virus 11 Beihai sesarmid crab virus 7 Hubei partiti like virus 22 0.9 1 Hubei reo-like virus 11 0.93 Beihai razor shell virus 4 len7927 Hubei toti like virus 6 Wuhan insect virus 28 0.99 1 Kadipiro virus 1 0.92 1 dsRNA virus environmental sample 1 0.84 0.98 dsRNA virus environmental sample 0.89 1 Rosellinia necatrix partitivirus 1 Hubei blood fluke virus 3 0.85 Hubei toti like virus 10 len7081 Pleurotus ostreatus virus 1 1 1 1 Wenling reo-like virus 2 Aedes camptorhynchus toti like virus 1 Fusarium poae virus 1 1 D Aedes alboannulatus toti like virus 1 Hubei partiti like virus 23 1 hypothetical protein 0.99 Rotavirus A Rhizoctonia solani virus 717 0.78 1 1 0.97 Osta virus Rotavirus F * Hubei partiti like virus 24 Salja virus Rotavirus C * Heterobasidion partitivirus 7 1 1 Hubei toti like virus 7 0.98 0.99 Adult diarrheal rotavirus strain J19 Ceratocystis polonica partitivirus Hubei toti like virus 8 1 Rotavirus G 1 1 Hubei toti like virus 9 1 Ceratocystis resinifera virus 1 Human rotavirus B Wenling toti like virus 1 0.98 Ceratocystis polonica partitivirus 1 Circulifer tenellus virus 1 1 1 1 Atkinsonella hypoxylon partitivirus 1 1 1 Human blood associated partitivirus 0.7

1 Vivastbo virus Fusarium graminearum dsRNA mycovirus 3 0.9 * 0.97 Wilkie partiti like virus 1 1 1 Camponotus nipponicus virus 0.99 Camponotus yamaokai virus 1 0.99 1 Hubei toti like virus 15 0.97 1 Hubei toti like virus 14 Beihai toti like virus 2 1 0.92 0.9 0.98 Hubei toti like virus 13 1 Hubei toti like virus 12 1 0.6 Jingmen toti like virus 1 Xingshan nematode virus 6 1 Lindangsbacken virus 1 * Murici virus 1 Australian totivirus * = Associated with moquitoes (Tab. 3) Hubei toti like virus 24 Beihai uca arcuata virus 1 = Found both in Cx. pipiens and Cx. torrentium 1 = Found only in Cx. pipiens = Found only in Cx. torrentium 0.7 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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 InternationalCx. torrentium license. (L12) Cx. torrentium (L9) Cx. torrentium (L1)

1 Cx. torrentium (L11) Cx. torrentium (L10) Cx. torrentium (L2) Cx. pipiens (L5) 0.99 Cx. pipiens (L6) Cx. pipiens (L3) Cx. pipiens (L4) 0.99 Cx. pipiens (L8) Cx. pipiens (L7) 0.81 Cx. coronator MF509890.1 1 Cx. coronator MF040162.1 0.85 Cx. surinamensis NC 037797.1 0.90 Cx. usquatissimus NC 036007.1 1 Cx. usquatus NC 036005.1 Cx. camposi NC 036008.1 0.80 0.95 Cx. declarator NC 037822.1 Cx. bidens NC 037809.1 1 Cx. chidesteri NC 037826.1 0.94 Cx. brami NC 037828.1 Cx. lygrus NC 037825.1 1 Ae. ochraceus KJ940764.1 1 Ae. ochraceus KJ940760.1 1 Ae. mcintoshi KJ940650.1 Ae. mcintoshi KJ940647.1

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Table 1. Overview of viral RdRp-motif library content compared to the total number of non-ribosomal RNA reads per library. Cx torrentium Cx pipiens Library L1 L2 L9 L10 L11 L12 L3 L4 L5 L6 L7 L8 Total virus reads 311,893 22,961,076 258,016 9,604,141 4,654,540 7,452,859 45,019 279,568 565,968 57,867 227,988 186,792 Host COX1 reads 586 265 322 2427 2254 3117 126 2529 317 2850 860 417 Total reads 34,150,856 62,820,620 43,914,132 52,916,282 62,936,342 59,016,596 39,231,440 41,210,662 41,328,330 40,762,624 44,703,752 46,526,884 Virus % 0.9133 36.5502 0.5875 18.1497 7.3956 12.6284 0.1148 0.6784 1.3694 0.1420 0.5100 0.4015 Host COX1 % 0.0017 0.0004 0.0007 0.0046 0.0036 0.0053 0.0003 0.0061 0.0008 0.0070 .,0019 .,0009 Other % 99.0850 63.4494 99.4117 81.8457 92.6008 87.3663 99.8849 99.3155 98.6298 99.8510 99.4881 99.5976

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Table 2. Individual abundance, measured as reads per million, of each virus, as well as Wolbachia bacteria, in comparison with the abundance of host COX1 gene. Cx torrentium Cx pipiens SINV+ SINV unscreened SINV+ SINV unscreened Location Dalälven Dalälven Dalälven Dalälven Dalälven Dalälven Dalälven Dalälven Dalälven Kristianstad Kristianstad Dalälven Threshold # mosq 10 10 50 50 15 15 10 15 15 15 15 50 Virus * ** Lenght L1 L2 L9 L10 L11 L12 L3 L4 L5 L6 L7 L8 Sindbis virus 0,672 1 11,688 671,784 417,443 0,387 0,151 0,540 0,474 2,523 0,995 0,339 2,012 0,380 0,107

Nam Dinh virus 303,146 1 20,240 73,673 303145,830 85,644 120821,017 28690,339 95,244 137,084 83,376 10006,671 116,626 87,174 106,154

Biggie virus 35,064 1 9207 16,720 15525,826 15,439 34543,772 23,405 35063,781 46,391 18,709 20,978 18,301 21,363 28,930 Negev virus 9,715 1 9493 1,171 9715,234 1,070 2,192 1,748 1,915 2,651 1,432 1,670 2,723 1,946 582,029 Rinkaby virus 0,706 1 14,498 0,000 0,159 0,068 0,151 0,127 0,034 0,280 0,000 0,387 0,196 706,361 0,172

Kerstinbo virus 1,825 1 11,280 0,381 0,207 0,250 10,734 117,913 0,136 156,456 1825,401 0,266 0,098 146,475 20,633 Forneby virus 0,138 1 8255 0,000 0,064 0,000 3,288 0,000 0,000 0,051 138,241 0,000 0,049 0,000 6,362 Osterfarnebo virus 0,087 1 5357 0,000 0,000 0,046 1,077 0,000 0,000 0,000 87,016 0,000 0,000 0,000 4,900 Hallsjon virus 0,014 1 2847 0,000 0,000 0,000 0,491 13,712 0,000 0,000 0,340 0,048 0,442 0,000 0,000

Tarnsjo virus (variant 1) 0,063 1 7890 49,047 0,000 11,955 27,629 63,064 2,864 0,051 0,000 0,024 14,253 0,045 0,086 Tarnsjo virus (variant 2) 0,076 1 7838 7,701 0,000 2,095 3,553 10,471 0,491 0,000 0,000 0,048 76,075 0,000 0,021

Culex associated luteo like 0,322 0,127 0,182 0,113 0,127 0,322 0,510 0,170 0,387 0,368 1101,496 109,421 virus 1,101 1 2790 Berrek virus 0,014 1 2807 0,000 0,000 0,046 0,000 0,000 0,034 0,000 0,000 0,000 0,000 0,000 13,906 Fagle virus 0,002 1 1453 0,000 1,544 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,000 Marma virus 2,602 1 3151 0,703 0,493 0,934 1,077 0,604 0,729 1,963 0,849 0,919 1,251 1737,371 2601,593

Merida virus 12,423 1 11,785 2327,789 2408,604 2230,876 7176,373 7,420 12423,082 16,084 9,803 2037,222 6,526 6,219 301,911 Culex mononega like virus 2 1,216 1 13,316 467,748 157,082 285,831 1216,374 553,242 311,692 1,096 467,088 87,857 13,296 0,761 23,513 Gysinge virus 0,260 1 9532 0,088 0,032 0,091 0,170 7,245 0,102 115,749 259,544 0,073 0,147 68,093 0,086 Culex mosquito virus 4 0,453 1 11,954 0,059 452,511 0,068 1,928 0,079 1,881 0,102 0,000 0,097 0,270 0,045 0,129 Culex mononega like virus 1 0,083 1 6604 0,000 0,000 0,046 0,038 0,000 0,000 0,000 0,000 0,000 0,000 0,000 83,113

Valmbacken virus 52,265 1 4315 19,092 32360,489 139,363 1158,301 29,172 52264,756 66,401 24,678 21,462 19,331 20,983 30,993

Jotan virus 20,309 1 9112 16,808 18,433 15,712 7758,198 41738,015 20308,796 50,878 91,457 912,788 18,718 18,119 22,224 Ista virus 5,605 1 9551 5605,247 576,435 2529,573 7361,250 2402,094 3661,343 8,131 8,129 4,065 3,950 4,094 116,771

Wuhan Mosquito Virus 4 1,900 1 2445 1,318 229,558 520,561 642,373 332,654 1679,358 677,569 1900,406 1,742 325,740 930,772 70,003 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Wuhan Mosquito Virus 6 0,636 1 2440 0,117 0,127 0,774 0,189 0,127 0,102 0,102 0,097 636,319 510,664 2,349 1,891

Vivastbo virus 1,338 1 2157 0,000 0,096 0,091 34,791 3,972 0,169 7,341 1338,440 0,266 0,393 119,475 26,974 Sonnbo virus 0,088 1 1737 0,000 0,032 0,000 0,038 0,064 0,034 0,000 87,720 0,048 0,000 0,000 15,561

Rasbo virus 0,017 1 5974 0,000 0,000 4,964 7,257 4,798 17,368 0,051 0,049 0,000 0,000 0,000 0,043 Kristianstad virus 0,022 1 5406 0,000 0,000 0,000 0,038 0,000 0,000 0,000 0,000 0,000 22,104 0,000 0,000 Asum virus 0,362 1 7184 0,000 0,032 0,410 0,397 0,016 0,051 0,051 0,218 0,097 361,949 0,045 0,043 Salari virus 0,014 1 6630 0,000 0,000 0,000 0,000 0,000 0,000 0,051 0,000 0,000 0,000 0,045 13,906 Anjon virus 0,407 1 6495 3,485 406,952 140,775 725,051 6,864 537,290 0,918 0,582 1,089 0,589 0,224 0,645

Gran virus 0,012 1 5622 0,000 0,032 9,473 5,858 3,559 11,912 0,000 0,000 0,048 0,000 0,000 0,021 Nackenback virus 0,559 1 6128 0,059 0,064 0,091 0,113 8,374 0,102 0,102 559,443 0,048 0,123 65,498 0,107 Vinslov virus 0,047 1 5590 0,000 0,000 0,046 0,000 0,000 0,000 0,000 0,340 8,082 47,445 0,000 0,000 Vittskovle virus 0,019 1 5671 0,000 0,684 0,046 0,000 0,000 0,000 0,000 0,000 0,000 19,380 0,000 0,000

Ahus virus 0,027 1 7732 0,000 0,032 0,000 0,038 0,000 0,000 0,000 0,243 4,404 26,691 0,000 0,000 Osta virus 0,019 1 5398 0,000 0,032 0,000 0,000 0,000 0,000 0,102 19,364 0,000 0,049 10,111 0,043 Lindangsbacken virus 0,105 1 6171 0,000 104,711 0,046 0,000 0,000 0,000 0,000 0,000 0,000 0,000 0,045 0,000 Salja virus 0,002 1 1286 0,000 0,000 0,000 0,000 0,000 0,000 0,000 1,626 0,000 0,000 1,611 0,000 Eskilstorp virus 0,210 1 2933 0,000 0,000 0,000 0,076 0,032 0,068 0,076 0,000 0,000 0,049 210,363 0,129

Wolbachia COX1 0,001 1 1573 0,00 0,99 0,00 0,00 0,00 0,00 0,00 0,05 0,05 0,00 0,00 0,00 Wolbachia WSP 0,000 1 614 0,00 0,00 0,00 0,00 0,03 0,20 0,10 0,34 0,22 0,00 0,00 0,09 Host COX1 0,067 1 1506 17,16 4,22 7,33 45,86 35,81 52,82 14,94 6,43 7,79 59,54 50,42 66,99 Number of virus species – – – 6 13 9 17 14 12 10 13 8 10 12 17 * = 0,1% of the most abundant library ** = Less than 1 per million readds Grey = not present Green = present, but not abundant Orange = present and >0.1% of total reads Red = present and more abundant than host

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Table 3. Indication of host associations for the discovered viruses. Host-association was assessed using (i) the abundance of viral contig per total amount of reads in a library, (ii) virus abundance in relation to the COX1 host gene, (iii) presence amongst the libraries, and (iv) phylogenetic clustering with other mosquito derived viruses. More Present in Clusters with abundant Mosquito >2 mosquito Virus family Cx torrentium Cx pipiens Abundant? than host associated? Virus libraries? viruses? RNA?

Sindbis virus Alpha P P Yes Yes No Yes Yes Nam Dinh virus Nido P P Yes Yes Yes Yes Yes Biggie virus Virga–Negev P P Yes Yes Yes Yes Yes Negev virus Virga–Negev P P Yes Yes No Yes Yes Rinkaby virus Virga–Negev NP P Yes Yes No No Yes Kerstinbo virus Endorna P P Yes Yes No No Yes Forneby virus Endorna P P No Yes No No No Osterfarnebo virus Endorna P P No Yes No No No Hallsjon virus Endorna P NP No No No No No Tarnsjo virus (variant 1) Tymo P P No Yes No Yes Yes Tarnsjo virus (variant 2) Tymo P P No Yes No Yes Yes Culex associated luteo like virus Luteo NP P Yes Yes No Yes Yes Berrek virus Luteo NP P No No No Yes No Fagle virus Luteo P NP No No No No No Marma virus Luteo NP P Yes Yes No Yes Yes Merida virus Mononega P P Yes Yes Yes Yes Yes Culex mononega like virus 2 Mononega P P Yes Yes Yes Yes Yes Gysinge virus Mononega P P Yes Yes Yes No Yes Culex mosquito virus 4 Mononega P NP Yes Yes No Yes Yes Culex mononega like virus 1 Mononega NP P No Yes No Yes Yes Valmbacken virus Reo P P Yes Yes Yes Yes Yes Jotan virus Picorna P P Yes Yes Yes Yes Yes Ista virus Picorna P P Yes Yes Yes No Yes Wuhan Mosquito Virus 6 Orthomyxo P P Yes Yes Yes Yes Yes Wuhan Mosquito Virus 4 Orthomyxo NP P Yes No No Yes Yes Vivastbo virus Partiti P P Yes Yes No No Yes Sonnbo virus Partiti NP P No Yes No No No Rasbo virus Bunya P NP No No No No No Kristianstad virus Bunya NP P No No No Yes No Asum virus Bunya NP P Yes No No No No Salari virus Bunya NP P No No No Yes No Anjon virus Phasma P P Yes Yes Yes Yes Yes Gran virus Qin P NP No No No No No Nackenback virus Qin P P Yes Yes Yes No Yes Vinslov virus Qin NP P No No No No No Vittskovle virus Qin NP P No No No No No Ahus virus Toti NP P No No No No No Osta virus Toti NP P No No No Yes No Lindangsbacken virus Toti P NP No Yes No Yes Yes Salja virus Toti NP P No No No Yes No bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. 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.

Eskilstorp virus Chryso NP P No Yes No Yes Yes P = Present NP = Not present >0.1% = abundant