Vertebrate animals of different ages also differ in their exposure and susceptibility to 35 microorganisms [1] . The importance of host age for the epidemiology and ecology of 36 microorganisms has been demonstrated by differences in the diversity and prevalence of parasites 37 [2], bacteria [3, 4] , and viruses [5-8] among hosts of different ages. In the case of wild birds, host 38 age is associated with a higher prevalence of viruses in juvenile compared to adult birds. These 39 include beak-and-feather disease virus in parrots [9], avian pox virus in albatross and tits [10, 11] , 40 avian avulavirus-1 in cormorants and waterfowl [12, 13] , infectious bronchitis virus in poultry [14] 41 and influenza A virus in wild waterfowl [8] . 42 Despite a similar potential for exposure, viral susceptibility varies among adult and juvenile birds. 43 This is believed to be due to an increase in the immune repertoire with age, with the age-related . In addition to varying susceptibility, 51 exposure to viruses may similarly vary in wild birds. For example, in migratory birds such as 52 shorebirds, adults and juveniles undertake southward migrations at different times, and in some 53 species use markedly different routes, resulting in different virus exposure potential [23, 24] . 54 As host age impacts the presence and prevalence of a range of specific viruses, we ask here whether 55 host age is also associated with modulating communities of viruses carried by birds? To this end, 56 we used the virome-scale data obtained from metagenomic next-generation sequencing as means to 57 go beyond the "one-host, one virus" model and provide key data on the abundance and diversity of 58 all the viruses present in a particular species. We used Ruddy Turnstones (Arenaria interpres) as a 59 model species as they are known to be important reservoir hosts for viruses such as influenza A 60 virus as well as for an abundance of other viruses [25, 26] . In the East Asian-Australasian Flyway, 61 this species breeds in Arctic Siberia and following migration, spends its non-breeding period along 62 the shorelines of Australia. Accordingly, we sampled individuals across three age groups at two 63 different points in their annual cycle, just after migration, upon arrival in Australia and again shortly 64 before departure to their Arctic breeding grounds. From these data we demonstrate that juvenile 65 birds have significantly higher viral abundance and diversity compared to adult birds and that 66 different time points in their migratory cycle had no apparent effect on virome strucutre. November) and (iii) 3+ -adult birds, classified as those that are 3 years and older (Fig 1) . A 82 combination of oropharyngeal and cloacal samples were collected using a sterile tipped applicator 83 and placed in virus transport media (VTM; Brain-heart infusion broth containing 2 × 106 U/L 84 penicillin, 0.2 mg/ml streptomycin, 0.5 mg/ml gentamicin, 500 U/ml amphotericin B, Sigma). 85 Sequence reads were demultiplexed and trimmed with Trimmomatic followed by de novo assembly 100 using Trinity [28] . No filtering of host/bacterial reads was performed before assembly. All 101 assembled contigs were compared to the entire non-redundant nucleotide (nt) and protein (nr) 102 database using blastn and diamond blast [29] , respectively, setting an e-value threshold of 1x10 -10 to 103 remove potential false-positives. 104 Abundance estimates for all contigs were determined using the RSEM algorithm implemented in 105 Trinity. All viral contigs that returned blast hits with paired abundance estimates were filtered to phylogenies were also estimated using the nucleotide sequences of complete or partial reference 122 genome sequences to better place viruses in context of currently described avian viral diversity. 123 Novel viral species were identified as those that had <90% RdRp protein identity, or <80% genome 124 identity to previously described viruses. Newly identified viruses were named after shipwrecks 125 surrounding King Island, Tasmania. Genome sequences have been deposited in NCBI GenBank 126 (accession numbers XX-XX). 127 Viral diversity and abundance across libraries 128 Relative virus abundance was estimated as the proportion of the total viral reads in each library 129 (excluding rRNA). All ecological measures were estimated using the data set comprising viruses 130 associated with birds and mammals, albeit with all retroviruses and retrovirus-like elements 131 6 removed (hereafter, "avian virus data set). Analyses were performed using R v 3.4.3 integrated into 132 RStudio v 1.0.143, and plotted using ggplot2. 133 Both the observed virome richness and Shannon effective (alpha diversity) were calculated for each 134 library at the virus family and genus levels using modified Rhea script sets [37] , and compared 135 between avian orders using t-tests. Beta diversity was calculated using the Bray Curtis dissimilarity 
Overall virus abundance and diversity 146 We characterized the total transcriptomes of Ruddy Turnstones from different age categories upon 147 arrival and prior to departure of migration (Fig 1, Table S1 ). All individuals were captured in 148 March/November, regardless of age category, and on the same beaches on King Island, Tasmania. 149 There was a large range in total viral abundance (0.16-9.91% viral reads) and putative avian viral 150 abundance (0.0071-1.13% viral reads) in each library (Table S1, Fig. S1 ). In addition to viral reads 151 that were likely associated with bona fide avian viruses, libraries had numerous reads matching 152 invertebrate, plant and bacterial viruses as well as retroviruses (Fig. S1 ). Although these 153 retroviruses are likely associated with birds, the challenge of differentiating between endogenous 154 and exogenous retroviruses meant that they were excluded from the analysis, as were those viruses 155 most likely associated with arthropods, plants, and bacteria. In total, 11 of the 14 viruses identified 156 in this study likely represent novel avian viral species ( viruses identified previously in metagenomic studies (Fig S2) . Cataraqui virus, an Orthoreovirus, 167 fell as a sister lineage to a recently described avian orthoreovirus -Tvarminne virus (Fig 3A) . 168 Cataraqui virus was detected in two libraries -juvenile (1o) and adolescent (2o) birds sampled 169 concurrently -with similar abundance levels (0.5% and 0.25% of reads in each library, respectively) 170 showing viral sharing across these age groups (Fig 3A, Fig 6) . The two genomes recovered were 171 highly similar, with only ~1 nucleotide difference in each of the lambda segments. While we could 172 not assemble this genome from adult 3+ birds, contigs of Cataraqui virus were also found, 173 comprising 0.0023% of the library. As this represents >0.1% of the read abundance of the library in 174 which this virus is at its highest abundance we tentatively exclude index-hopping. (Fig 3B, Fig S3) . 181 We also identified five picornaviruses (family Picornavirdae). Three of these -Waterwitch virus, 182 Whister virus, and Loch Leven virus -fall as sister lineages to members of the genus Hepatovirus 183 and Tremovirus and were found exclusively in 1o birds, with Waterwitch virus and Whister virus 184 forming a distinct clade (Fig S4) . In contrast, British Admiral virus fell as a divergent sister-group 185 to the Gallivirus genera, while Netherby virus, the only picornavirus found in a 3+ bird, fell into a 186 currently unassigned clade comprising a chicken and a quail phacovirus (Fig S4) . 187 We also identified a novel virus in the family Phenuiviridae, Bruthen virus. This virus fell in a 188 group of unclassified viruses that are the sister-group to members of the genus Phlebovirus that 189 contains numerous tick-borne viruses (Fig 4) Deltacoronavirus) (Fig 5, Fig S5-S6) . These viruses are common in wild waterbirds, although most 199 sequence data is limited to short fragment of the RdRp (Fig S8, S9) The variant of avian avulavirus 2 identified here (AAvV-2/Ruddy turnstone/Tasmania/2015) was a 213 (weakly supported) sister-group to a clade of viruses described in wild birds in China (Fig S7) , and 214 we speculate that this likely represents a lineage that is more common in wild birds than poultry. Regardless of sampling period, juvenile (1o) birds had higher viral abundance and diversity than 225 adult (3+) birds at the viral family, genus and species levels. First, viral abundance was higher in 226 juvenile than adult birds, with a 10-fold difference in November (1.12% compared to 0.007% avian 227 viral reads) and March (0.2% compared to 0.02% avian viral reads). Adolescent (2o) birds, which 228 overwintered on the island rather than making a return trip to the Arctic breeding grounds like the 229 9 3+ birds did, also had very high viral abundance in November (0.7% avian viral reads) (Fig 6A) . 230 Second, this trend remained when assessing diversity within each sample (i.e. alpha diversity). 231 Observed richness and Shannon diversity was higher in juvenile than adult birds, in which observed 232 richness was significantly higher in 1o birds at the viral genus level (Fig S8) (Fig 2, Fig 6) . Although this study is clearly limited in sample size, the overall 238 trend remains across our biological replications (here different time periods). Sampling period was 239 not a significant predictor of either observed richness or Shannon diversity at the virus family, 240 genus and species levels. For example, when assessing the number of viral species, we observed the 241 same number of viruses revealed (excluding the adolescent 2o birds as they were only sampled in 242 November) in both November (n=6) and March (n=7) (Fig 2) . 243 To ensure this trend was not due to a sequencing artefact we tested for differences in viral (Fig S9) . 254 No obvious trends were observed with respect to beta diversity (Fig S10) . Specifically, neither age 255 (R 2 =0.49, p=0.33) nor sampling time point (R 2 =0.2, p=1) was associated with avian virome 256 structure at the viral genus or family levels. There was very limited viral sharing (i.e. connectivity) 257 among birds of different age categories even though they were sampled on the same beaches at the 258 same time. The only viral species that we were able to annotate from more than one library was 259 Cataraqui virus, identified in juvenile and adolescent birds at the November time point: however, 260 reads for this virus were also present in the adult birds in November (Fig 2, Fig 3A) . While the full 261 genome of avian avulavirus-2 was only identified in the adult birds in March, reads were present in 262 juvenile birds from March. Despite this, due to cohabitation, virus connectivity was far lower than 263 10 expected. We suggest that adult birds may already have antibodies against many of these viruses, 264 resulting in no detectable infections in this age category. Further, we found no difference in viral abundance or diversity of adult or juvenile birds when 297 comparing those captured in November (recently arrived) or the subsequent March (imminently 298 prior to departure of the 3+ adult birds). This not only shows the lack of a migratory cycle effect, 299 but also that the impact of age is strong, despite potentially large differences in physiology of 300 Ruddy Turnstones at these sampling events. Prior to migration, birds go through a period of 301 impressive mass increases; some species may more than double their body mass, with a 1-3% 302 increase per day [51, 52] . Indeed, birds captured in March had an additional 50% in mass compared 303 to November. Studies in the microbiome of migratory birds found that the majority of bacterial taxa Where two libraries share a virus species, the networks between the two libraries are linked. 535 Placement of libraries is arranged by sampling date on the y-axis and age on the x-axis. Virus 536 colour corresponds to virus taxonomy. A list of viruses from each library is presented in Table S2 , 537 and phylogenetic trees for each virus family and species can be found in Fig 3-5, Fig S2- 