Microbial composition of enigmatic bird parasites: Wolbachia and Spiroplasma are the most important bacterial associates of quill mites (Acari: Syringophilidae)

The microbiome is an integral component of many animal species, potentially affecting behaviour, physiology, and other biological properties. Despite this importance, bacterial communities remain vastly understudied in many groups of invertebrates, including mites. Quill mites (Acariformes: Syringophilidae) are a poorly known group of permanent bird ectoparasites that occupy quills of feathers and feed on bird subcutaneous tissue and fluids. Most species have strongly female biased sex ratios and it was hypothesized that this is caused by endosymbiotic bacteria. Their peculiar lifestyle further makes them potential vectors for bird diseases. Previously, Anaplasma phagocytophilum and a high diversity of Wolbachia strains were detected in quill mites via targeted PCR screens. Here, we use an unbiased 16S amplicon sequencing approach to determine other Bacteria that potentially impact quill mite biology. We performed 16S V4 amplicon sequencing of 126 quill mite individuals from eleven species parasitizing twelve bird species (four families) of passeriform birds. In addition to Wolbachia, we found Spiroplasma as potential symbiont of quill mites. Interestingly, consistently high Spiroplasma titres were only found in individuals of two mite species associated with finches of the genus Cardfuelis, suggesting a history of horizontal transfers of Spiroplasma via the bird host. Furthermore, there was evidence for Spiroplasma negatively affecting Wolbachia titres. We found no evidence for the previously reported Anaplasma in quill mites, but detected the potential pathogens Brucella and Bartonella at low abundances. Other amplicon sequence variants (ASVs) could be assigned to a diverse number of bacterial taxa, including several that were previously isolated from bird skin. We observed a relatively uniform distribution of these ASVs across mite taxa and bird hosts, i.e, there was a lack of host-specificity for most detected ASVs. Further, many frequently found ASVs were assigned to taxa that show a very broad distribution with no strong prior evidence for symbiotic association with animals. We interpret these findings as evidence for a scarcity or lack of resident microbial associates (other than inherited symbionts) in quill mites, or for abundances of these taxa below our detection threshold.


Introduction
There is abundant evidence that microbial taxa are an essential component of many 2 animal species [1]. Bacteria-encoded traits may significantly impact host phenotypes, 3 e.g. through providing essential nutrients [2,3], defending against pathogens [4,5], but 4 also affecting ecological features of their hosts, such as mate choice [6] and life history 5 traits [7]. Because of their potential importance in understanding the biology of many 6 organisms, the number of microbiome studies has been soaring [8]. This popularity is 7 owed to methodological advances (high-throughput sequencing technologies) allowing 8 comprehensive investigation of the microbial communities [9], but also to the decreasing 9 costs of these approaches [10]. However, the main focus of microbiome studies so far has 10 been vertebrates [11]; while in invertebrates, the focus has been on taxa of medical, 11 veterinary, or economical importance. For example, in mites, microbiome studies have 12 been conducted on the pests of stored food products [12,13], dust mites producing 13 allergenic agents [14][15][16], and mites transmitting pathogens, such as sheep scab mites 14 [17] red poultry mites [18,19], and the honey bee parasite Varroa [20]. 15 In the present study, we have focussed on quill mites (Acariformes: Syringophilidae). 16 These obligatory bird ectoparasites live and reproduce inside the quills of feathers where 17 they feed on subcutaneous fluids such as lymph and blood. Quill mite dispersion has 18 been observed on the same individual (from infected to uninfected feathers), between 19 individuals of the same species (e.g., from parents to hatchings) and occasionally by 20 transfer between gregarious bird species [21][22][23][24]. This mode of feeding and dispersion 21 makes quill mites potential vectors for bacterial pathogens, similar to ticks or lice [25]. 22 However, only two bacterial taxa were recorded in quill mites so far: 1) Anaplasma 23 phagocytophilum (Alphaproteobacteria, Rickettsiales) was detected in two quill mite 24 species from three bird species [26]; 2) Multiple genetically distinct lineages of 25 Wolbachia (Alphaproteobacteria, Rickettsiales) were found in five species of quill mites 26 [27]. As these studies were targeted PCR screens, it remains unclear what other 27 Bacteria populate quill mites. Furthermore, the importance of quill mites for bird 28 pathogen dynamics is not known. 29 To address these questions, we here assess the bacterial composition of 126 quill mite 30 individuals encompassing eleven species with a more unbiased 16S rRNA amplicon 31 sequencing approach. We find that the symbionts Wolbachia and Spiroplasma are 32 2/17 among the most commonly taxa associated with quill mites. Other taxa include 33 Bacteria that were previously found in association with arthropods, and Bacteria with a 34 very broad distribution. Strikingly, neither quill mite taxonomy nor bird host taxonomy 35 significantly influences bacterial composition in quill mites. Furthermore, we find that 36 despite the detection of Bartonella and Brucella, quill mites do not seem to be a major 37 pathogen vector in birds.

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Animal collection and DNA extraction. A summary of collected quill mite 40 species and their bird hosts can be found in Table 1. All quill mites used in this study 41 were collected in Kopan, Poland during spring migration of birds monitored by the Bird 42 Migration Research Station, University of Gdansk, April 2009. One secondary flight 43 feather was completely removed from each bird specimen and dissected under a stereo 44 microscope (Olympus ZS30). Individual mites were washed twice and preserved in 96% 45 ethanol and total genomic DNA was extracted from single specimens using DNeasy 46 Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) as described previously [28]. 47 This procedure leaves the exoskeletons intact, and the specimens were subsequently 48 mounted on microscopic slides in Faure medium, and determined using the key from 49 Skoracki et al. (2016) [29]. All morphological observations were carried out with an 50 Olympus BH2 microscope with differential interference contrast (DIC) optics and a 51 camera lucida. All DNA samples and corresponding voucher specimens are deposited in 52 the collection of the Department of Animal Morphology, Faculty of Biology, Adam 53 Mickiewicz University in Poznań, Poland. To identify potential contaminants, in 54 addition to sequencing a negative control alongside all samples, we further extracted 55 DNA from reagents and materials commonly used in the laboratory this work was 56 carried out in. One library each was created from extraction buffer (ALT), millipore 57 water, microscope swabs, pipette swabs, and swabs of other equipment (pincettes, 58 scalpels, benches, etc). These five libraries were processed and sequenced separately 59 from the other samples, but by using identical procedures. 60 Library preparation and sequencing. We amplified and sequenced the V4   Read processing and statistical analyses. Reads were trimmed of adaptors and 77 primer sites by using cutadapt version 1.16 [30]. The remaining reads were dereplicated, 78 denoised, and chimeras eliminated using the DADA2 package version 1.8 [31] within the 79 R statistical programming environment [32]. Taxonomic assignment of the ASVs 80 (amplicon sequence variants), to species level where possible, was also performed within 81 DADA2 using the SILVA database version 132 [33]. Next, contaminant taxa were 82 identified from the sequenced extraction control using the 'prevalence' algorithm 83 implemented in the R package decontam [34]. Further potential contaminants were 84 identified by processing the five libraries derived from reagents and materials as 85 described, and then excluding all ASVs that were found in any of these control libraries 86 from subsequent analyses.

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To reduce the impact of ASVs with very low abundance, we removed all ASVs that 88 were present in only a single sample and also discarded ASVs from Bacterial Phyla that 89 only occurred once in total. To account for potential biases between samples with groups. First, we plotted the abundance of the most frequently found bacterial families 97 using the R packages phyloseq and ggplot2 [35,36]. Next, ordination analyses were 98 performed with phyloseq using Bray distances and non-metric multidimensional scaling 99 (NMDS). Differences in abundances of particular taxa between groups (quill mite species, 100 bird host species, developmental stage, Wolbachia positive and negative samples) were 101 determined with Kruskal-Wallis rank sum tests, and p-values were adjusted to these 102 multiple comparisons to control for the false discovery rate [37]. These tests were done 103 separately for differences in abundance of bacterial phyla, orders, families, and genera. 104 Furthermore, we calculated Jensen-Shannon distances between the aforementioned 105 groups and used adonis tests (analysis of variance using distance matrices) implemented 106 in the R package vegan [38] to test if they differed significantly. The phyloseq object file 107 containing all data used in the described analyses is available as Additional file 1. 108

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We have investigated the microbial composition of 126 individuals belonging to eleven 110 quill mite species that parasititze twelve bird host species of passeriform birds. Supplementary Figure S1). However, when trying to identify differential abundance 125 patterns of microbial composition between groups using analysis of variances, we found 126 that bacterial composition was more similar between samples from the same quill mite 127 species or genus and bird host species or genus than expected by chance. Furthermore, 128 six bacterial families were found to be differentially abundant between quill mite species 129 with a Kruskal-Wallis test (p<0.01, Fig. 3), one of which (Xanthobacteraceae) was also 130 found to differ between samples of different bird host genera.

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Out of 912 detected ASVs, the ten most abundantly encountered genera were   Figure 3. Abundance of five bacterial families that were found to be differentially abundant between quill mite species analysed. Counts for the symbionts Wolbachia and Spiroplasma were excluded.
As opposed to the general trend in the microbiome composition data, there was 141 strong evidence for differential abundance of the symbionts Wolbachia and Spiroplasma 142 between the bird hosts from which the mites were collected. For example, high 143 Spiroplasma titres were only observed in two mite species collected from the host genus 144 Carduelis (Fig. 4a, Supplementary Table S4). Further, although Wolbachia was present 145 in mites sampled from all bird hosts, it was especially prevalent in mites collected from 146 Turdus sp., Erithacus sp., and Fringilla sp. In contrast, it was absent or at very low 147 titres in mites parasitizing Luscinia sp. (Fig. 4a). On average, the abundance of 148 Wolbachia was lower in samples that also contained Spiroplasma (Figs. 1a, 4b). Notably, 149 this was not an effect of Spiroplasma presence reducing the amount of available reads 150 for Wolbachia (Fig. 4b). For mites harbouring both symbionts (eleven samples in total), 151 we found that the abundances for Wolbachia and Spiroplasma seemed to be positively 152 correlated (Fig. 4c).    Table  S3 and Table S4. 155 We here have sequenced microbial taxa from quill mites, an enigmatic group of bird 156 ectoparasites. The taxa detected through 16S sequencing may be 1) resident symbionts 157 of quill mites, 2) environmentally acquired, transient bacteria, or 3) contaminants from 158 reagents and materials. Each of these options comes with a number of assumptions that 159 can be tested with our data. are not found anywhere else [40]. The composition of these taxa is correlated with from other arthropods, and are unable to permanently live outside their hosts [41,42]. 170 Further, we document a very high abundance of these taxa in at least some of the 171 investigated samples (Fig. 4), which is in line with the assumptions above. In a previous 172 study, Wolbachia strains of quill mites were investigated with a multi locus approach 173 and it has been shown that quill mite associated strains are genetically very different to 174 any other Wolbachia strains described so far [27]. Here, we have found 8 different ASVs 175 annotated as Wolbachia, each of which is 100% identical to at least one Wolbachia 16S 176 sequence previously isolated from quill mites. For Spiroplasma, we found a single ASV 177 that is only 92% identical to the next closest match in the Silva database. This implies 178 that Spiroplasma in quill mites might be genetically distinct from Spiroplasma of other 179 arthropods, as is the case for Wolbachia. However, sequencing data of more loci are 180 needed to establish the phylogenetic placement of Spiroplasma from quill mites. Weeksellaceae) [45,46]. Despite these similarities, and some statistical support for bird 202 hosts shaping the microbiome community in our study, the lack of clustering in 203 ordination analysis indicates that environment is not the major determining factor of 204 quill mite microbiome composition. 3) Importantly, contaminants from reagents and kits may significantly impact 206 microbiome compostion estimates, especially when using low biomass samples such as 207 quill mites [47][48][49]. This is problematic in any microbiome study, and is very difficult to 208 exclude with certainty. Here, we removed contaminants statistically in silico based on 209 the microbial composition of the sequenced extraction control [34]. Further, we removed 210 all ASVs present in independently sequenced controls derived from reagents and 211 9/17 equipment commonly used in the laboratory where this study was performed (see 212 Materials and methods). However, a number of ASVs we recovered correspond to 213 common kit contaminants in 16S microbiome studies (e.g., Ralstonia, Kocuria), human 214 skin Bacteria (Corynebacterium) or ubiquitous taxa with no strong evidence for 215 symbiotic associations with arthropods (Pseudomonas, Acinetobacter). These taxa 216 might constitute true associates, but we cannot exclude the possibility that they 217 originate from contaminating sources.

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In summary, we found a diverse range of Bacteria associated with quill mites. The 219 lack of differentiation between different mite species or between species collected from 220 different bird hosts leads us to conclude that there are no strong associations with 221 typical gut bacteria as observed in other arthropods. However, we cannot exclude that 222 we missed such potential associates due to the limited amount of DNA that can be 223 extracted from the minute hosts. finding is of potential importance in understanding this pathogen's dynamics.

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Bartonella are gram-negative Bacteria that are typically transmitted by blood 240 sucking arthropods, and are infectious in mammalian hosts [56][57][58]. There are also 241 reports on Bartonella incidence in birds [59,60], and it is conceivable that the Bacteria 242 originate from the birds, rather than from the mites. That would suggest that the host 243 range for Bartonella spp. is broader than previously reported and here we expand the 244 list of potential sources for this zoonotic infection. However, Bartonellaceae can be 245 symbiotic in other hosts, such as honey bees and ants [61,62]. Further, Bartonella-like 246 symbionts were recently found in a number astigmatid mites [63], indicating that the 247 Bartonella detected here might be quill mite symbionts, rather than pathogens. With 248 our data, it is not possible to rule out either possibility. 249 Finally, we found the symbionts Spiroplasma and Wolbachia in quill mites. Both of 250 these are common across a range of arthropod species [41,64], are typically transmitted 251 intraovarially, and may cause sex-ratio distorting phenotypes [65,66]. Whereas 252 Spiroplasma was so far not reported from quill mites, Wolbachia was previously detected 253 and our findings confirm that this is a common symbiont of quill mites [27]. The 254 observed presence and abundance of both taxa are not uniform across the sampled taxa 255 (Fig. 4a). For example, Wolbachia is most abundant in mites parasitizing birds of the 256 genera Turdus, Erithacus, and Fringilla, whereas Spiroplasma is most strongly 257 associated with mites parasitizing Carduelis. One reason for this may be that some taxa 258 are more susceptible than others for endosymbiosis with certain Bacteria, and this 259 phylogenetic effect has been reported for other host taxa as well [67,68]. Strikingly, 260 very high Spiroplasma abundances were only found in two investigated mite species that 261 are specialised parasites of three bird species of the genus Carduelis (Figs. 1a, [72,73]. Although the potential mechanism of horizontal symbiont transmission via 271 feather quills is unclear, our data suggest that the bird-parasite interactions may be 272 important for endosymbiont transmission dynamics in quill mites. 273 Interestingly, we found that Spiroplasma presence leads to reduced Wolbachia titers, 274 although this is based on a small sample size for samples that are both Wolbachia and 275 Spiroplasma positive (N=11, Fig. 4b). Furthermore, in these eleven samples, 276 Spiroplasma and Wolbachia titers seem to be positively correlated (Fig. 4c). It is 277 conceivable that sharing of hosts leads to competition for finite resources the host can 278 provide [74], and thus the growth of one symbiont might limit that of another. In 279 Drosophila for example, Spiroplasma seem to limit the proliferation of Wolbachia [75] 280 and in aphids, competition between co-occuring secondary symbionts appears to be 281 harmful to the host [76]. Such negative fitness impacts can also expected when both 282 symbiont titres are very high, as found here in quill mites. Although purely speculative, 283 this may be the reason why we only observed simultaneously high Spiroplasma and 284 Wolbachia titres in very few of the 126 investigated quill mites (Fig. 4c).

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Summary 286 We find a diverse, but relatively uniform set of bacterial taxa within quill mites that 287 includes arthropod endosymbionts, pathogens, and bird associated bacteria. The 288 importance of most of these microbes for quill mite biology is unclear, but abundances 289 and distribution patterns suggest that Spiroplasma and Wolbachia are the most 290 important quill mite associates.

Table S1
Fusion PCR primers sequences used in this study. Unique random barcode sequences are highlighted in bold.

Table S2
Overview on the impact of filtering and decontamination on the number of retained ASVs and samples in this study. For details on each of the steps please refer to the Materials and methods section.

Table S3
List of all ASVs detected in this study, ordered by abundance (relative abundances summed over all samples).

Table S4
Average abundance of Spiroplasma and Wolbachia across sampled bird and mite species.

Additional file 1
Phyloseq object including all ASVs, sample and metadata information.