bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452008; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Genome-resolved metagenomic analyses reveal the presence of a bacterial endosymbiont in an avian nasal mite (Rhinonyssidae; Mesostigmata) Carolina Osuna-Mascaró 1,, Jorge Doña 2,3, Kevin P. Johnson 2, Manuel de Ro as !," 1 De#ar$%en$ of Biology, )niversi$( of *evada, Reno+ [email protected] 2 -llinois *atural His$ory /urve(, Prairie Research -ns$i$u$e, )niversi$( o& -llinois at )r0ana-Cha%#aign, Cham#aign, )/1+ [email protected] 2 -llinois *atural His$ory /urve(, Prairie Research -ns$i$u$e, )niversi$( o& -llinois at )r0ana-Cha%#aign, Cham#aign, )/1+ 2# ohnso,illinois.edu 3De#ar$amen$o de Biología Ani%al, )niversidad de 4ranada, 4ranada, /#ain 4 De#ar$%en$ of Micro0iology and Parasi$olog(, 5acul$y of Phar%ac(, )niversidad de /evilla, /evilla, /#ain+ dero as,us.es Abstract: Rhinon(ssidae (Mesos$igmata) is a &amil( o& nasal mites onl( &ound in 0irds. 1ll species are hema$o#hagous endo#arasites, 8hich ma( damage $he nasal cavities o& 0irds, and also could 0e #o$ential reservoirs or vec$ors o& o$her infec$ions. Ho8ever, $he role o& me%0ers o& Rhinon(ssidae as disease vec$ors in 8ild 0ird #o#ulations remains uninves$igated, 8ith s$udies o& $he micro0iomes o& Rhinon(ssidae 0eing almos$ non-exis$ent. -n $he nasal mite 6Tinaminyssus melloi7 fro% roc2 doves 6Columba livia), a #revious s$ud( &ound evidence o& a highl( abundant #u$ativel( endosy%0io$ic 0ac$eria fro% Class 1lphapro$eo0ac$eria. Here, 8e e9#anded $he sam#le size o& $his species, incor#orated contamination controls, and increased se;uencing de#$h in sho$gun se;uencing and genome-resolved me$agenomic anal(ses. Our goal 8as $o increase $he information regarding $his mite species 8i$h its #u$ative endosy%0iont. Our results su##or$ $he endosy%0io$ic nature o& $his 0ac$erial $axon, 8hich is $he <rs$ described &or 0ird's nasal mites $o date, and im#rove $he overall unders$anding o& $he micro0io$a inhabiting these mites. *Corres#onding au$hors> COM> cosuna,unr.edu+ MR, e%ail> dero as,us.es Ke(8ords> Rhinonyssidae+ endos(%0ion$+ %e$ageno%ic; Brucella bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452008; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1. Introd!ction Mites are one of the mos$ diverse grou#s of eu2ar(o$es on ear$h [1]. Mites are u0i;uitous, occu#(ing aquatic, terres$rial, and ar0oreal habitats [2-8]. On some occasions, mites have an intimate association (sy%0iosis) with a different organism, which, in the mos$ e9$re%e scenario (permanent sy%0ionts), re#resents the habitat in which the( undergo their entire life-c(cle [9]. For exam#le, a single species of insec$, mammal, or bird can o&$en hos$ several mite species and have different t(#es of interac$ions with the% [9, 10-12]. The nature of these interac$ions can range from mu$ualism (e.g., mites inhabiting and cleaning birds' feathers; [13]) to parasitism (e.g., mites inhabiting the nasal passages and lungs of seals causing illness $o the%+ [14, 15]). -n some cases, mites can affec$ the hos$'s heal$h and be responsible for transmit$ing zoono$ic diseases [9]. For exam#le, the itch mite Sarcoptes scabiei has caused the po#ulation decline of different mammal species b( transmit$ing scabies [16-19]. In ano$her case, Varroa destructor, a parasitic mite of the Asian hone(0ee (Apis cerana) has caused the decline of the Huro#ean hone(0ee b( transmit$ing a virus [20]. In some cases, disease transmission ma( be mediated b( endosy%0io$ic pa$hogens that inhabit the mites. One exa%#le of this mechanism is Leptotrombidium scutellare, a mite tha$ parasitizes mice and carries the endosy%0iont 0ac$eria Orientia tsutsugamushi, responsible for the scru0 t(#hus disease [21]. Endosy%0ionts as causal agents of diseases have also been widel( re#or$ed in tic2s [22-24]. Ho8ever, exa%#les of endosy%0ionts in mites are s$ill rare. Rhinon(ssidae (Mesos$igmata) is a famil( of nasal mites with more than 500 species described world8ide [25-28]. The( have been descri0ed from birds [29]. Almos$ all species of 0irds are inhabited b( nasal mites, which usuall( live in the nasal cavit( on vascularized e#i$helial tissue [9]. All rhinon(ssid species are hema$o#hagous endo#arasites [30]. /#ecificall(, rhinon(ssid mites damage the nasal cavities of birds, which, in rare cases, ma( lead to the death of the hos$s (Rhinon(ssidosis aviu% disease7 [28]. Moreover, it has been bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452008; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. sugges$ed that these mites could be po$ential reservoirs or vec$ors of o$her infec$ions (such as Ies$ Nile fever, Q fever, avian influenza, and L(me diseases), as demons$rated in mites of the &amil( Derman(ssidae [31]. Ho8ever, the role of species of Rhinon(ssidae as disease vec$ors in wild bird po#ulations is ye$ to be unders$ood. In addition, s$udies on rhinon(ssid micro0iomes more generall( are almos$ non-exis$ent. -n a preliminar( previous s$ud(, the micro0iome of t8o different s#ecies of rhinon(ssid mites (Tinaminyssus melloi and Ptilonyssus chlori) from t8o different avian hos$ species 6Columba livia and Chloris chloris, respec$ivel(7 was charac$erized [32]. The results of that s$ud( sugges$ed that the nasal mite Tinaminyssus melloi har0ored a po$ential endosy%0iont 1lphapro$eo0ac$eria (Famil(: Bar$onellaceae) in a high abundance. Ho8ever, the lo8 se;uencing coverage, small sam#le size, and lac2 of control sam#les did no$ allo8 de<nitive conclusions to be made. In par$icular, onl( a par$ial (~26%) asse%0l( of the bac$erial genome 8as achieved, and it could no$ be ruled ou$ co%#le$el( if this bac$erial taxon came from the 0ird hos$ ins$ead of the mite. Here, we focused on e9#anding kno8ledge of the micro0iome of this mite species 6Tinaminyssus melloi), e%#hasizing increasing the information of its association wi$h this #u$ative endosy%0iont. In the current s$ud(, we e9#anded the sam#le size, used contamination controls, and increased se;uencing de#$h. In par$icular, we collec$ed t8o mite #ools (five mite individuals per pool) plus a control bird saliva sam#le from t8o different individual Roc2 doves (Columba livia). Then, we conduc$ed sho$gun and genome-resolved me$agenomic anal(ses to charac$erize the miteOs micro0iome along with evaluating the genome pro#er$ies of this bac$erial taxon, which ma( be informative regarding its endosy%0io$ic s$atus. 2. Materials and Methods 1 to$al of ten nasal mites were collec$ed from t8o different freshl( dead Columba livia individuals (i.e., 5 mites per individual hos$). The nasal cavities of the birds were dissec$ed bioRxiv preprint doi: https://doi.org/10.1101/2021.07.12.452008; this version posted July 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. under a s$ereomicrosco#e, and the mites were taxonomicall( identified. A saliva sam#le was also collec$ed from each individual. Mite and saliva sam#les were preserved at -20°C in tu0es 8i$h 100% e$hanol. 'e&ore DN1 isolation, sam#les were washed three times with e$hanol to remove #ossible e9$ernal contaminants follo8ing [13] and [33]. To$al genomic DN1 was isolated fro% all sam#les using the Quic2-DN1 MicroPre# kit (Z(mo), specificall( designed to isolate DN1 from small sam#les. A sam#le that did no$ contain tissue was included and treated as a regular sam#le to chec2 for cross-contamination during the DN1 isolation procedure. To$al DN1 was quantified using the Qu0it High Sensitivit( dsDN1 Assa( (Thermo Fisher Scientific). Libraries were pre#ared using the Ne9$era DN1 Flex Librar( Pre# kit (Illumina), s$ric$l( follo8ing the manu&ac$urer's ins$ruc$ions. BrieK(, DN1 was enzymaticall( cu$, and ada#$ers were added in a single s$e#. The ligated DN1 was am#lified, and oligonucleo$ide indices were added to bo$h ends of the frag%ents for pos$-se;uencing de%ultiplexing. The cons$ruc$ed libraries were quantified with the Qu0it dsDN1 HT Assa( kit (Ehermo Scientific), and qualit( chec2ed on an Agilent 2100 Bioanal(zer (Agilent Technologies). According to the Ju0it results, the libraries were pooled in e;uimolar amounts, and this pool was se;uenced on a NovaSe; PH150 single lane frac$ion (Illumina), aiming for a to$al ou$#u$ of 30 gigabases. Ehe DN1 isolation, am#lification, librar( pre#ara$ion, and whole-genome se;uencing were carried ou$ in AllGene$ics & Biolog( SL (w88.allgene$ics.eu). 5or the genome-resolved me$agenomic anal(ses, we trimmed the ra8 reads using fas$# [34].