Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information.Species in the genus TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry.We sequenced the ITS regions and the chloroplast DNA regions We found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades.Two species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution. Morus spp.), in the family Moraceae , are deciduous woody plants of great economic importance. The leaves of mulberry are the main food for silkworms  in the Moraceae , and the phylogenetic relationships among clades were determined . We constructed phylogenetic trees using ITS sequences and the chloroplast DNA (cpDNA) sequences M. alba, M. mongolica, M. cathayana, M. wittiorum, M. nigra, M. yunnanensis, and M. notabilis) for this study  and cpDNA sequences (MG434693).We selected 33 mulberry accessions , M. indica (DQ226511.1), M. cathayana (KU981118.1), M. mongolica (KM491711.2), M. alba var. multicaulis (KM491711.2), and M. alba var. atropurpurea (KU355276.1)) using VISTA viewer (http://genome.lbl.gov/vista/index.shtml) . These sequences were amplified using GoTaq Flexi DNA Polymerase  according to the manufacturer’s instructions. All the polymerase chain reaction (PCR) cycles consisted of initial denaturation of 95 °C for 5 min; followed by 32 cycles of 95 °C for 30 s, annealing for 30 s, 72 °C for 1 min; and then final extension for 7 min. The amplified fragments were isolated by electrophoresis on 1% (w/v) agarose gels. The purified sequences were cloned into the pMD19-T vector, and then 7–20 positive clones were sequenced for each accession.Genomic DNA was extracted from all mulberry accessions using the CTAB method . Chloropx.shtml) , we selelsewhere . We desihttp://www.bioinfo.rpi.edu/applications/mfold) . All the sequences were aligned using Clustal X 1.81 software . The bous/mfold) .M. mesozygia (HM747171), M. insignis (HM747169), M. serrata (HM747176), M. rubra (HQ144180), M. celtidifolia (HM747168), M. macroura (HM747170), M. mongolica (KF784879), M. wittiorum (AY345154), and M. australis (KT002555)) reported previously  and Bayesian inference (BI) methods. The first phylogenetic analysis was conducted using the ITS sequences of the 33 mulberry accessions determined in this study and those from another nine species (eviously . The beseviously . MrBayeseviously . Four Maeviously . A discreviously . All chatrnL-trnF and trnT-trnL regions. The best-fit model (GTR) for BI and M-L analyses for cpDNA sequences was selected by the lowest AIC scores in MrmodelTest 2.3 , M. insignis (clade D2), M. serrata (clade A4), M. rubra (clade A3), M. celtidifolia (clade A1), M. macroura (clade B), M. mongolica (clade B), M. wittiorum (clade B), and M. australis (clade B) in the phylogenetic analyses, as they represented six of the eight clades classified in a previous study , two accessions of species M.mongolica (Jimengsang and Mengsang) , three accessions of species M. cathayana , four accessions of species M. wittiorum , and one accession of species M. nigra  formed a subclade, and M. nigra was separated from the other mulberry accessions on a long branch  ; Table 1M. nigra ; Table 1M. nigra . Speciesg branch .ITS pseudogenes have been detected in many plants , and shoWe conducted phylogenetic analyses of ITS functional sequences and pseudogenes. The main topologies of the phylogenetic trees were the same as that reported elsewhere . In the M. wittiorum. However, Yun6muben had long-type ITS sequences that clustered in clade 3, while Yun7muben had short-type ITS sequences that clustered in clade 2. As the morphological characteristics were very complex, no details are discussed here. These phenomena are indicative of concerted evolution or introgression of the ITS region, and of hybridization  and M. alba (B clade) has been detected in previous studies  were used for phylogenetic analyses to study the concerted evolution of mulberry. The mulberry accessions in clade B were separated into two clades in the phylogenetic tree based on cpDNA sequences. The sequences in clade I were almost identical, sequences in clade II showed more variations and a subclade was clustered  and a long-type sequence clade (clade 3). Two pseudogroups were also divided in the ITS phylogenetic tree. These findings suggest that there is a shallow level of phylogeny among 29 mulberry accessions, consistent with the two clades detected in the cpDNA phylogenetic tree . Thus, cLeucaena, it was proposed that some ITS pseudogene sequences are inherited from the maternal progenitor (Morus genus.On the basis of a study of the genus ogenitor . In thisogenitor  and 4. CaAA) existed at a certain time and showed stronger ecological potential. Those resources then hybridized with mulberry with long-type sequences (bBB). The ITS sequences gradually became short-type sequences through concerted evolution or hybridization. Finally, long-type ITS sequences (B) were left as pseudogene sequences (B) in the offspring, and most mulberry had short-type functional ITS sequences . Thus, mulberry with long-type ITS sequences (bBB) are the original mulberry resources, and are being polluted by those with short-type ITS sequences during evolution. The actual evolutionary process of mulberry will be more complex than this evolutionary pattern, and dominated by hybridization.The three connection patterns and the incongruent distribution patterns between the cpDNA and the ITS phylogenetic trees imply that hybridization has contributed to the evolution of mulberry. Based on the discussion above, a simple evolutionary pattern is proposed for mulberry . First, n = 12x = 84) belonged to clade 2 and clade I). These results indicate that hybridization and polyploidy have played important roles in the evolution of mulberry, even in the accessions with higher ploidy levels. Clade 2 of the phylogenetic tree constructed from ITS sequences contained mulberry accessions with different high ploidy levels, identical ITS sequences, and successively evolved cpDNA sequences, implying that concerted evolution has played a key role in the evolution of this clade. In conclusion, polyploidy, hybridization, and concerted evolution have all played important roles in the evolution of ITS sequences in mulberry.In the phylogenetic trees constructed from ITS and cpDNA sequences  and 5, aBased on phylogenetic analyses of ITS sequences and cpDNA sequences, clade B was separated into two species. We found ITS pseudogenes in mulberry, and detected a concerted evolutionary process in the direction of short-type ITS sequences. We detected three connection patterns between ITS pseudogene sequences and cpDNA sequences, suggesting that the ITS pseudogene sequences connect with genetic information from the female progenitor. Combining the three connection patterns and incongruent distribution patterns between phylogenetic trees constructed from cpDNA and ITS sequences, hybridization is recent or still occurring, and has played important roles in mulberry evolution. The proposed evolutionary pattern, in which hybridization is a key feature, helps us to understand the evolutionary history of mulberry and highlights the importance of conserving local resources. Finally, polyploidy, hybridization, and concerted evolution have all played roles in the evolution of ITS sequences in mulberry.10.7717/peerj.8158/supp-1Supplemental Information 1M. alba var. atropurpurea was using as reference. trnL-trnF and trnT-trnL in 48k–52k region were selected for further analyses.Chloroplast genome sequences Click here for additional data file.10.7717/peerj.8158/supp-2Supplemental Information 2Phylogenetic tree was constructed using SYM+G model in MrBayes. Differently colored clades correspond to those in Click here for additional data file.10.7717/peerj.8158/supp-3Supplemental Information 3Phylogenetic tree was constructed using GTR model in MrBayes. Mulberry accessions are labeled with clade name shown in Click here for additional data file.10.7717/peerj.8158/supp-4Supplemental Information 4Click here for additional data file.


AbstractChironomidae (Diptera), are dominant components of most freshwater ecosystems. Many chironomids construct tubes or cases as larvae out of various materials bound together with silk. The structures of tubes show a wide range of variation, and some are morphologically comparable to those of caddisflies. Herein a new species is described, Eukiefferiellaendobryoniasp. nov., which exhibits a very unusual behavior in which it constructs tubes from aquatic mosses. This species’ fourth-instar larvae construct their cases exclusively from the leaves of Fontinalis mosses  and exhibit a stereotyped behavior in which they remain attached to the apical shoot of the moss stem. The larvae then pupate within the case. The case of E.endobryoniasp. nov. represents one of only a few examples of chironomid tubes made exclusively out of plants. Based on the species delimitation analyses using the partial COI sequences, together with some morphological and behavioral characteristics, this species is hypothesized to be a member of devonica group, and especially may have a close affinity to E.dittmari (Lehman). A provisional typology for the diversity of chironomid tube structures is provided, with a summary of different tube structures, which can be used for future research.The non-biting midges,  Protozoa, Mollusca, Annelida, Polychaeta, Crustacea, Echinodermata, fishes, and Nematoda , and Ephemeroptera (family Polymitarcyidae). Tube morphology and ultrastructure vary significantly among taxa, and these tubes may serve various functions . The life history of this species was clarified with the aid of DNA barcoding, and a description of it is given herein, including an account of its larval tube construction behavior. As the taxonomy of Eukiefferiella can be problematic, the genetic differentiation of the new species in comparison to some congeners which are hypothetically closely related is estimated using methods for delimitation of species. Additionally, a provisional typology of chironomid tube morphology is provided, to highlight the diverse morphology of tube structure among chironomids.While searching for arthropods that interact with aquatic bryophytes in North America, I discovered chironomid larvae that were notably distinct from other tubicolous chironomids due to their unique tube-constructing behavior. Specifically, the fourth-instar larvae of these chironomids make cases exclusively using the leaves of Fontinalis have been recorded from the lake itself and the brooks in its vicinity, F.dalecarlica B. S. G. and F.novae-angliae SullF.dalecarlica is common in gently flowing water bodies, including in the brook that I sampled. Additional samples were collected during 4–9 April and 11–13 November in 2018 in a flowing-water stream, Rowans River, along Sparks Lane in Cades Cove, in the northwest part of the Great Smoky Mountains National Park (GRSM), Tennessee, USA, ca. 430 km away from Mountain Lake. Fontinalisnovae-angliae with rigid stems and concave leaves occur from the streambed. Samples were collected in GRSM under research permit GRSM-2017-SCI-2389.Chironomids were collected during 24–27 February and on 10 November in 2018 in a stream connected to Mountain Lake, Virginia, USA. Mountain Lake is an oligotrophic lake that is located at an elevation of 1181 m above sea level near the summit of Salt Pond Mountain, and is the only natural lake of substantial size in the unglaciated part of the Southern Appalachian Mountain Range . MountaiInsects were searched for underwater in the sampled streams and brooks and were collected together with the host plants occurring in their habitats. At Mountain Lake Biological Station, the clumps of mosses were detangled from detritus, and sediments were washed out of them. The larvae were placed in small plastic cases and observed with a microscope. The plastic chambers were constantly cooled with a refrigerant to keep their temperature in the range between 10–22 °C. Fourth-instar larvae were observed for 27 h in total, between 08:00 hrs and 18:00 hrs during 4–25 March 2018. Rearing and observations of chironomid larvae were performed at the National Museum of Natural History, Smithsonian Institution.Chironomidae  gene sequences were determined. Total genomic DNA was extracted from 23 specimens at different stages, including adults (from a single adult leg or abdomen), larvae , and pupae (the whole abdomen) or pupal exuviae, using a NucleoSpin Tissue kit  and following the protocol provided by the manufacturer, with some modifications. The protocol was modified as follows: (i) tissue was digested for 48 h at 58 °C; (ii) after digestion with proteinase K, tissues were removed, washed in distilled water and used for morphological assessments; and (iii) the final elution volume was 30 μL. The primer pair used for the COI region consisted of primers 911 and 912 of ae e.g., . AmplifiPCR) products was performed using the ABI Big Dye Terminator 3.1 cycle sequencing kit  while following the manufacturer’s instructions and was carried out in an ABI 3130 Capillary Electrophoresis Genetic analyzer. Both DNA strands were sequenced. Sequences were deposited in the GenBank database (Table Direct sequencing of polymerase chain reaction (nucleobytes.com). Nucleotide sequences were aligned with Clustal W implemented in MEGA 7  and to assess the intra- and interspecific genetic distances, the species delimitation plug-in in the software Geneious Prime 2019.2.3 (www.geneious.com) was used , although the sequence data identified at the species level were available only for five species , E.ilkleyensis (Edw.), E.claripennis (Lundbeck), E.minor (Edw.), E.dittmari (Lehman)). In the dataset, 23 sequence data representing five species were included (Table BI). Trees were rooted with two species of the genus Cardiocladius , C.fuscus Kieff.). Evolutionary model was selected with MrModeltest v. 4.0b10 (BI trees were constructed with MrBayes v.3.2.6 (PP). At the point of burn-in, the chains had all converged to a stable standard deviation of split frequencies lower than 0.01.The COI sequences of a rich record of . 4.0b10 . The bes. 4.0b10 . For the v.3.2.6 , using tAB (AB is a test for taxonomic distinctiveness of a clade based on the null hypothesis that monophyly is a chance outcome of random branching. Rodrigo’s P(Randomly Distinct) (‘Rodrigo’s P (RD)’) is the probability that a focal clade has the observed degree of distinctiveness  due to random coalescent processesAB and Rodrigo’s P (RD): the average pairwise tree distance, among members of the focal species/populations (‘D Intra’) and between the members of the focal species and members of the next closest species (‘D Inter’); as a measure of genetic differences between the focal species and its closest neighboring species, the ratio of D Intra to D Inter (‘Intra/Inter’); as the measures for evaluating diagnosability of each species/population, the mean probability of making a correct identification of a hypothetical sample of the focal species using placement on a tree under two different criteria, ‘P ID(Strict)’  and ‘P ID’ ; the mean distance between the most recent common ancestor of a species and its members (‘Av(MRCA)’). Accession number, voucher ID, and information on the localities for each specimen are shown in Table The species delimitation plug-in in the Geneious Prime 2019.2.3  was usedAB  Rodrigo’AB  were calAll specimens of adult abdomens, pupae, and larvae were digested with proteinase K, which made it relatively easy to examine the specimens morphologically. When necessary, the apical portion of the adult abdomen was macerated with warm (ca. 90 °C) 5% KOH and rinsed with distilled water. Each body part sample was mounted on a microscopic slide with Euparal.AR), leg ratios , wing ratio (L/WR) and hypopygium ratio (HR), and other morphological features were measured for adult male specimens following The terminology of morphological features used herein followed AR antennal ratio: length of last flagellomere / length of remaining flagellomeres;LR leg ratio: length of first tarsal segment/ length of tibia;BV “Beinverhältnis”: length of femur, tibia plus first tarsal segment/ length of tarsal segments 2–5;SV length of femur plus tibia/ length of tarsal segments 1–3;L/WR wing length/ wing width ratio;HR, hypopygium ratio: length of gonocoxite/length of gonostylus.NMNH . For each specimen, voucher ID is given as ‘YI-CR-##’.The type specimens are deposited in Taxon classificationAnimaliaDipteraChironomidae9358422A-D137-5BA3-8BC4-629764467C1Fhttp://zoobank.org/2EFD6644-44B9-4CF2-8C5F-6E2523DE6CF6Adult male with squama with few (two or three) setae; gonostylus with crista dorsalis; hind tibial comb and tibial spurs reduced, outer spur absent. Pupa lacks precorneal setae and respiratory horns; three anal macrosetae consisting of two thinner inner macrosetae and a normal outer macroseta. Larval body setae short; seta interna with five branches deeply divided to the base; mentum with four pairs of lateral teeth and single, wide, truncate median tooth.Holotype: USA, VA • 1 adult male (YI-CR-013); Mountain Lake (37.357627 N 80.534448 W); 24-II-2018 (as larva); Y. Imada leg; emerged as adult on 12-III-2018; NMNH.Paratypes: USA, VA • 2 adult males  and 3 adult females ; Mountain Lake (37.357627N 80.534448W); 24-II-2018 (as larvae); Y. Imada leg; emerged as adults between 12-III-2018 and 28-IV-2018; NMNH.35.600894N, 83.794004W); 13-XI-2018 (as larvae); Y. Imada leg; NMNH; VA • 1 male pupa (YI-CR-012), 2 female pupa , 1 pupal exuviae (no voucher), 4 larvae ; Mountain Lake ; 9-XI-2018; Y. Imada leg, NMNH.USA, TN • 2 female pupae , 2 larvae ; Sparks Lane (Unknown.Unknown.N = 4) Body length 3.0 mm. Head capsule dark brown. Body yellowish. Head capsule with frontoclypeal apotome with clypeus without divided by strong suture. Antenna nonretractile, 5-segmented; fourth segment twice as long as third segment; lauterborn organ small; blade as long as flagellum; ring organ in basal third. Premandible with one broad, blunt apical tooth. Mandible with apical tooth longer than first lateral tooth; inner margin smooth, without serrations; seta subdentalis short, peg-like; five very long seta interna with five branches divided nearly to the base, each branch similar in length and width to each other; mola with four long spines. Maxilla without pecten galearis; chaetulae of palpiger lacking; lamellae of galea short; anterior lacinial chaeta apparently short, broad-based, more or less differentiated from other chaetae. Mentum with single median tooth and four pairs of lateral teeth; ventromental plates inconspicuous, without beard beneath. Parapods well developed. Claws of anterior parapods all smooth. Procercus unsclerotized, less than 1.5 times as long as wide, without tooth, spur, or seta; anal setae 5–7. Supraanal seta absent. Anal tubules developed, longer than posterior parapods. Body setae very short and inconspicuous, shorter than one-quarter the length of abdominal segments.(N = 8) Frontal apotome without frontal seta and warts. Thoracic horn and precorneal seta absent. Dorsocentrals four. Thorax nearly smooth. Wing sheath smooth, without pearl row. T I–II, T VIII, S I and S VIII without shagreen. T II–IX with strong anterior shagreen. S II–VII with weak posterior shagreen. Pedes spurii A and B absent. Caudal spines absent on T II–VIII. S IV–VII female at most with very weak caudal spines. Orally curved hooklets present in uninterrupted rows posterior to caudal spines on T III–V. Apophyses and O setae absent. Segments IV–VIII with very short and weak L-setae. Anal lobe with three unequal anal macrosetae, consisting of two, thinner inner macrosetae and a normal outer macroseta; without median seta, fringe, apical spine. Body length 2.9–3.0 mm without antenna. Body color dark brown. Antennal length 0.8 mm. Flagellum plumose, with 13 flagellomeres; apex spatula-shaped, without a strong straight seta; antennal groove in male reaching flagellomere 3; AR 1.1. Eye bare. Temporal setae 2, not clearly separated into inner and outer verticals and postorbitals. Postocular setae present in a single row, only behind eyes. Palpus 5-segmented; palpomere lengths: 55–72, 86–90, 96, 159–159 (N = 1); palpomeres with 3, 4, 5, 0 setae, respectively. Antepronotum well developed with lobes meeting medially at anterior margin of scutum; dorsal anterpronotals absent; four lateral antepronotals; acrostichals absent; six dorsocentrals in a single row. Approximately three prealars. Scutellum smooth with nine scutellars in single row. Supraalar setae present. Wing length 2.3 mm; L/WR 3.01. Wing membrane glabrous, unmarked. Anal lobe small. Costa not extended. Crossvein m-cu absent. Cu1 straight. R4+5 only fused with C at apex. R2+3 present, ending at middle of distance between R1 and R4+5. Cu1 very slightly curved apically at wing margin. Squama with two or three setae. Sensilla campaniformia ca. eight at base of brachiolum, three above setae and eight at apex of brachiolum; 1 on Sc, one basally on R, one near base of R1; and one on FR. Calypter without marginal setae; calyptral fringe absent. First tarsomere of foreleg shorter than fore tibia. Fore coxa not enlarged. Hind tibial comb and tibial spurs reduced; outer spur absent. Pulvilli very faint. Gonostylus hinged to gonocoxite and folded inward. Anal point absent. Anterior margin of transverse sternapodeme convex, phallapodeme and aedeagal lobe normal. Virga absent. Gonocoxite with well-developed inferior volsella. Gonostylus with crista dorsalis; apical spine absent. HR 1.99. Lengths of leg segments and leg ratios as in Table  Body length 2.8 mm. Antenna with five flagellomeres; flagellomere lengths (in µm): 56.7, 35.8, 38.2, 45.2, 101.2; with 2, 3, 2, 3, 3 setae, respectively (N = 1). Eye bare. Clypeus with 8 setae. R with two setae, squama with 4–6 setae. Scutellum as in male. Gonocoxapodemes not jointed mesally, well sclerotized. Gonocoxite long, with long and short setae. Tergite IX with two unseparated distinct lobes. Triangular floor under vagina present. Gonapophysis VIII pointed caudally, with two apodeme lobe. Membrane T-shaped. Labia small, bluntly quadrangular, void of microtrichia. Seminal capsule ovoid, darker sclerotized in oral half, without microtrichia. Spermathecal ducts with triangular bulb before separate openings. Cercus normal, length twice as long as width..endo- (ἔνδον), a prefix meaning within, bryon (βρύον), meaning moss, and the suffix -ia (-ία), forming abstract nouns of feminine gender. It alludes to the biology of this species, which live within the case made of mosses.The species name is a compound word in which three words from Ancient Greek are combined, Eukiefferiella in that its pupae lack the precorneal seta. This species can also be distinguished from others in the genus by the following combination of traits: pupa lacks respiratory horns, and has the unique configuration of pupal anal macrosetae ; and larva has a mentum with four pairs of lateral teeth and a single, wide, and truncate median tooth. Any geographic variation in this species’ characters was detectable between the populations sampled in VA and TN.This species is unique among species of Eukiefferiella sp. voucher BIOUG01648-H02’ in GenBank (accession No. KR660601.1)  together with the above-mentioned sequence data. A Bayesian tree indicated that E.endobryonia sp. nov. was sister to E.dittmari among four species of Eukiefferiella in the dataset with significantly high BPP support  and thus the reciprocal monophyly of the clade of E.endobryonia sp. nov. was not supported. Two geographic populations sampled, Great Smoky Mountains (GRSM) and Mountain Lake (ML), formed separate clades and between which genetic divergence among population was substantial (Intra/Inter = 0.12)  ; thus, teny Fig. , as wellort Fig. . Values ce Table . Likewisce Table . However2) Table , of whicda Table .Chiloscyphus , as well as some pleurocarpous moss species, such as Brachythecium spp. , were abundant there on the upper sides of boulders and cobbles that were exposed to spray and occasionally submerged in water. Fontinalisdalecarlica, a host plant species of E.endobryonia sp. nov., occurred at high densities on the lateral sides of submerged boulders in the stream. As a matter of fact, this seemed to be the only aquatic moss species of which conspicuous amounts were found in this particular stream. I was able to find some white-bodied insect larvae occupying some of the apical shoots of Fontinalis moss plants . The foraging areas were therefore mainly restricted to the region immediately surrounding the tube. This territorial feeding behavior has been reported for many tube-dwelling chironomids  Edw. (The life cycle of this new species between late spring and early autumn (May–October) is unknown. This species is likely multivoltine because fourth-instar larvae and pupae were found together at both sites in both April and November. It appears that the larvae were collector-gatherers at the third instar, but became scrapers at the fourth instar sensu. The thibehavior  within tbehavior . The foubehavior ; simultads e.g., . The larK.) Edw. . The larK.) Edw. , which mK.) Edw. . JudgingN = 7)  Fig. . The pupEukiefferiella represents a large and widespread genus of Orthocladiinae , E.brevicalcar (Kieff.), E.claripennis, E.coerulescens (Kieff.), E.cyanea Thienemann, E.devonica, E.gracei (Edw.), E.minor, E.unicalcar (Sæth.), and E.pseudomontana Goetghebuer. However, there is also evidently a large number of undescribed speciesThe study of the taxonomy of p, level . Ten spep, level : E.brevEukiefferiellaendobryonia sp. nov. was assigned to Eukiefferiella on the basis of diagnostic characters proposed by previous studies . Some morphological and behavioral traits of E.endobryonia sp. nov. show affinity to those of E.ilkleyensis in the E.devonica group, specifically: the larval mentum medially truncate with four lateral teeth; and the larva of E.ilkleyensis is reported to make a case at the shoot tips of the moss Eurhynchiumriparoides . The immature stages of E.dittmari is not described, but the larvae live in mosses (E.endobryonia sp. nov. was estimated to be distinctive and a significant level of genetic differentiation is detected between the geographic populations sampled (ML and GRSM). However, Rosenberg’s PAB for each species used herein suggests that the null hypothesis that monophyly of each taxonomic group occurs due to the random coalescent process is not rejected. To clarify the phylogenetic status of the species and to trace the evolutionary history of biology of this group, especially the specialized relationship with mosses, it is necessary to elucidate the biology for the species of which immature stages are unknown and to accumulate morphological and genetic data of the related groups.The molecular phylogeny Fig.  clarifiees group  but may ca group . E.endon mosses . Based oEukiefferiella species are generally abundant in the riffles of rivers and streams , which may result in a higher mortality rate of the host   and E.calvescens (Edw.), which construct loose tubes incorporating rocky particlesE.clypeata Thienemann, which inhabits the exposed regions of rocks swept by water currents and builds solid sheets of silkE.claripennis  ; Fig. 4Ecylidae) ; Fig. 4Hcylidae) . Some spripennis .Eukiefferiella are frequently found among aquatic mosses, including Fontinalis spp.   in its later larval instars  and transportable cases . Fixed shelters are much more common than transportable cases. Second, fixed shelters can be categorized into three groups (see below) based on the substratum to which the tube is attached. In fact, chironomids as a whole are able to colonize a broad spectrum of substrates. Third, these tube morphotypes can be further subdivided based on the materials out of which the tube is made, but only if the larva has a preference for certain types of particles e.g., , and mosChironominaeOrthocladiinae and Chironominae) due to the influence of the striated ventromental plates, which are only present in the Chironominae  rock material-bound tubes. Rock materials with various grain sizes are used to make tubes, ranging from very coarse  to fine . This type can therefore be subdivided by the grain size of particles used. Soil-dwelling chironomids often occur in lentic or lake environments, where they make soft, cryptic tubes with various lengths and forms, from short, cylindrical tubes to meandering, non-blindly ending tubes, in mud. The shapes of the silk-laden burrows or tubes of species dwelling in soft sediments are associated with their feeding strategies, as these are often deposit- and filter-feeding animals (Chironomusplumosus (Linn.) (Chironominae: Chironomini)  Fig. , makes tChironomus, which correspond to the different feeding strategies used by these species. The first method is found in the filter-feeding C.plumosus: the larva spins a funnel-shaped net across the lumen of the tube for filtering fine organic particles out of the water  rivulorum Kieff.  rivulorum are coated with diatoms, including Hannaea Patrick and Reimer (Fragilariaceae), and exert a considerable effect on the periphytic diatom floraTanytarsini (Chironominae) make rigid tubes, such as Rheotanytarsus Thienemann & Baus and Lithotanytarsus Thienemann. Rheotanytarsus  tubes on symbiotic animals. Various chironomid lineages  exhibit a wide range of symbiotic associations with aquatic animals, ranging from ectoparasitism to phoresis and commensalism, which can be either obligate or facultative associations   or sessile  organisms. Information on the tubes formed by symbiotic chironomids is often limited, and their construction behaviors were not described in many previous studies. The second major group of tubes is: ciations . Their hciations ; Fig. 5Eciations , mayflieciations ; Fig. 5Fciations ; Fig. 5Gciations , spongesciations , and bryciations . Host seciations . Some spchanisms . Some sychanisms . For exa(3) tubes on/within plants, algae, cyanobacteria. Some chironomids in the Orthocladiinae and Chironominae often facultatively make tubes on the external surfaces of plants or algaeTelmatogon Schiner (Telmatogetoninae) inhabit intertidal zones and make their tubes within the green algae occurring on rocks (Clunio spp. (Orthocladiinae) are marine and make irregular silk tubes dotted with debris on seaweeds, and feed on encrusting diatoms and organic matter  Kieff.) are filter-feeders and spin a conical net inside the mine to filter out floating particles . Species of Stenochironomus Kieff. feed either on living or sequestered plant leaves and tend to show weak preferences for host plants (Cricotopusnostocicola Wirth (Orthocladiinae)  Fig.  has a sythe host . For manov. Fig. , as desctruction .(4) portable cases. Among chironomids, transportable cases are far less common than fixed shelters. This is in sharp contrast to the myriad examples of portable cases seen among the Trichoptera  is the only species so far known to the make portable cases among the species in the Orthocladiinae , Calopsectra Kieff. and Micropsectrapharetrophora Fittkau & Reiss (Chironominae: Tanytarsini)   Fig.  build ‘cni) Fig. . A few garticles . Lauterbf.) Fig.  makes a f.) Fig. . The conIntegripalpia  are ectoparasites of caddisflies, and supposedly do not manipulate silk (Podonominae and Tanypodinae, are free-living (Telmatogetoninae is likely to be one of the earliest-diverging clades of case-builders (Chironominae and Orthocladiinae, which split during the mid-Jurassic. Interestingly, the spinning mechanism and silk properties are apparently different among the three clades of tube-making chironomids (Herein, I made the simplistic assumption that the evolution of case-construction behavior in chironomids could be estimated based on the results of a previous molecular phylogenetic study , althougubfamily , althougubfamily . Importaate silk . The eare-living . Among ebuilders . Tube dironomids . The difronomids . Furtherronomids . The behronomids . It woulronomids .Trichoptera (Wiggins and Mackay 1978The tube morphology of chironomids does indeed show significant diversity. It is likely that lability in silk production, particle and substrate selection, and construction behavior has made it possible for chironomids to use a broad array of ecological niches, as is the case for the


Klebsiella pneumoniae (K. pneumoniae) is a common cause of health-care associated infections (HAIs) and has high levels of antibiotic resistance. These bacteria are well-known for their ability to produce biofilm. The purpose of this study was to identify the antibiotic resistance pattern and biofilm-producing capacity of K. pneumoniae isolated from clinical samples in a tertiary care hospital in Klaten, Indonesia.K. pneumoniae was isolated from inpatients in Soeradji Tirtonegoro Hospital Klaten from June 2017 to May 2018. Identification of K. pneumoniae isolate was done by analyzing colony morphology, microscopic examination, and by performing biochemical testing. Testing of antibiotics susceptibility and biofilm-producing capacity used the Kirby-Bauer disk diffusion method and adherence quantitative assays, respectively.K. pneumoniae isolates were isolated from 962 total clinical bacterial isolates during the study. Most of them were collected from patients aged more than 60 years old and were mainly obtained from respiratory specimens (51.50%). Most of K. pneumoniae isolates were extensively resistant to antibiotics. A more favorable profile was found only towards meropenem, amikacin, and piperacillin-tazobactam, showing 1.20%; 4.79% and 10.53% of resistance, respectively. The overall proportion of multidrug-resistant K. pneumoniae isolates was 54.49%. In addition, 148 (85.63%) isolates were biofilm producers, with 45 (26.95%) isolates as strong, 48 (28.74%) isolates as moderate, and 50 (29.94%) isolates as weak biofilm producers.A total of 167 (17.36%) K. pneumoniae isolates demonstrated resistance to a wide range of antibiotics and are biofilm producers.Most of the  Klebsiella pneumoniae are quickly developing multidrug resistant (MDR) strains and commonly pose a serious threat to the patients because of an increased fatality rate due to the reduced effectiveness of therapy options. K. pneumoniae is known to be responsible for community acquired infections although recently it is routinely observed as a major cause of hospital acquired pathogens. K. pneumoniae has been observed to develop resistance to antibiotics more easily than most bacteria through the production of enzymes such as Extended Spectrum β-Lactamase (ESBLs) and Carbapenemase [. Some of the main contributors in the emergence and spread of highly resistant bacteria for health-care associated infections (HAIs) are the intensive and prolonged use of antibiotics in the hospital setting [The emergence of antimicrobial resistance (AMR) is a rapidly increasing challenge in today’s healthcare institutions worldwide. Pathogenic bacteria, for example, penemase –3. The m setting .K. pneumoniae also plays an important role in spreading antimicrobial resistance genes from bacteria in the environment to clinically important bacteria [bacteria . There abacteria .K. pneumoniae is a Gram-negative encapsulated bacteria that thrives in the mucosal surfaces of mammals and in the soil, vegetation, and water. In humans, K. pneumoniae typically will colonize the oropharynx and gastrointestinal tract, from where it can easily enter the circulation and other tissues causing infections such as bacteremia, septicemia, surgical site infection, urinary tract infection, hospital acquired pneumonia, and ventilator-associated pneumonia. It also contributes to the high frequency of opportunistic infections that occur among patients with immunocompromised situations, such as bladder neuropathy or diabetes mellitus [mellitus , 7.K. pneumoniae is also known for its capability to form biofilms, which are communities of bacteria embedded in an extracellular matrix. This matrix consists of proteins, exopolysaccharides, DNA, and lipopeptides [K. pneumoniae and the biofilm-forming phenomenon [K. pneumoniae has some virulence factors such as capsule polysaccharide, lipopolysaccharide, type 1 and type 3 fimbriae, outer membrane proteins and determinants for iron acquisition and nitrogen source usage. K. pneumoniae used these virulence factors for survival and for evade from immune system during infection as well as biofilm formation itself [K. pneumoniae can produce a thick layer of extracellular biofilm that supports the bacterial attachment to living or non-living surfaces, protecting antibiotics penetration and reducing its effects [peptides . In 1988n itself , 11. K.  effects .K. pneumoniae isolates from 1105 bacteria isolated in Soeradji Tirtonegoro hospital in Klaten, Indonesia. In 2005, a study conducted in a hospital in Surabaya, Indonesia reported the prevalence of ESBLs was 28% among clinical isolates of K. pneumoniae [. These antimicrobial-resistant bacteria have become a worldwide problem and there is still very limited data regarding biofilm producing capacity and antimicrobial resistence of K. pneumoniae in Indonesia. Biofilm forming capacity is demonstrated to have a greater resistance to antibiotics. Further studies are needed to describe the pattern of antibiotic resistance and the capacity to produce biofilm of K. pneumoniae clinical isolates in Indonesia. Thus, the purpose of this study was to identify the antibiotic resistance patterns, and the biofilm-producing capacity of K. pneumoniae clinical isolates in the Soeradji Tirtonegoro hospital in Klaten, Indonesia.In 2017, there were 213 (19.28%) K. pneumoniae clinical isolates from inpatients in Soeradji Tirtonegoro Hospital, a tertiary care hospital Klaten, Indonesia, during June 2017 – May 2018.Using a cross-sectional design, this observational research studied K. pneumoniae isolate was conducted by culturing on McConkey agar, Gram staining and biochemical testing using Microbact™ GNB 24E . Kirby Bauer method was used to perform antibiotic susceptibility tests. The Clinical and Laboratory Standards Institute 2015 was used to classify as sensitive, intermediate or resistant bacteria [K. pneumoniae isolates that showed résistance to three or more different classes of antimicrobials were classified as multidrug-resistant (MDR) K. pneumoniae [Identification of bacteria . K. pneuA quantitative adherence assay was employed to perform biofilm formation assay . An overK. pneumoniae were tested by Chi-square tests using STATA 13 software . The results were presented as prevalence ratios with a 95% confidence interval. Statistical significance was set if p-value < 0.05.Frequencies and percentages were used to describe the variables in this study. Associations between the antibiotic sensitivity pattern and the biofilm producing capacity of The Medical and Health Research Ethics Committee, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia permitted this study with number: KE/FK/0862/EC/2017.K. pneumoniae isolates were examined from 962 total clinical bacterial isolates at Soeradji Tirtonegoro Hospital, Klaten, Indonesia. K. pneumoniae isolates were isolated from 107 male (64.07%) and 60 female (35.93%) patients. Most of K. pneumoniae were isolated from patients aged more than 60 years old (38.10%) (Table K. pneumoniae samples were mostly isolated from respiratory specimens (51.50%) and pus (16.17%)  K. pneumoniae were resistant to a wide range of antibiotics. Among biofilm producer isolates, K. pneumoniae had only a good sensitivity to meropenem (98.60%), amikacin (95.80%), and piperacillin-tazobactam (90.00%). In contrast, among non-biofilm producer isolates, K. pneumoniae showed good sensitivity to meropenem, levofloxacin, amikacin, piperacillin-tazobactam and ciprofloxacin with 100.00%; 95.83%; 91.67%; 87.50%, and 86.67% of sensitivity respectively  isolates as biofilm producer and 24 (14.37%) isolates that were not biofilm producers. Among biofilm producers, there were 45 (26.95%) isolates as strong, 48 (28.74%) isolates as moderate, and 50 (29.94%) isolates identified as weak biofilm producers (Table K. pneumoniae MDR isolates were found in 91 (54.5%) isolates and 76 (45.5%) isolates were non-MDR  Program [The overall proportion of MDR s et al.  informeda et al.  showed ti et al.  which re Program .K. pneumoniae tested, 70 isolates (64.7%) were identified as high or moderate biofilm producers and 40 isolates (35.3%) were identified as weak biofilm producers. In another study, Cepas et al. [K. pneumoniae strains were biofilm producers. Yang and Zhang [K. pneumoniae isolated from urine, sputum, wound swabs and blood were biofilm producers. Seifi et al. [K. pneumoniae (93.6%) were biofilm producer and only 6.4% were not biofilm producers. Among biofilm producer strains, 33% were categorized as strong biofilm producer, 52.1% as moderately biofilm producers and 8.5% as weak biofilm producers. The capacity to form biofilms was different from each isolate because in general several factors are influencing the capacity such as the physicochemical characteristic of K. pneumoniae, physical interaction between the constituents, the type of surface where the biofilm attaches, temperature, pH, etc. [K. pneumoniae biofilm-producing ability correlate to antibiotic resistance profile.In this study, 148 85.63%) of 167 isolates were biofilm producers. A similar study reported by Hassan et al. [s et al.  reportednd Zhang  reported3% of 167pH, etc. . Vuotto pH, etc.  highlighE. coli, K. pneumoniae, and P. aeruginosa. There was no statistically significant relationship between general MDR and biofilm formation in three Gram-negative species because the MDR isolates did not show any greater disposition to becoming a stronger biofilm producer compared to non-MDR isolates. However, they reported some correlations between biofilm formation and resistance to specific antibiotics. Resistance to gentamicin and ceftazidime was correlated with biofilm formation in E. coli, as well as ciprofloxacin in P. aeruginosa and piperacillin-tazobactam and colistin in K. pneumoniae. This pattern of resistance should raise grave concerns because colistin is now considered to be the last line treatment choice for K pneumoniae [Cepas et al.  looked feumoniae .K. pneumoniae which is biofilm producer than non-biofilm producer. This finding has been reported in many studies. A study by Saha et al. [K. pneumoniae, the clear relationship between these factors has not been fully identified and elaborated. This finding is confirmed by de Campos et al. [This study showed that antibiotic resistance was greater among a et al.  demonstra et al. , 34. Anoa et al.  proposeds et al.  explainiIt has been confirmed by several studies that in the majority of cases, a single antibiotic therapy is inadequate to annihilate biofilm-forming infections. In consequence, managing infections with currently accessible antibiotics and evaluating the outcomes have become important and urgent protocols for the successful treatment of biofilm-associated infections . SeveralOur study showed the antibiotic-resistant bacteria problems in hospital setting which are also reported by some studies , 39. ThiResistance mediated by ESBLs consists of penicillins, cephalosporins  and aztreonam. Dumaru et al.  performeThe major cause of inappropriate antibiotic prescribing is due to a lack of education about infection and antibiotic usage. One of the most relevant steps in antibiotic prescribing is an adjustment of initial antibiotic therapy based on the clinical microbiology result . TherefoK. pneumoniae isolates showed resistance to a wide range of antibiotics. Most of these were also shown to be biofilm producers of various capacities. Global efforts should be intensified to prevent the spread of multi-drug resistant bacteria and eliminate the hospital-born microbes that are causing a dramatic rise in mortality.Most of the


Myxidium giardi Cépède, 1906 was described infecting the kidney of the European eel, Anguilla anguilla (L.), having spindle-shaped myxospores and terminal sub-spherical polar capsules. Since then, numerous anguillid eels globally have been documented to have similar Myxidium infections. Many of these have been identified using the morphological features of myxospores or by the location of infection in the host, and some have been subsequently synonymised with M. giardi. Therefore, it is not clear whether M. giardi is a widely distributed parasite, infecting numerous species of eels, in multiple organs, or whether some infections represent other, morphologically similar but different species of myxosporeans. The aim of the present study was to assess the status of M. giardi infections in Icelandic eels, and related fish hosts in Malaysia and to use spore morphology and molecular techniques to evaluate the diversity of myxosporeans present.The myxosporean Megalops cyprinoides (Broussonet), were noticeably smaller. However, the SSU rDNA sequences from the different tissues locations in eels, were all very distinct, with percentage similarities ranging from 92.93% to as low as 89.8%, with the sequence from Malaysia being even more dissimilar. Molecular phylogenies consistently placed these sequences together in a clade that we refer to as the Paramyxidium clade that is strongly associated with the Myxidium clade (sensu stricto). We erect the genus Paramyxidium n. g. (Myxidiidae) to accommodate these histozoic taxa, and transfer Myxidium giardi as Paramyxidium giardi Cépède, 1906 n. comb. as the type-species.The morphologies of the myxospores from Icelandic eels were very similar but the overall dimensions were significantly different from the various tissue locations. Myxospores from the kidney of the Malaysian tarpon, Myxidium (M. giardi) causing systemic infections in eels in Iceland. There are three species, confirmed with a robust phylogeny, one of which represents Paramyxidium giardi n. comb. Additional species probably exist that infect different tissues in the eel and the site of infection in the host fish is an important diagnostic feature for this group (Paramyxidium n. g. clade). Myxospore morphology is generally conserved in the Paramyxidium clade, although actual spore dimensions can vary between some species. Paramyxidium spp. are currently only known to infect fishes from the Elopomorpha.There is not a single species of  The importance of the shape of myxospores and its relative usefulness in myxosporean taxonomy has been scrutinised in recent years, and it has been unambiguously demonstrated that numerous genera are now polyphyletic in molecular phylogenetic analyses due to the use of spore morphology as the principal taxonomic measure –3. HowevMyxidium giardi Cépède, 1906 was originally described from the kidney of the European eel, Anguilla anguilla (L.), in France, with myxospores described as spindle-shaped with terminal sub-spherical polar capsules [Myxidium spp. infections, many of which have been distinguished using morphological features such as spore size and number or lack of valvular striations [Myxidium spp. infecting eels have subsequently been synonymized with M. giardi, as less emphasis was placed on details such as the site of infection [M. giardi was considered to have an almost worldwide distribution [M. giardi is a widely distributed parasite infecting numerous species of eels, in multiple organs, or whether some infections represent other, morphologically similar, species of myxosporeans. One short rDNA sequence exists for M. giardi from the urinary system of A. anguilla from Scotland, and phylogenetic analyses place this in the freshwater urinary clade, with numerous other myxosporeans found infecting the urinary systems of fish, but none with a similar spore morphology [capsules , but havcapsules . Globallriations , 9 or thriations . Howevernfection  and M. gribution . Currentrphology .Anguilla Garsault, distributed in all the world’s oceans. Two species inhabit the Atlantic Ocean, the European eel, A. anguilla, and the American eel, Anguilla rostrata (Lesueur) [Megalops cyprinoides (Broussonet) (Order Elopiformes), inhabits tropical coastal and brackish waters of the Indo-Pacific, migrating between the open sea and inland rivers and mangroves [Anguillid eels (Order Anguilliformes) are catadromous fish consisting of 17 species in a single genus, Lesueur) . A dramaLesueur) . The Indangroves . Elopifoangroves .Myxidium giardi-like parasites from different infection sites in the hosts. In addition, we aim to compare these with morphologically similar parasites previously observed by one of us (MAF) in tarpon from Malaysia.In the present study, we aimed to examine eels from Iceland and genetically characterise A. anguilla,  were collected from Lake Vifilsstadavatn in Iceland during May 2013 using fyke nets. The fish were transported to the laboratory, placed in tanks with freshwater and kept alive until examination. Prior to examination, the fish were euthanized in MS-222. All eels were dissected and examined for myxosporeans in the gills, kidney and stomach wall using dissecting and compound microscopes. Images were taken of twenty spores from each tissue location for size estimations using ImageJ. In addition, fourteen mature Pacific tarpon, Megalops cyprinoides total length range 22–34 cm, were caught with gill nets in Kilim mangroves on Langkawi Island, Malaysia. Tarpon were examined for gill and kidney myxosporeans, using dissecting and compound microscopes. In Malaysia, myxospores were photographed in the field using a portable compound microscope and a Dino-Eye eyepiece camera. Infected tissues from eels and tarpon were fixed in 10% buffered formalin and processed for standard histology, 3 μm thick sections prepared, stained with Giemsa and mounted in resin-based medium.Thirty-one live European eels, n = 100) were taken from sediment samples from the lake Vifilsstadavatn in Iceland, isolated and incubated at 10 °C in filtered (pore size 0.45 μm) lake water in 24-well tissue culture plates. Oligochaetes were examined daily using a dissecting microscope for the presence of actinospore production, typically characterized by a cloudy secretion from the anus of the worms, and confirmed using a compound microscope. Every other day, the water in the wells was exchanged. The oligochaetes that produced actinospores were used for DNA analysis and images of the actinospores taken as detailed above.Oligochaetes (SSU rDNA) was amplified using the general myxosporean primers and methodology previously described [SSU rDNA including the phylogenetically informative variable V1-V5 regions; PCR conditions were the same as in [SSU rDNA sequence alignments of the novel sequences to other related myxosporean sequences identified from the BLAST searches and from relevant literature [Infected tissue  or spore-filled cysts (gills), equal to approximately 20–40 mg, were placed directly into DNA lysis buffer for molecular analysis. Total DNA was extracted using a GeneMATRIX DNA isolation kit  following the tissue protocol and used as templates in subsequent PCR reactions. Parasite small subunit ribosomal DNA  was set as the outgroup. Posterior probability distributions were generated using the Markov Chain Monte Carlo (MCMC) method with four chains being run simultaneously for 1,000,000 generations. ‘Burn-in’ was set at 2500 and trees were sampled every 100 generations making a total of 7500 trees used to compile the majority rule consensus trees.Phylogenetic analyses were performed using the maximum likelihood methodology in PhyML  with theMeasurements of myxospores from each organ  were tested for normality using the Shapiro-Wilk test. To test whether there was a significant difference in analogous measurement between the different myxospores, a Student’s t-test was applied. Both tests were performed in RStudio (version 0.98.1062).Myxidium giardi  using a dissection stereoscope, ranging between 2–10 cysts per gill arch. Infections in the kidney and the stomach wall were only detected using a compound microscope. Spores were detected in the kidney of 10 fish (32%) and from stomach scrapings from 6 eels (19%). Infections were light in most cases, being more apparent in the gills. All myxospores detected had a similar morphology that conformed to the currently accepted shape and size range for Myxidium giardi from eels in France . The plasmodia were most commonly found as intratubular, both in the proximal and distal kidney tubules, although they were also, to a lesser extent, observed in the renal interstitium. The associated histopathology was mostly due to mechanical disruption of the tissue as the large plasmodia often caused extensive widening of the kidney tubules followed by atrophy and degeneration of the tubular epithelial cells  in the gastric gland interstitium . Infections cause separation of the lamellar epithelial cells from the basement membrane and disruption of pillar cells  and develop as intratubular, both in proximal and distal tubules. They are seen at various stages in development, some of which are fully mature, and commonly attached to the brush border of the proximal kidney tubule epithelium. In the distal kidney tubules, the proportion of mature plasmodia seem higher  and for the three species recognised here as new, i.e. P. magi n. sp. (2070 bp), P. branchialis n. sp. (2082 bp), P. bulani n. sp. (2056 bp) and the Aurantiactinomyxon (865 bp), which have been assigned accession numbers in GenBank: MH414925-MH414929. BLAST searches confirmed a myxosporean origin for all sequences with closest matches in the database being to numerous actinosporean sequences and also Myxidium lieberkuehni Bütschli, 1882, the type-species of the genus Myxidium. The SSU rDNA sequences from infected tissues from eels, were all very distinct, with percentage similarities ranging from 92.93% to as low as 89.8%, over > 2000 bp of sequence data  shown as the lilac box in Fig. Paramyxidium bulani n. sp. from a tarpon in Malaysia, represented the basal sequence of the clade. The Myxidium clade (sensu stricto), that includes the type-species M. lieberkuehni, was also fully supported  / Chloromyxum grouping. Basal to this is the hepatic biliary clade, a well-supported clade which contains numerous taxa currently assigned to the Myxidiidae, some of which infect vertebrates other than fish [Molecular phylogenetic analyses produced trees with very similar topologies irrespective of the methodology used Fig. . All Parhan fish .Fig. 3MaClass Myxosporea Bütschli, 1881Suborder Variisporina Lom & Noble, 1984Family Myxidiidae Thélohan, 1892GenusParamyxidiumn. g.Myxospore length width ratio between 1.4–1.6:1 resulting in a lemon-shaped form in valvular aspect and oval to bluntly-rounded form in sutural view. Valvular striations present, with sigmoidal sutural line. Polar capsules two, almost spherical. Two nuclei in the sporoplasm positioned between polar capsules. Capsular foramina open sub-terminally. Histozoic polysporous plasmodia develop in various host tissues with different species infecting different host tissues.Type-species:Paramyxidium giardi  n. comb.Other species:Paramyxidium branchialis n. sp.; Paramyxidium bulani n. sp.; Paramyxidium magi n. sp.Etymology: The new genus is named Paramyxidium n. g. as it is placed next to the type-species of the genus Myxidium (s.s.) in phylogenetic analyses.ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [Paramyxidium is: urn:lsid:zoobank.org:act:27B9FBC6-345A-4DD9-93D6-A13EACB82A18.e (ICZN) , detailsParamyxidium spp. are sufficiently different with respect to either myxospore morphology/ characteristics, tissue location, host type or DNA sequences to necessitate the erection of a novel genus within the family Myxidiidae Thélohan, 1892 to accommodate them. The family Myxidiidae currently contains five genera: Myxidium Bütschli, 1882; Coccomyxa Léger & Hesse, 1907; Cystodiscus Lutz, 1889; Enteromyxum Palenzuela, Redondo & Alvarez-Pellitero, 2002; Soricimyxum Prunescu, Prunescu, Pucek & Lom, 2007; and Zschokkella Auerbach, 1910. Species of Cystodiscus and Soricimyxum are only found in non-fish hosts [Enteromyxum spp. have very large and elongated polar capsules, spores that lack valvular striations, and are phylogenetically associated with morphologically similar forms from the gastrointestinal tract of marine fishes [Coccomyxa spp. are typically found in the gall-bladder of marine fishes, are more ellipsoidal in morphology, and are phylogenetically distantly placed in the marine gall-bladder (marine Myxidium) clade [Myxidium and Zschokkella are more complicated to decipher for as both are polyphyletic, with many species likely being wrongly attributed to each genus [Paramyxidium taxa in a robust clade which is sister to the Myxidium clade (s.s.). In addition, host differences and tissue tropism are apparent, with Paramyxidium taxa infecting the Elopomorpha in numerous organs and Myxidium spp. (s.s.) infecting the renal system of members of the Protacanthopterygii.sh hosts . Enterome fishes . Coccomych genus . HoweverParamyxidium to accommodate these myxosporeans, and transfer Myxidium giardi as Paramyxidium giardi Cépède, 1906 n. comb. as the type-species. The annelid hosts for Paramyxidium spp. are likely to be oligochaetes. Currently known species are restricted to freshwater environments and fish hosts from the Elopomorpha, presumably becoming infected during migratory periods into fresh water.Therefore, we erect the genus Type-host:Anguilla anguilla (L.) (Anguilliformes: Anguillidae).Type-locality: Lake Vifilsstadavatn , Iceland.Type-specimens: Hapantotypes  were deposited at the Natural History Museum, London, UK, under the accession number NHMUK 2018.235.Site in host: Stomach.Prevalence: 19% (6/31).Representative DNA sequences: A partial SSU rDNA sequence (2070 bp) was deposited in the GenBank database under the accession number MH414927.ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [Paramyxidium magi is: urn:lsid:zoobank.org:act:FEC23203-00E5-4862-8D24-FA5D6D8FFB0C.e (ICZN) , detailsEtymology: Specific name derived from Icelandic for stomach ‘magi’Spore. Mature spores lemon-shaped in valvular aspect . Polar capsules almost spherical, 3.6–4.6 × 3.1–4.0 (4.0 × 3.6) (n = 20), opening sub-terminally  in the gastric gland interstitium .Type-locality: Kilim mangroves, Langkawi Island, Malaysia.Other localities: Pangkor Island, Malaysia.Type-specimens: Hapantotypes  were deposited at the Natural History Museum, London, UK, under the accession number NHMUK 2018.237.Site in host: Kidney.Prevalence: 29% (4/14).Representative DNA sequences: A partial SSU rDNA sequence (2059 bp) was deposited in the GenBank database under the accession number MH414929.ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [Paramyxidium bulani is: urn:lsid:zoobank.org:act:DA7B51B0-590F-45A5-88CF-32A015B99593.e (ICZN) , detailsEtymology: Specific name refers to the local Malay name for the fish host (ikan bulan).Spore. Mature spores oval to bluntly-rounded in sutural view, lemon-shaped in valvular aspect, 6.1–6.9 × 4.2–5.1 (6.7 × 4.7) (L × W) (n = 20). Polar capsules almost spherical, 2.1–2.5 × 1.5–2.0 (2.3 × 1.8) (n = 20), opening sub-terminally. Sutural line sigmoidal and inconspicuous  and develop as intratubular, in both proximal and distal kidney tubules. Differs from other ei Figs.  and 2dType-host:Anguilla anguilla (L.) (Anguilliformes: Anguillidae).Type-locality: Lake Vifilsstadavatn , Iceland.Type-specimens: Hapantotypes  were deposited at the Natural History Museum, London, UK, under the accession number NHMUK 2018.236.Site in host: Gills.Prevalence: 83% (25/31).Representative DNA sequence: A partial SSU rDNA sequence (2082 bp) was deposited in the GenBank database under the accession number MH414926.ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [Paramyxidium branchialis is: urn:lsid:zoobank.org:act:2481F7F4-5B9F-42C8-B2EB-FED264FA7283.e (ICZN) , detailsEtymology: Specific name refers to site of infection in the host.Spore. Mature spores oval to bluntly-rounded in sutural view, lemon-shaped in valvular aspect, 10.7–12.3 × 6.6–7.8 (11.6 × 7.3) (L × W) (n = 20). Polar capsules almost spherical, 3.9-4.5 × 3.2-4.2 (4.2 × 3.8) (n = 20), opening sub-terminally. Sutural line sigmoidal and inconspicuous. Fine spore striations visible across both spore valves.c.35–142 μm), and develop in the gills . The clade is also comprised of numerous sequences generated from actinospores that probably represent a hidden diversity of unidentified myxosporeans infecting various tissues from anguillid eels; as the sequences have all been generated from European freshwater systems that lack elopiform fish other than eels and Myxidium-like infections have been noted in numerous tissues from the European eel [Morphological features, such as myxospore size and number of valvular striation, have been used to identify or differentiate between species of the past –10. Howethe past , 4. The the past –7, 14 anpean eel , 30. Howescribed –12, or hpean eel , 32.Paramyxidium group is robustly supported as a sister clade to the Myxidium clade (s.s.) / Chloromyxum grouping that form as fully supported subclades  are described from the renal systems of either pike or salmonids (Protacanthopterygii), whilst the adjacent Chloromyxum sister group are all described from Cypriniformes, when infecting the gall-bladder , being from the urinary tract of an Atheriniformes fish. This suggests that these two clades have evolved from a common ancestor that infected the renal system of fish potentially having a Chloromyxum-like form. The Paramyxidium, as a sister to these two groups, have only been described from the Elopomorpha, with the basal species in the clade again coming from a renal infection, further indicating that the common ancestor for the whole grouping was likely to have been a renal myxosporean. A deeper extension of this phenomenon can also be applied to our tree topology. The node from which the Platysporina clade forms as a sister to the Paramyxidium/Myxidium (s.s.) clade, could also represent a common ancestor that infected the renal system of fish. Recently the Myxobilatidae  have been shown to be phylogenetically placed within the platysporinids [Chloromyxum-form. This outcome is not unexpected, as it has been previously demonstrated that the Chloromyxum morphotype is important in phylogenetic studies and appears to represent a basal morphotype for numerous myxosporean clades [Our phylogenies show that the des Fig. . These tporinids , therefon clades , 23, 34.Myxidium clade (s.s.) currently contains myxosporeans that infect the urinary system of fish from the Protacanthopterygii, a superorder of more basal teleosts, which includes the Salmoniformes  and Esociformes (pikes). The sequence in the tree representing Sphaerospora oncorhynchi Kent, Whitaker & Margolis, 1993 (GenBank: AF201373) is, therefore, more likely to be from a fish that was co-infected with a Myxidium, likely related to the Myxidium sp. described as the CKX myxosporean from coho salmon in Canada [S. oncorhynchi and Myxidium salvelini Konovalov & Shul’man, 1966, in the renal systems from the same Oncorhynchus spp. [Myxidium spp. have been reported from the urinary systems of salmonids: Myxidium minteri Yasutake & Wood, 1957; M. salvelini Konovalov et Shul’man, 1966; Myxidium noblei Konovalov, 1966; Myxidium sp. of Mavor & Strasser, 1918.Certain anomalies do exist in our phylogenetic tree, which can be explained as follows. The n Canada . Indeed,hus spp. , 37 and Zschokkella sp. (GenBank: AJ581918), Chloromyxum schurovi (GenBank: AJ581917) and Myxidium giardi (GenBank: AJ582213)  in some derived species could explain why evolved chloromyxid forms are present in some clades, such as the Myxobilatidae clade (C. schurovi) and the hepatic biliary clade (Chloromyxum trijugum). Indeed, Holzer et. al. [C. schurovi, whilst being genetically similar to members of the clade it occupied (Myxobilatidae), it shares less than 70% identity to other more basal Chloromyxum species and concluded it had evolved further than all other sequenced chloromyxids [C. schurovi with the Myxobilatidae can potentially be explained in terms of DNA sequence, spore morphology and tissue tropism. However, the presence of Myxidium giardi (GenBank: AJ582213) and Zschokkella sp. (GenBank: AJ581918) still cannot, as spores with these morphologies share no synapomorphies with any members of the clade or the predicted ancestral morphotypes, which are assumed to be chloromyxids. One possibility is that the sequences generated for M. giardi and Zschokkella sp. either do not correspond to the myxospores that were observed in the tissue samples, or that they were misidentified. They may represent variants of a sequence for Zschokkella stettinensis Wierzbicka, 1987, described from the urinary bladder of the European eel [Z. stettinensis was a possible identity for this myxosporean [Z. stettinensis look superficially like those of M. giardi but are also very similar to the Neomyxobolus morphotype , being described as spores having two delicate streaks running parallel to the suture [Neomyxobolus spp. [Z. stettinensis or genetically related forms, which are better placed in the genus Neomyxobolus Chen et Hsieh, 1960. Myxidium streisingeri Whipps, Murray & Kent, 2015 also groups in the Myxobilatidae and is another species showing several traits in common with the genus Neomyxobolus [Myxidium. Furthermore, these species also have the tissue location  and spore characteristics to allow placement in the Myxobilatidae.The positioning of the sequences ysporina . This co, Whipps  demonstr of fish . Chlorom et. al. , reporteromyxids . Therefopean eel . Indeed,osporean . The myxe suture , a featulus spp. . Therefoyxobolus , indicatParamyxidium morphologies were observed. However, the Pacific tarpon is known to have other myxosporean parasites [In the present study, coinfections with other myxosporeans were not observed in Icelandic eels, only arasites . Therefoarasites , 43 (MAFParamyxidium giardi n. comb. (syn. Myxidium giardi) does not cause systemic infections in eels. In Iceland there are three species now confirmed, using morphological and molecular data. Additional species probably exist that infect different tissues, such as the skin, and a further member of this clade was identified here from an Aurantiactinomyxon sequence. Therefore, the site of infection in the host eels is an important diagnostic feature for this group (Paramyxidium clade). Myxospore morphology is highly conserved in the Paramyxidium clade, and although some myxospore dimensions are noted as significantly different between isolates, differentiating species based on spore dimension alone is not recommended. Despite very similar myxospore morphologies being present across the group there are relatively low genetic similarities between SSU rDNA sequences. The Paramyxidium clade is a well-supported sister to the Myxidium (s.s.) and Chloromyxum clades, each demonstrating a degree of fish host group specificity.


Bartonella spp., we conducted tick microbiome analysis and determination of the infection status of wild animals, pets and pet owners in the state of Hesse, Germany.Arthropod-borne diseases remain a major health-threat for humans and animals worldwide. To estimate the distribution of pathogenic agents and especially Bartonella spp. were detected in 9.5% of all ticks and in the blood of 17 roe deer. Further data reveal the presence of the human and animal pathogenic species of genera in the family Spirochaetaceae (including Borrelia miyamotoi and Borrelia garinii), Bartonella spp. (mainly Bartonella schoenbuchensis), Rickettsia helvetica, Francisella tularensis and Anaplasma phagocytophilum in ticks. Co-infections with species of several genera were detected in nine ticks. One dog and five dog owners were seropositive for anti-Bartonella henselae-antibodies and one dog had antibodies against Rickettsia conorii.In total, 189 engorged ticks collected from 163 animals were tested. Selected ticks were analyzed by next generation sequencing (NGS) and confirmatory PCRs, blood specimens of 48 wild animals were analyzed by PCR to confirm pathogen presence and sera of 54 dogs, one cat and 11 dog owners were analyzed by serology. Bartonella spp., especially Bartonella henselae, was detected. The high number of various detected pathogens suggests that ticks might serve as an excellent sentinel to detect and monitor zoonotic human pathogens.This study provides a snapshot of pathogens circulating in ticks in central Germany. A broad range of tick-borne pathogens are present in ticks, and especially in wild animals, with possible implications for animal and human health. However, a low incidence of  Globalization and climate change both contribute to the spread of infectious diseases often transmitted by insects and arthropod vectors. The monitoring and control of vector-borne diseases is an important task in public health, consolidating medical, veterinary and environmental research to realize potential health threats and to avert and reduce them .Borrelia spp. and Bartonella spp.  and B. henselae in humans [In ticks, the prevalence of  to ~40%  and, alt to ~40% , ticks alla spp. . The veclla spp.  and by ulla spp. . Severallla spp. , 38, 39,n humans . As co-iBartonella spp. and other pathogens in feeding ticks and blood of pets and wild animals in central Germany  by combining NGS and conventional PCRs for pathogen detection. Moreover, we attempted to detect pathogen-specific antibodies in the serum of pets and their owners.We investigated the presence of Ticks and serum from pets were collected by veterinarians located in the state of Hesse, Germany, and tick and blood samples from wild animals were collected by hunters directly after shooting and by employees of the Landesbetrieb Hessisches Landeslabor, Gießen, Germany. All locations are given in Table Bartonella by the Robert Koch Institute, Berlin, Germany. There was no increase of Bartonella-positive cases during this study; therefore, the possibility of DNA contamination from non-study sources is highly unlikely.The laboratories of the Institute for Medical Microbiology and Infection Control at the University Hospital of the Goethe University in Frankfurt (Germany) undergo a strict and externally reviewed quality control management with all required positive and negative controls  and are appointed as National Consiliary Laboratories for Bartonella spp. were never detected (data not shown). DNA from whole-blood was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and eluted in 200 μl of elution buffer as recommended by the manufacturer.DNA extraction from ticks was conducted as previously described . All ticBartonella spp. in addition to other pathogens. Therefore, all ticks and EDTA-blood were analyzed for Bartonella spp. DNA using two different PCR methods [16S ribosomal DNA (rDNA) nested PCR [B. henselae Houston ATCC 49882, 1 ng) and negative (water) intra-assay controls were always included. DNA was amplified in a Biometra T3000 thermocycler . Products were separated on agarose gels, stained with ethidium bromide and visualized under UV light. PCR products were sequenced by a commercial provider , analyzed using the Chromas software  and compared to Bartonella spp. strains deposited in the NCBI databank using the BLAST online tool for species level identification.In this study, we focused on the detection of  methods : 16S ribsted PCR , 44 usinsted PCR  using thBartonella spp. 16S-rDNA nested PCR and the Bartonella spp. 16S-23S rDNA ITS region PCR, the Rickettsia spp. 23S-5S ITS region [Coxiella burnetii IS1111 region [16S rDNA of Anaplasma spp. [Leptospira spp. LipL32 gene [Borrelia spp. 16S rDNA [ospA gene of Borrelia spp. in ticks and of Francisella tularensis were conducted using LightMix Kits  according to the manufacturer’s instructions. Positive controls (each containing 1 ng of DNA) were the following: Rickettsia helvetica , Coxiella burnetii , Anaplasma phagocytophilum Webster , Leptospira interrogans  and Borrelia miyamotoi HT31 .Whenever potentially pathogenic bacterial species were detected by NGS in the microbiome analysis (see below), subsequent PCRs for species determination were conducted from ticks and from EDTA-blood of the animals, if available. Primers for the detection of the S region , the Cox1 region , 16S rDNsma spp. , the LepL32 gene  and Borr16S rDNA  are giveBartonella ruminant-lineage, a PCR protocol specific for a 406 bp internal fragment of the rpoB gene  was performed [Bartonella rpoB DNA was amplified by using 5 μl of starting material. Positive  and a negative (water) controls were always included. Primers are given in Table Bartonella species-discriminatory nucleotide polymorphism of the 403 bp rpoB fragment, the obtained sequences were aligned to sequences of Bartonella schoenbuchensis , B. capreoli , Bartonella chomelii , B. bovis  and Bartonella melophagi  using Clone Manager Professional Suite 8 . As shown in Fig. rpoB fragment allows species discrimination of B. chomelii, B. bovis and B. melophagi based on multiple nucleotide positions, as well as species discrimination of B. schoenbuchensis and B. capreoli based on a single nucleotide position (position 391). Co-infections with B. schoenbuchensis and B. capreoli were detected by analyzing double peaks at the discriminatory nucleotides.To increase the species-discriminatory power of the rDNA-sequences within the erformed . Bartone16S rRNA gene of each tick was amplified using previously described primers [16S rDNA nested PCR and the 16S-23S-rDNA-ITS region PCR were always run in parallel to the NGS were compared. Furthermore, whenever human pathogens were detected by NGS, all positive results were confirmed by conventional PCR methods (see above).The V4 region of the  primers . Amplifi16S rRNA gene (~ 1.5kb) of selected samples was amplified using the native 16S Barcoding Kit SQK- RAB204 . Library preparation and sequencing were done following the manufacturer’s instructions. Each PCR reaction was performed in a total of 50 μl of reaction solution comrpising 14 μl of nuclease-free water, 10 ng of input DNA, 1 μl of barcode and 25 μl of LongAmp Taq 2X Mastermix . The following PCR protocol was performed: 95 °C for 1 min; 25 cycles at 95 °C for 20 s, 55 °C for 30 s and 65 °C for 2 min, and a final extension step at 72 °C for 5 min. PCR products were purified using 30 μl of AMPure XP DNA beads (Beckman Coulter). Samples were eluted in 10 μl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl. Quantification of the libraries was performed using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific); subsequently they were pooled together, prepared for loading and eventually sequenced on a R9.4.1 FLO-MIN106 flowcell.In brief, the entire fastq files. The microbiome analysis was done following the MiSeq standard operation procedures [MiSeq Software v.2.6 was used to split the sequences by barcode and to generate the ocedures  using Moocedures . Qiime  from Oxford Nanopore Technologies.The paired-end reads were joined and the primer sequences were removed. We filtered for the expected amplicon length and removed reads with ambiguous base calls or with homopolymers longer than eight nucleotides. Duplicate sequences were merged. The unique reads were aligned against the SILVA-bases bacterial reference alignment . NucleotB. henselae-antibodies by indirect immunofluorescence assay (IIFA) using a B. henselae/B. quintana (IgG) kit  according to the manufacturer’s instructions with slight modifications [Bartonella dog/cat IgG antibodies. Signals were evaluated as positive when specific fluorescence was detected at a titer of ≥ 1:64 [All serum samples were screened for the presence of anti-ications . Pet serf ≥ 1:64  for animAnaplasma phagocytophilum , Rickettsia conorii (IIFA) (cut-off: ≥ 1:256), R. rickettsii , Borrelia spp. , Leptospira spp. , Coxiella burnetii  and Francisella tularensis .Whenever potentially pathogenic genera were found in ticks by NGS or PCR, the corresponding animal sera and, if available, sera of the respective pet owners were analyzed for antibodies against the most common pathogenic bacterial species of these genera. Animal sera were sent to the veterinarian diagnostic laboratory Laboklin  for the detection of pet antibodies against Anaplasma phagocytophilum was IIFA , for Rickettsia typhi and Rickettsia rickettsii it was IIFA , for B. burgdorferi (sensu lato) it was ELISA , for Leptospira spp. it was ELISA  and for Coxiella burnetii it was IIFA . Testing for anti-Francisella tularensis antibodies was conducted at the Institut für Mikrobiologie der Bundeswehr, München, Germany by ELISA .Serodiagnostics of human sera was performed in the diagnostic laboratories of the Institute for Medical Microbiology and Infection Control at the University Hospital of the Goethe University in Frankfurt am Main (Germany) under fully certified conditions . The method used for the detection of antibodies against Capreolus capreolus; 39 whole-blood samples and 95 ticks), 6 boar , 1 wisent , 1 raccoon  and one red fox . The number of sampled animals is summarized in Fig. Ixodes ricinus ticks (identified by standard taxonomic keys). Among the 189 ticks, two were nymphs and 6 were male adults. Two tick samples (1 dog and 1 cat) were identified as Rhipicephalus sanguineus.From March to October 2017, 189 engorged ticks from 103 animals were collected at four collection sites in the federal state of Hesse, Germany shown in Fig. Bartonella spp. since there is a broad spectrum of Bartonella spp. that can lead to severe infections in humans and animals [Bartonella spp. were detected in ticks and full blood of roe deer. DNA of two different Bartonella spp. was found in roe deer blood: B. schoenbuchensis in 10 (25.6%) animals, B. capreoli in 4 (10.3%) animals and a co-infection of both Bartonella spp. was detected in the blood of 3 (7.7%) roe deer. Among the roe deer with blood positive for Bartonella spp., nine did not have Bartonella spp.-positive ticks. DNA of Bartonella spp. was found in 9.5% of all ticks. Bartonella schoenbuchensis DNA was detected in 14 ticks  from 10 roe deer, B. capreoli DNA was found in one tick  and B. henselae DNA was detected in three ticks  of three roe deer. Moreover, Bartonella spp. DNA was found in ticks of three animals whose blood contained no Bartonella spp. DNA.In addition to the detection of multiple pathogens by microbiome analysis, we focused on the detection of  animals . BartoneBartonella spp., Anaplasma spp. and Rickettsia spp., all known to be harmful to humans. Thus, we were interested to analyze and identify the microbial composition of blood-fed ticks from wild animals and pets sampled throughout Hesse. For this, a 16S rRNA gene amplicon-sequencing (V4 region) and bioinformatics analysis workflow was established. Overall, 136 ticks sampled from 97 animals were sequenced on the MiSeq Illumina platform, resulting in a minimum sequencing depth of 5000 reads per sample for further analysis. After performing the initial NGS sequencing of 136 ticks we continued tick collection, resulting in the fact that not all of the total collected ticks (n = 189) underwent microbiome analysis.It is widely known that ticks act as vectors for various pathogens including The alpha diversity of ticks sampled from wild animals showed a dominant higher number of operational taxonomic units (OTUs) compared to ticks collected from pets, indicating greater species richness in wild animals Fig. . Here, wMidichloriaceae, a common tick endosymbiont, is represented as the most dominant OTU for pets (~ 80%) and wild animals (~75%)  Fig. a. As seel groups , 59. As 16S rRNA gene using Oxford Nanopore Technology was applied. Three samples revealing a higher percentage of unclassified bacteria (relative abundance > 15%) were selected in addition to two samples indicating the presence of the genus Borrelia. Our results revealed that the previously identified OTU “Bacteria unclassified” was resolved to Spiroplasma ixodetis, another known tick endosymbiont [Borrelia miyamotoi was found in the selected two tick samples formerly by Illumina sequencing and PCR confirmation, and was also identified by Nanopore sequencing to species level.In order to identify bacteria to the species level, full length sequencing of the symbiont . BorreliBartonella spp. was detected in four ticks (5.3%) from three roe deer. Three were confirmed to be B. schoenbuchensis by PCR. DNA of Spirochaetaceae was found in 13 ticks (17.1%) from six roe deer, four dogs and one boar. The presence of Borrelia garinii was confirmed in one tick from a dog and that of Borrelia miyamotoi in one tick from another dog and two ticks from two roe deer. Rickettsia DNA was detected in 15 ticks (19.7%) from 10 roe deer, one raccoon, two dogs and one boar. The presence of Rickettsia helvetica was confirmed in nine of those ticks. Coxiella DNA was detected in three ticks (3.9%) from two dogs and one raccoon. Confirmation PCRs for Coxiella burnetii remained negative so, potentially, those Coxiella spp. detected by NGS and not confirmed in Coxiella burnetii-specific PCRs represent Coxiella-like tick endosymbionts [Francisella tularensis DNA was detected in one female adult Rhipicephalus sanguineus tick from a dog. This result was confirmed by real-time PCR. Anaplasma DNA was found in 15 ticks (19.7%) from nine roe deer, one dog and one boar and Anaplasma phagocytophilum was confirmed to be present in 12 of those ticks. Staphylococcus DNA was detected in eight ticks (10.5%) from five dogs, two boars and one raccoon. Table Confirmation PCRs were conducted for all pathogenic species Table . DNA of ymbionts . Francis ticks 5.% from thAnaplasma phagocytophilum was detected in the blood of eight roe deer.Whenever potentially pathogenic genera were found in the microbiome analysis, animal whole-blood (when available) was also analyzed by PCR. Here, Spirochaetaceae, Anaplasma spp. and Staphylococcus spp. In another tick from a raccoon, Rickettsia spp., Coxiella spp. and Staphylococcus spp. DNA were found. Spirochaetaceae, Bartonella spp. and Anaplasma spp. were detected in three ticks from three roe deer. Spirochaetaceae, Rickettsia spp. and Anaplasma spp. were found in one tick from a roe deer. Furthermore, we detected three ticks from three roe deer with co-infections with two pathogens: one tick was co-infected with Bartonella spp. and Anaplasma spp. DNA, another tick was co-infected with Spirochaetaceae and Anaplasma spp. and the third tick was co-infected with Rickettsia spp. and Anaplasma spp. DNA.Furthermore, co-infections of ticks with various bacteria were also detected. NGS revealed that nine ticks (11.8%) were co-infected with more than one pathogen Table . One ticB. henselae-antibodies (IgG) (titer: 1:640); in the respective tick, no B. henselae DNA was detected. One dog serum was found to be positive for Rickettsia conorii-antibodies (titer: 1:128); in the respective tick, Rickettsia helvetica DNA was detected. For all other dogs, antibodies against Anaplasma phagocytophilum, Borrelia spp., Leptospira spp., Coxiella burnetii, Francisella tularensis and Rickettsia rickettsii were not detected.One of 54 dogs (1.9%) was seropositive for anti-B. henselae-antibodies (IgG) were found . All of the respective dogs, however, were seronegative, and, moreover, the remaining six dog owners did not show antibodies against B. henselae. No antibodies against Anaplasma phagocytophilum, Rickettsia typhi, Rickettsia rickettsii, Borrelia burgdorferi, Francisella tularensis, Coxiella burnetii or Leptospira spp. were found in the sera of the dog owners whose dogs were infested with ticks harboring a particular pathogen’s DNA.In five dog owners, anti-Bartonella spp., Spirochaetaceae, Anaplasma spp., Rickettsia spp., Coxiella spp., Francisella spp. and Staphylococcus spp.) were found in ticks feeding on pets and wild animals in Hesse, Germany, and identified using NGS and classical PCR diagnostics for microbiome profiling.Ticks and tick-borne diseases remain a remarkable health threat for humans and animals in modern days. In this study, seven zoonotic potentially human pathogenic genera  suggesting a similar prevalence of Bartonella spp. in ticks collected at different locations in the state of Hesse, Germany. DNA of the ruminant-associated B. schoenbuchensis and B. capreoli was detected in 7.4% and 0.5% of the ticks collected from roe deer, respectively. Furthermore, DNA of B. schoenbuchensis (25.6%), B. capreoli (10.3%) and DNA from both Bartonella spp. (7.7%) were found in the blood of roe deer. In ticks collected from nine of those Bartonella-positive animals, no Bartonella spp. was detectable indicating that these negative ticks cleared the infection or that there were not enough pathogens in the ticks to be detected by the applied PCRs. The deer ked (Lipoptena cervi) is suspected to be the main vector for B. schoenbuchensis [B. schoenbuchensis, B. capreoli, B. chomelii and B. bovis [Bartonella spp. can cause diseases in their reservoir or accidental human hosts but, in general, Bartonella spp. are known to cause chronic asymptomatic infections in their mammalian reservoir hosts [Bartonella schoenbuchensis is suspected to cause unspecific symptoms like muscle pain and fever in humans as it was isolated from one patient with a history of tick bites in which no other causative agent was detected [Bartonella DNA was detected in ticks collected from three animals whose blood samples were free from Bartonella DNA, indicating that the infection might have been acquired elsewhere. This result is in-line with another study where B. henselae DNA-positive and negative ticks were removed from dogs at the same time, with this observation leading to the hypothesis that the DNA-positive ticks had already acquired the B. henselae infection before feeding on these dogs [By conducting a literature search including 19 published studies, iewed in ). In theuchensis , 63–65 auchensis 5.6%, B. B. bovis –29, 66. ir hosts , 31. Bardetected . Bartoneese dogs .Bartonella spp. in ticks can occur so that even larvae can possibly transmit these pathogens. Bartonella DNA was detected in unfed adult I. persulcatus [Dermacentor variabilis and I. scapularis ticks [I. ricinus adults and nymphs [I. ricinus ticks collected from France [Bartonella spp. are co-infections of animals with multiple tick-borne pathogens. Chomel et al. [B. clarridgeiae endocarditis was not only Bartonella seropositive but also seropositive for Anaplasma phagocytophila which is usually transmitted by ticks [Ehrlichia spp., Babesia canis, Bartonella vinsonii and Rickettsia rickettsii were found in dogs which were heavily exposed to ticks [Bartonella spp. were found to have high antibody titers against several tick-borne pathogens . HoweveBartonella spp., six other potentially pathogenic genera were detected by NGS. Spirochaetaceae (17.1%), Rickettsia spp. (19.7%), Coxiella spp. (3.9%), Francisella spp. (1.3%), Anaplasma spp. (19.7%) and Staphylococcus spp. (10.5%) were also identified in ticks. These results coincide with several studies investigating the microbiome of ticks worldwide. Francisella spp., Coxiella spp., Rickettsia spp. and Shigella spp. were detected in two tick species collected from humans in Turkey [Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp., Wolbachia spp., Mycobacteria spp., Pseudomonas spp., Staphylococcus spp., Acinetobacter spp., Klebsiella spp. and Leptospira spp. were found in ticks removed from domestic animals in Malaysia [Francisella spp., Rickettsia spp. and Bartonella spp. were detected in ticks removed from small rodents [Anaplasma spp., Borrelia spp., Coxiella spp., Ehrlichia spp., Francisella spp. and Rickettsia spp. were found in questing ticks collected by flagging in France [Rickettsia spp., Coxiella spp. and Bacillus spp. [The microbiome analysis presented here reveals that, besides n Turkey  and Coxietaceae 1.1%, Rick rodents , Anaplasia spp. 1.7%, Coxilus spp. . All thelus spp. , 7 and oBorrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Francisella tularensis and Anaplasma phagocytophilum; these results were widely congruent with those from the NGS approach. All of these bacterial species can cause unspecific febrile illnesses in humans and probably also in pets [Anaplasma phagocytophilum was detected in the blood of eight roe deer. Compared to the average prevalence of Borrelia burgdorferi (sensu lato) in questing ticks in Europe , the DNe  are not available.Anti-d donors . In fiveBartonella spp. in animals and their ectoparasites. Members of several pathogenic genera were found in ticks and also in wild and domestic animals by PCR and NGS techniques with Illumina short read and Nanopore long read sequencing, and this indicates a potential infection risk for humans and animals. Even though the number of samples used herein was too small to evaluate the epidemiology of different tick-borne diseases, knowledge of the presence of pathogens in ticks might allow to monitor circulating pathogens that could harm humans and animals. Ectoparasite control and an increased attention toward possible tick-borne infection are crucial to the prevention of (or at least early diagnosis of) tick-borne infections in humans and animals.In summary, our data provide an overview of different pathogens circulating in ticks in central Germany  and suggest a low incidence of


Aspergillus fumigatus is the primary cause ofinvasive aspergillosis. In patients with high-risk conditions, including stem cell andorgan transplant recipients, mortality exceeds 50%. Triazole antifungals have greatlyimproved survival . In 2016, CDCreceived the first TR34/L98H isolate through this passive monitoring system,and an additional four have been identified to date among approximately 2,300 totalisolates received  have beenreported (A. fumigatus colonization are not reportable in any state andfew laboratories perform susceptibility testing for Aspergillusspecies. Four of the seven patients with TR34/L98H were not treated withantifungal therapy following culture; these four isolates, all from sputum orbronchoalveolar lavage, likely reflected A. fumigatus colonizationrather than infection. However, the presence of highly resistant A.fumigatus strains in patient isolates suggests that U.S. cliniciansneed to be aware of the risk for triazole-resistant aspergillosis. Notably, fourpatients had no known exposure to antifungal medications before culture of theresistant isolate, supporting possible environmentally acquired resistance.A.fumigatus can undergo selection for antifungal resistance duringtriazole fungicide exposure in the environment, and spores of resistant strainsmight be transmitted through the air and inhaled, further exploration of triazolefungicide use and presence of triazole-resistant A. fumigatus inthese areas is warranted.The five isolates identified at CDC during 2016–2017 were collected frompatients who did not share health care facilities, procedures, or county ofresidence, arguing against shared health care acquisition. Given that A. fumigatus isolates with the TR34/L98H mutationsidentified in the United States to date, four were collected in Pennsylvania, two inVirginia, and one in California. These three states contributed only 28% of all CDCA. fumigatus isolates collected during 2015–2017,raising the possibility of geographic localization. Second, because isolates werecollected through passive monitoring and not systematic surveillance, caution mustbe exercised when interpreting these findings.The findings in this report are subject to at least two limitations. First, among theseven 34/L98H triazole-resistant A.fumigatus detected in the United States, systematic surveillance,detailed geographic data, and data on triazole fungicide use could be important forassessing the scope of the problem and trends in resistance. Exploration of riskfactors for patient acquisition might provide opportunities to prevent exposure andmitigate risk for invasive infection in susceptible populations. Clinicians andmicrobiologists need to be aware of the possibility of triazole-resistant A.fumigatus infections, even in triazole-naïve patients. Expandedcapacity to test for antifungal susceptibility in A. fumigatuscould help inform clinical and public health decisions.With environmentally derived TRAspergillus fumigatus is the primarycause of invasive aspergillosis. In patients with high-risk conditions,mortality exceeds 50%. A. fumigatus isolates resistant tomedical triazoles have recently been identified in the United States inclinical and environmental specimens. The resistance markerTR34/L98H causes resistance to all triazoles and is associatedwith agricultural and environmental fungicide use.The environmental mold 34/L98H A. fumigatusisolates were identified during 2010–2017 from three states; fourwere collected from patients with no known previous triazole exposure.Seven U.S. clinical TRA. fumigatus can occur in patientsnot previously exposed to these medications.U.S. clinicians and public health personnel should be aware that infectionswith triazole-resistant