Christopher D Lowe
email: clowe@liv.ac.uk

Patrick J Keeling

Phill Laura E Martin

C Watts

David J S Montagnes

Claudio Slamovits

Phillip C Watts

Who is Oxyrrhis marina? Morphological and phylogenetic studies on an unusual dinoflagellate

Keywords: Protist diversity, phylogenetic species, intraspecific variation Protist diversity, phylogenetic species, intraspecific variation

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INTRODUCTION

Oxyrrhis marina is an extensively studied morphospecies (Montagnes et al., this issue-a), exhibiting a wide geographic distribution (Watts et al., this issue). Much study of O. marina has been motivated by the recognition that it possesses unusual cytological and genetic features (e.g. [START_REF] Leander | Early evolutionary history of dinoflagellates and apicomplexans (Alveolata) as inferred from hsp90 and actin phylogenies[END_REF][START_REF] Slamovits | The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both apicomplexan and dinoflagellate mitochondrial genomes[END_REF]; accordingly, O. marina has become a significant target for the study of evolutionary patterns and genome organisation within the Alveolata [START_REF] Slamovits | The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both apicomplexan and dinoflagellate mitochondrial genomes[END_REF][START_REF] Zhang | Spliced leader RNA trans-splicing in dinoflagellates[END_REF]Slamovits and Keeling, this issue). Despite this, the study of variability within the O. marina morphospecies has been ignored.

Recent work indicates that levels of genetic divergence within this taxon may be extensive, which coupled with substantial physiological variation, is potentially sufficient to infer that O. marina represents more than one species (e.g. [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF]. Such diversity is alarming, as researchers around the world continue to isolate strains and conduct experiments on this "species" (Fig. 1). Here, we provide a brief historical guide to the morphological and phylogenetic literature that has defined Oxyrrhis -we highlight why O.

marina is an important model organism, but also indicate that this taxon harbours extensive cryptic diversity, which has remained poorly described. Superficially, O. marina is easily recognised (e.g. [START_REF] Dodge | Marine Dinoflagellates of the British Isles. Her Majesty's Stationary Office[END_REF] and easy to isolate from the natural environment (Lowe et al., this issue); while such characteristics make O. marina simple and practical to study, they also present significant problems. Approximately 40 O. marina isolates are reported in the literature. However, most of these are poorly characterised beyond their gross morphology. Consequently, the bulk of studies are not interpretable in a comparative context, and despite recent evidence of substantial genetic variation [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF], there are limited molecular, physiological, morphological, or ultrastructural data to corroborate such diversity or aid the delineation of potentially multiple species in the genus. This presents a dilemma: O. marina is commonly employed as a 'model' to examine a broad range of ecological, physiological, and behavioural responses (see other papers in this special issue). However, different research groups employing different isolates of O. marina are potentially working on highly divergent strains or even different species. Thus, the context in which comparisons between isolates can be made is difficult to assess. Indeed, a review of the literature since 1950 (Fig. 1a,b; Table 1) indicates that ~160 studies examined various aspects of O. marina biology and reveals that: 1) most studies examine a single strain (74 examined one strain, 64 provided no isolate information, 14 were not traceable by the authors); 2) many isolates are reported only once in the literature (38 isolates are reported, 30 of which are reported once or twice); and 3) most laboratories (research groups) work on only a single strain. As a result, there are few comparative studies, and our ability to resolve potential strain and species differences are limited. The articles in this special issue expand on various aspects of O. marina biology. To place these, and future work, in a taxonomic context, it is essential that we first explore our current understanding of who O. marina is and the extent of diversity in this taxon.

In this paper, two major issues are examined. First, we explore the morphological, cytological, and molecular literature that has contributed to the definition of O. marinahighlighting in particular the historical and contemporary evidence for multiple Oxyrrhis species. In parallel, we review the unusual characteristics possessed by O. marina that have motivated much of the study on this organism and informed its key phylogenetic position within the alveolates. We do not purport to have conducted an exhaustive review of the literature (which is extensive and covers >160 years); instead, we highlight key studies and provide sufficient guidance to allow researchers to further explore the topic. Ultimately, we indicate that while the phylogenetic position of O. marina is now reasonably well established,

MORPHOLOGICAL STUDIES OF OXYRRHIS MARINA

Gross morphology

Oxyrrhis marina Dujardin, 1841 (Fig. 2a, Table 2) was originally described as oblong, oval bodied, with pointed anterior, obliquely notched anteriorly, possessing "several" flagella protruding sideways from the notch centre. Diagnostic features were: colourless, subcylindrical, rough bodied cell, with rounded posterior, 0.05 long (no units, but remarks on magnification of the original figure indicate 44 µm long). The type location was the Mediterranean (likely on the French coast), but, as was typical of protistan studies of the time, no type material was deposited.

The first main revision by Saville-Kent (1880) provided further details (Fig. 2b, Table 2), based on the literature and observations of isolates from Jersey (UK). The revision provided information on: two flagella, one extending and the other coiled within the oral aperture; swimming and feeding behaviour (e.g. the longitudinal flagellum being responsible for trapping prey, while the transverse flagellum pushes it into the oral cavity); division by transverse fission; an anterior contractile vacuole; and, in illustrations, a posterior ventral bulge (or tentacular lobe) within the posterior ventral depression.

Several other older O. marina reviews exist. [START_REF] Senn | Oxyrrhis, Nephroselmis und einige Euflagellaten, nebst Bemerkungen über deren System[END_REF] extensively reviewed the literature and provided new details (Fig. 2c), indicating: no observable contractile vacuole; the flagella insert on either side of the ventral bulge; O. marina was a dinoflagellate, possibly F o r P e e r R e v i e w related to Gymnodinium; and there was only one Oxyrrhis species. [START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF] provided a later review, which included new observations of binary fission (Fig. 2d). However, of the older literature we suggest that [START_REF] Kofoid | The free-living unarmored dinoflagellata[END_REF] provide the best synthesis and most rigorous diagnosis of the genus and species (for veracity, reported in full below); they also supported the notion that there is only one species of Oxyrrhis, placed within the Gymnodinioidae.

Diagnosis of Oxyrrhis

"Body subovoidal, asymmetrically contracted on the left posteriorly; girdle postmedian, incomplete distally, lacking postmargin; sulcus spreading posteroventrally, divided anteriorly by pendant tentacular lobe; transverse flagellum originating to the left and the longitudinal to the right of the lobe; nucleus with beaded chromatin; marine." [START_REF] Kofoid | The free-living unarmored dinoflagellata[END_REF].

Diagnosis of O. marina

"Body elongate oval, asymmetrical posteriorly; girdle imperfect on right side, without a postmargin; flagella midventral; stout tentacle-like lobe pendant between the two flagella, dividing the broad undeveloped ventral sulcus; colourless; length, 10-37 µm; marine." [START_REF] Kofoid | The free-living unarmored dinoflagellata[END_REF].

Three other free-living Oxyrrhis species have been described: O. phaeocysticola [START_REF] Scherffel | Phaeocystis globosa nov. spec., eiuigen Beti-achtungen iiber die Phylogenie niederer, insbesondere brauner Organismen[END_REF]O. tentaculifera Conrad, 1939;and O. maritima van Meel, 1969 (Fig. 3, Table 2). Oxyrrhis phaeocysticola (Fig. 3a) was distinguished as Oxyrrhis-shaped, including possessing a ventral bulge, but its swimming pattern was flagella first, in contrast to O. marina , which swims with the flagella in the posterior (e.g. [START_REF] Scherffel | Phaeocystis globosa nov. spec., eiuigen Beti-achtungen iiber die Phylogenie niederer, insbesondere brauner Organismen[END_REF]. Oxyrrhis maritima (Fig. 3b) and O. tentaculifera (Fig. 3c) were both isolated from Belgian coastal waters. Oxyrrhis maritima was ambiguously distinguished as larger and rounder than O. marina, while O. tentaculifera was defined as possessing a long tentacle (probably a longer version of the ventral bulge indicated above), extending from the notch, but otherwise, it was superficially similar to O. marina. Oxyrrhis phaeocysticola was moved to the genus Hemistasia [START_REF] Elbrächter | Hemistasia phaeocysticola (Scherffel) comb. nov., rediscription of a free-living, marine, phagotrophic kinetoplastid flagellate[END_REF], thus creating the new combination Hemistasia phaeocysticola. Oxyrrhis tentaculifera and O. maritima were synonymised with O. marina by [START_REF] Dodge | Marine Dinoflagellates of the British Isles. Her Majesty's Stationary Office[END_REF], whose reasoning was that as O. marina exhibits considerable morphological variation, these two species were insufficiently different from O. marina. We suggest that the description by [START_REF] Conrad | Notes protistologiques IX sur trois dinoflagellates de l'eau saumatre[END_REF] of O. tenticulifera is sufficiently distinct (particularly the presence of a conspicuous, long tentacle) to stand as a distinct species, though the lack of corroborating observations of this morphotype limits further consideration.

Regardless, the question of multiple Oxyrrhis species has not been revisited until relatively recently (see below); thus the current genus Oxyrrhis contains only the original species, O.

marina.

Other observations of gross morphology are distributed throughout the literature (Fig. 2). For example, isolates of O. marina collected from Venice were illustrated with a ventral bulge and were noted to vary extensively in cell size and shape in response to a range of osmotic conditions (Diskus, 1955;Fig. 2e). Cell shape and size appear to be highly variable in O. marina [START_REF] Triemer | A unique mitotic variation in the marine dinoflagellate Oxyrrhis marina (Pyrrophyta)[END_REF] noted changes in shape following food ingestion; concurrently, in our own experience of culturing large numbers of O. marina isolates, variation in size occurs depending on food concentration and culture status (e.g. [START_REF] Kimmance | Do temperature-food interactions matter? Responses of production and its components in the model heterotrophic flagellate Oxyrrhis marina[END_REF]. Furthermore, our own recent observations on clonal isolates collected across Europe suggest clone-specific variation in cell size (C. Lowe, unpublished data), though whether these differences are systematic and correlated with phylogenetic identity is not yet clear. As noted above, early studies recognised O. marina to be a dinoflagellate, though a somewhat unusual one. Virtually all of the subsequent morphological and ultrastructural work has focused on providing data to characterise O. marina and to assess its affinity within the alveolates. Below, we briefly review the morphology of O. marina, moving from larger to smaller structures, and highlight significant findings.

Oxyrrhis -an unusual dinoflagellate

Cysts

There are two independent descriptions of cyst formation in Oxyrrhis. [START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF] noted thin-membrane covered cysts induced by both excess and lack of food -to our knowledge this is the only report of cyst formation in O. marina. A more recent study indicated that Oxyrrhis sp. formed robust adherent cysts [START_REF] Jonsson | Tidal rhythm of cyst formation in the rock pool ciliate Strombidium oculatum Gruber (Cilophora, Oligotrichida): a description of the functional biology and analysis of the tidal synchronization of encystment[END_REF]. In this work Oxyrrhis sp., from intertidal rock pools of Brittany, France, formed 10 µm spherical cysts that adhered to the substrate ~0.5 h prior to pools being covered by the incoming tide; excystment occurred 18 h later as the tide receded a second time, and Oxyrrhis sp. were then free swimming for 6 h. Though gross morphology, based on scanning electron microscopy, suggested that the cells observed in this study were identical to O. marina, [START_REF] Jonsson | Tidal rhythm of cyst formation in the rock pool ciliate Strombidium oculatum Gruber (Cilophora, Oligotrichida): a description of the functional biology and analysis of the tidal synchronization of encystment[END_REF] conservatively referred to 'Oxyrrhis sp.' as circatidal adherent cysts have not been reported in O. marina.

The ventral bulge or tentacle

This medial, ventral structure is well documented in the earlier literature, and as indicated above, its length was used to diagnose O. tentaculifera. In O. marina it is relatively small (~5 µm), is constricted proximally, and is located below the horizontal ridge (Dodge and Crawford, 1971a, b). See Cortical microtubular arrangement, below for more details of this structure. The structure and function of the O. marina flagellar apparatus is well documented (Dodge and Crawford, 1971;[START_REF] Cachon | Ultrastructure of the flagellar apparatus of Oxyrrhis marina[END_REF][START_REF] Cachon | Nanofilament dependent motility in dinoflagellates[END_REF]Godart et al., 1992;[START_REF] Roberts | The flagellar apparatus of Oxyrrhis marina (Pyrrophyta)[END_REF][START_REF] Roberts | The flagellar apparatus and cytoskeleton of the dinoflagellates -a comparative overview[END_REF] and differs from those of other dinoflagellates, in that the transverse flagellum lacks a broad striated strand (Cachon et al., 1988a,b). Unique features are that the transverse flagellum of O. marina possesses a row of complex mastigonemes, while the longitudinal flagellum possesses simple mastigonemes; both flagella are covered with scales, except at the proximal ends (see Scales below, [START_REF] Clarke | Flagellar scales in Oxyrrhis marina Dujardin[END_REF]Fig. 4a). The flagellar root system has been studied in detail in two strains (from Newport, Rhode Island, and Texas Culture Collection strain LB1974; [START_REF] Roberts | The flagellar apparatus of Oxyrrhis marina (Pyrrophyta)[END_REF]; this and other studies indicate that O. marina differs from other dinoflagellates in several respects, including: the breadth of the posteriorly directed microtubular root; the orientation of connective structures and electron dense core of the ventral microtubular root; and the occurrence of fibres that parallel the flagella [START_REF] Roberts | The flagellar apparatus of Oxyrrhis marina (Pyrrophyta)[END_REF][START_REF] Roberts | The flagellar apparatus and cytoskeleton of the dinoflagellates -a comparative overview[END_REF].

The two flagella of O. marina differ in function, as recognised by early researchers, e.g. Saville Kent (1880) and [START_REF] Senn | Oxyrrhis, Nephroselmis und einige Euflagellaten, nebst Bemerkungen über deren System[END_REF]. Structure and function of the two flagella were rigorously investigated using an isolate from Villefranche-sur-Mer [START_REF] Cachon | Ultrastructure of the flagellar apparatus of Oxyrrhis marina[END_REF]; this study indicated that the waves produced by the longitudinal flagellum are planar and symmetrical, and the transverse flagellum, which is coiled, produces helical waves and is responsible for the cork-screw like swimming motion, propelling the cell forward (see Boakes et al., this issue).

Scales

Oxyrrhis marina is distinguished from other dinoflagellates by possessing flagellar and body scales (Clarke andPennick, 1972, 1976). Scales cover both flagella of two isolates, LB1133/1 (isolated from Långskar, Finland -now culture CCAP1133/5, maintained by the Scales also occurr on the whole cell surface of three isolates: LB1133/2, LB1133/4 (isolated from Bahrain -now CCAP1133/4, maintained by CCAP, Oban), and a sample from Norfolk [START_REF] Clarke | Occurrence of body scales in Oxyrrhis marina Dujardin[END_REF].

Both flagellar and body scales are ellipsoidal or circular spiral plates 0.15-0.175 x 0.2 µm, (Fig. 4d), each having a tight spiral of two to five turns (Fig. 4b,c, Clarke andPennick, 1972, 1976). The scales cover the flagella, although they may be absent close to the cell; they are arranged lengthways around the flagella, forming a helix, with rows overlapping.

On the transverse flagellum the row of mastigonemes runs parallel to a row of scales (Fig. 4b). Imbrication of body scales appear in some (but not all) areas of the cell surface [START_REF] Clarke | Occurrence of body scales in Oxyrrhis marina Dujardin[END_REF].

Trichocysts

As noted in some of the earliest descriptions, the cell surface is rough, with staggered double rows of projections running along the cell length [START_REF] Clarke | Occurrence of body scales in Oxyrrhis marina Dujardin[END_REF]; these are concentrated at the anterior end and appear to be associated with the region where trichocysts abut the cell surface. Dodge and Crawford (1971) suggest that the trichocysts of O. marina are similar in structure to those of other dinoflagellates, though possibly more rigorous study will reveal differences, as more recent detailed studied of extrusomes of other alveolates show unique structures (e.g. [START_REF] Cavalier-Smith | Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF].

Cortical microtubular arrangement

The cortical structure of O. marina is similar to that of other alveolates (see Dodge and Crawford, 1971a); however, its microtubular cytoskeleton differs from that of dinoflagellates (see Roberts et al., 1993 Fig. 17 transverse band associated with the ventral ridge, which may be homologous to the structures underlying the anterior ridge of the cingulum in other dinoflagellates. In contrast, there appears to be no transverse microtubules, homologous to those of the lower region of the cingulum [START_REF] Roberts | The cortical microtubular cytoskeleton of Oxyrrhis marina (Dinophyceae) observed with immunofluorescence and electron-microscopy[END_REF]. The ventral bulge (see above) has been suggested to be a reduced hyposome (i.e. the postior half of the cell below the cingulum; Fig. 5; [START_REF] Brown | The cytoskeletal microtubular system of some naked dinoflagellates[END_REF], but the lack of associated transverse microtubules prevents comparison to the same structures in other dinoflagellates.

Mitotic apparatus, division, and chromatin structure

Dinoflagellates exhibit unusual nuclei, with a range of structural and molecular modifications that distinguish them from the 'typical' eukaryote model of nuclear and chromosomal organisation (Hausmann et al., 2003). For some time, it has been recognised that the nuclear structure of O. marina differs from that of the typical dinoflagellate (e.g. [START_REF] Hall | Binary fission in Oxyrrhis marina Dujardin[END_REF], but see Keeling and Slamovits this issue). Oxyrrhis marina generates an intranuclear mitotic spindle during mitotic cell division, in contrast to the extranuclear spindle of most dinoflagellates [START_REF] Cachon | The nuclear division in Oxyrrhis marina: an example of the role played by the nuclear envelope in chromosome segregation[END_REF]Treimer, 1982;[START_REF] Gao | Nuclear division in the marine dinoflagellate Oxyrrhis marina[END_REF]. As in other dinoflagellates, the nuclear envelope of O. marina persists throughout mitosis.

However, unlike dinoflagellates, plaques (from which the mitotic spindle is generated) appear on the nuclear envelope during prophase [START_REF] Triemer | A unique mitotic variation in the marine dinoflagellate Oxyrrhis marina (Pyrrophyta)[END_REF][START_REF] Gao | Nuclear division in the marine dinoflagellate Oxyrrhis marina[END_REF]. Chromosomal and chromatin structures in dinoflagellates are also atypical of eukaryotes and exhibit birefringent periodic banded or arched structures [START_REF] Cachon | The nuclear division in Oxyrrhis marina: an example of the role played by the nuclear envelope in chromosome segregation[END_REF][START_REF] Triemer | A unique mitotic variation in the marine dinoflagellate Oxyrrhis marina (Pyrrophyta)[END_REF][START_REF] Gao | Nuclear division in the marine dinoflagellate Oxyrrhis marina[END_REF]); these features have not been reported in O. marina, although in other respects its condensed chromosomal structure is dinoflagellate-like. Additionally, the typical eukaryote complement of DNA-associated histones are absent in dinoflagellates, and again O. marina differs from dinoflagellates by possessing a single 23 KDa histone-like DNA associated protein [START_REF] Kato | Isolation of the major basic nuclear protein and its localization on chromosomes of the dinoflagellate, Oxyrrhis marina[END_REF].

Of final note, to our knowledge there are no data to indicate whether O. marina is haploid, diploid, or polyploid. Most dinoflagellates are haploid (Hausmann et al., 2003), although diploidy occurs in some genera (e.g. Noctiluca, [START_REF] Zingmark | Sexual reproduction in the dinoflagellate Noctiluca miliaris Suriray[END_REF][START_REF] Pfiester | Dinoflagellate reproduction[END_REF]Montagnes et al., this issue-b).

Oxyrrhis taxonomy and phylogeny

The above morphological, ultrastructural, and cytological studies provide extensive data to infer the taxonomic position of O. marina relative to other alveolates; indeed, much of the ultrastructural study of O. marina has been stimulated by the recognition that it is a somewhat aberrant dinoflagellate. Two conflicting taxonomic positions have been proposed for O. marina: either basal to (or an early branch of) the dinoflagellate lineage, suggesting an ancestral state; or derived, occurring within the Gonyaulaceae. Several authors (e.g. [START_REF] Cachon | The nuclear division in Oxyrrhis marina: an example of the role played by the nuclear envelope in chromosome segregation[END_REF][START_REF] Taylor | On dinoflagellate evolution[END_REF][START_REF] Loeblich | Dinoflagellate evolution[END_REF][START_REF] Kato | Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina[END_REF], support the basal position for O. marina based on the argument that the flagellar apparatus, reduced sulcus and girdle, cortical microtubular structure, and the apparent intermediate nuclear and chromosomal organisation are all primitive. Contrastingly, other authors (see Cavalier-Smith and Chao, 2004) infer a highly derived position for O. marina, based on rDNA phygenies and the subsequent argument that the presence of histone-like proteins, an intranuclear mitotic spindle, and the reduction of sulcus and cingulum grooves support this derived position (see Cavalier-Smith and Chao, 2004 for their reasoning).

Two striking conclusions arise from a summary of the morphological literature: 1) O. marina is a dinoflagellate, but its position within the group is uncertain; and 2) despite early descriptions of multiple Oxyrrhis species, most studies accept the opinions of Kofoid and Sweezy (1921) and [START_REF] Dodge | Marine Dinoflagellates of the British Isles. Her Majesty's Stationary Office[END_REF] that only the single species O. marina exists. In the next section, we examine molecular phylogenetic data to further consider these two issues.

Molecular phylogenetic studies of Oxyrrhis marina

Genetic data have inevitably been applied to examine the taxonomic and phylogenetic affiliation of O. marina. Molecular genetic studies provide support for both proposed positions of O. marina, though the majority of recent studies support that it is an early branching dinoflagellate, or a close ancestral lineage, branching after perkinsids. The first phylogenetic study to include O. marina [START_REF] Lenaers | A molecular phylogeny of dinoflagellate protists (Pyrrhophyta) inferred from the sequence of 24S ribosomal-RNA divergent domain-D1 and domain-D8[END_REF], assessed phylogenetic relatedness within the dinoflagellates based on sequence data for two divergent domains of the 24S rRNA gene in 12 species. This study supported a basal position, placing O. marina as an early emerging dinoflagellate, preceding the Peridiniales. A subsequent study [START_REF] Saldarriaga | Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage[END_REF], based on SSU rDNA and sequences for actin, alpha-tubulin, and beta-tubulin highlighted the two opposing phylogenetic positions. Phylogenies based on SSU rDNA sequences indicated a derived branching position within the Gonyaulacales but noted that the affiliation should be interpreted cautiously as a result of the highly divergent O. marina rDNA sequence [START_REF] Saldarriaga | Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage[END_REF]. Conversely, in the same study actin, alpha-tubulin, and beta-tubulin genes of O. marina were not noticeably divergent, and in phylogenetic trees based on all three proteins individually and in combination O. marina branched at the base of the dinoflagellate lineage. A study using these genes plus HSP90 achieved similar results [START_REF] Leander | Early evolutionary history of dinoflagellates and apicomplexans (Alveolata) as inferred from hsp90 and actin phylogenies[END_REF]. Furthermore, a recent analysis including an extensive dataset with 30 protein-coding genes strongly supported the basal position of Oxyrrhis [START_REF] Slamovits | The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both apicomplexan and dinoflagellate mitochondrial genomes[END_REF]. Finally, studies of mitochondrial genome structure and RNA editing mechanisms also lend support to an early branching position for O. marina relative to the dinoflagellates; in particular since Oxyrrhis was found to completely [START_REF] Slamovits | The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both apicomplexan and dinoflagellate mitochondrial genomes[END_REF][START_REF] Zhang | mRNA editing and spliced-leader RNA trans-splicing groups Oxyrrhis, Noctiluca, Heterocapsa, and Amphidinium as basal lineages of dinoflagellates[END_REF]. Although some authors (e.g. [START_REF] Cavalier-Smith | Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF] have maintained a derived position for O. marina, this hypothesis has not received further support and in the light of the current wealth of data (i.e. multiple protein phylogenies, mitochondrial genome structure, and RNA editing mechanisms) we assume that the basal position of Oxyrrhis relative to dinoflagellates is the correct interpretation.

Contemporary evidence for cryptic Oxyrrhis species

The above review highlights that the taxonomic and phylogenetic affiliations of O. marina are well described, if not entirely agreed upon. In contrast, our understanding of genetic, physiological, and morphological variability within O. marina is limited. Indeed, while early morphological studies argue for multiple Oxyrrhis species, assessments of variability between different O. marina strains and isolates are rare. Given the increasing number of examples of cryptic diversity in a broad range of free-living protist taxa (e.g. [START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to quaternary climate dynamics[END_REF][START_REF] Slapeta | The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes[END_REF], this lack of study represents an important oversight. In fact, recent studies of O. marina suggest that high levels of genetic diversity occur within the current O. marina morphospecies [START_REF] Cavalier-Smith | Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF][START_REF] Lowe | There are high levels of functional and genetic diversity in Oxyrrhis marina[END_REF][START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF].

In the following section we examine assessments of variability within O. marina, highlight that current observations of morphological and cytological variation are scarce and indicate that genetic studies reveal extensive diversity. Based on the strength of the molecular phylogenetic data, we propose that there are two Oxyrrhis species -O. marina and O. maritima -for which we provide new diagnoses (the existence of a third species, O. tenticulifera, is also discussed below). Ultimately, this re-designation reflects the extent of diversity within the genus and provides an important framework to direct future comparative morphological, physiological, and genetic studies.

Combining morphological and molecular data

Six studies have examined variation between O. marina isolates (Table 1). Of the morphological and cytological studies, only Clarke andPennick (1972, 1976) and [START_REF] Roberts | The flagellar apparatus of Oxyrrhis marina (Pyrrophyta)[END_REF] compared O. marina isolates, based on scales and flagellar structure, respectively, and neither noted variation. The most extensive assessments of diversity within O. marina are phylogenetic, though these too are limited. Three studies have quantified the level of genetic variation between O. marina isolates based on a single gene (rDNA) and a small number of isolates (n = 2, 3, and 11 for [START_REF] Saldarriaga | Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage[END_REF][START_REF] Cavalier-Smith | Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF]Lowe et al., 2005, respectively). These studies indicate: 1) an exceptionally high level of divergence in the basal O. marina branch [START_REF] Saldarriaga | Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage[END_REF] and 2) two divergent lineages that have been proposed as separate species [START_REF] Cavalier-Smith | Protalveolate phylogeny and systematics and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF][START_REF] Lowe | There are high levels of functional and genetic diversity in Oxyrrhis marina[END_REF]. Following this, a recent assessment of diversity within O. marina examined 5.8S ITS rDNA and mitochondrial cytochrome c oxidase I (COI) in 58 O. marina isolates; this work supported two highly divergent lineages, each composed of two distinct clades (Fig. 6; [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF]. Based on the COI gene, sequence divergence between lineages was 10.5 % (within lineage divergence was <1% in both cases). Mitochondrial COI sequences in particular are now commonly used to aid species delineations across a broad range of organisms [START_REF] Hebert | Biological identifications through DNA barcodes[END_REF][START_REF] Sites | Delimiting species: a Renaissance issue in systematic biology[END_REF][START_REF] Frezal | Four years of DNA barcoding: Current advances and prospects[END_REF], with for example, 3-11% divergence (at COI) used to delineate species across a range of protist taxa (e.g. Evans et al., 2007;[START_REF] Chantangsi | Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena[END_REF][START_REF] Gentekaki | High-Level genetic diversity but no population structure inferred from nuclear and mitochondrial markers of the peritrichous ciliate Carchesium polypinum in the Grand River Basin (North America)[END_REF][START_REF] Lin | High-level diversity of dinoflagellates in the natural environment, revealed by assessment of mitochondrial cox1 and cob genes for dinoflagellate DNA barcoding[END_REF]. Comparisons of these divergence estimates strongly support the occurrence of two Oxyrrhis species.

Oxyrrhis marina is more than one species

Based on the molecular evidence detailed above, we propose two Oxyrrhis species: O. A third species, O. tentaculifera, may also occur. As noted, in our opinion the description of O. tentaculifera [START_REF] Conrad | Notes protistologiques IX sur trois dinoflagellates de l'eau saumatre[END_REF] is sufficiently distinct to stand as a separate species -though contemporary observations and DNA sequence data for this species are clearly required to support its existence and assess its precise relationship to the two other Oxyrrhis species. For completeness, and to highlight this species as a subject for future study, a diagnosis of O. tenticulifera is included below.

Amended Diagnoses

Diagnosis of Oxyrrhis

Cell subovoidal, asymmetrical posteriorly; girdle postmedial, not extending to dorsal surface; sulcus spreading posteroventrally; flagella midventral; tentacular lobe occurs between two flagella, dividing the broad undeveloped ventral sulcus; brackish to marine; generally intertidal but occasionally open water.

Diagnosis of Oxyrrhis marina Dujardin, 1841

Length, 20-30 µm, but occasionally twice this size; appears colourless but with pink pigmentation that is apparent in concentrated cultures; tentacular lobe never extends beyond cell posterior; for mitochondrial cytochrome c oxidase I > 97% identity to accession number FJ853710 (Strain CCMP604).

Diagnosis of Oxyrrhis maritima van Meel, 1969

Length, 20-30 µm, but occasionally twice this size; appears colourless but with pink pigmentation that is apparent in concentrated cultures; tentacular lobe never extends beyond cell posterior; brackish to marine; for mitochondrial cytochrome c oxidase I > 97% identity to accession number FJ853706 (strain CCAP1133/5). Diagnosis of Oxyrrhis tentaculifera [START_REF] Conrad | Notes protistologiques IX sur trois dinoflagellates de l'eau saumatre[END_REF] Length, 16-24 µm; cell ~ 2 times as long as wide; compressed dorso-ventrally; tentacular lobe extends beyond cell posterior, used for prey capture and at times adhesion to surfaces; cell colourless. Type location: brackish marsh, Belgium (51º 17' N, 3º 12' E).

Reasoning for diagnosis of three species of Oxyrrhis

High levels of cryptic genetic diversity are now documented for many free-living protist taxa.

Such variety raises important questions -is extensive genetic variation paralleled by functional diversity, and does this need to be accounted for in evaluations of physiological responses and ecological interactions? Clearly, the use of experimentally tractable model organisms, such as O. marina, is an important strategy to address these questions. However, failure to recognise the sources and extent of cryptic variation in these organisms is problematic.

For Oxyrrhis, the designation of two species highlights for future studies that: 1) a more cautious approach must to be taken in selecting and characterising Oxyrrhis isolates for experimental study (i.e. it is inappropriate to report assessments concerning poorly characterised isolates) and 2) comparative studies of multiple isolates are required to assess individual, population, and species level variation in the Oxyrrhis genus. Such recommendations are clearly relevant to all protist species and it should now be exceptionally clear that new species designations should include morphological and genetic data, and where possible examination of multiple isolates to assess variability.

Our reasoning for the designation of lineage i and lineage ii (Fig. 6, Table 3; [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] as O. marina, and O. maritima, respectively, follows (Table 3). As indicated above, there are no morphological data to tie the proposed molecular-based species to the original description of O. marina, nor does type location (i.e. coastal Mediterranean, France) provide a criterion to assign species names (as representatives from all clades occur in the French Mediterranean; [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF]Lowe, unpublished data). The original description of O. maritima as larger and rounder than O. marina offers a potential distinguishing morphological characteristic; however, our observations to date (unpublished) do not suggest a difference in cell size between Oxyrrhis lineages. Furthermore, the small amount of work that recognises ecophysiological differences between isolates [START_REF] Lowe | There are high levels of functional and genetic diversity in Oxyrrhis marina[END_REF] offers no guidance on defining "ecotypes". Therefore, we have chosen to designate species based on the least disruptive classification, using occupied names of junior synonyms. In this respect, there are a range of criteria that suggest that O. marina should be represented by lineage i -it is the most prevalent, has the widest distribution, and has the highest number of confirmed isolates, and therefore changing its name would be most disruptive (Table 3) -the overriding reason, however, is simple: there are only two well-studied [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] isolates of Oxyrhis that are available from commercial culture collections in lineage ii, while there are six, well-studied, commercially available isolates in lineage i. Thus, by assigning the specific epithet maritima to lineage ii, we minimise the need to reassign names to past work and minimise future confusion.

Recommendations for future studies

Our recommendations for species designation are based on phylogenetic data only. Clearly then, there is scope to re-visit, in a comparative context, many morphological studies conducted on single O. marina isolates to better define the extent of diversification within the genus. We suggest that studies of flagellar scales, tentacular structure and size, cyst formation, and potentially flagellar rootlet structure will be fruitful directions for such work.

In addition, the recognition of several species in the genus provides further avenues of research for which these model organisms may be applied. For example, as a novel intermediate taxon at the base of the dinoflagellate lineage, Oxyrrhis is increasingly a target (1955); f. an illustration from a guide to protozoa of Woods Hole (Calkins 1902); g. the two general illustrations presented in Dodge and Crawford (1972a); h. a simple schematic presented in [START_REF] Roberts | The flagellar apparatus of Oxyrrhis marina (Pyrrophyta)[END_REF]; i. a schematic, indicating ultrastructure and microtubules [START_REF] Brown | The cytoskeletal microtubular system of some naked dinoflagellates[END_REF]; j. our own general illustration. All illustrations presented to be associated with the scale bar. nov. [START_REF] Elbrächter | Hemistasia phaeocysticola (Scherffel) comb. nov., rediscription of a free-living, marine, phagotrophic kinetoplastid flagellate[END_REF]; b. O. maritima [START_REF] Van Meel | Etudes hydrobiologique sur les eaux saumatres en Belgique 10 especes de Protists rare ou nouvelle pour la cote Belge[END_REF]c. O. tentaculifera Conrad, 1939. Scale bar applies to all illustrations. , micrographs, anddescriptions in Clark andPennick, 1972, 1976); d. a scale (modified from [START_REF] Clarke | Occurrence of body scales in Oxyrrhis marina Dujardin[END_REF]. The scale bar is associated with b and c, only.

For the size of cells and scales see the text.

F o r P e e r R e v i e w

  it remains poorly characterised. Most critically, we assert that O. marina, sensu lato, actually represents more than one species, for which we provide new diagnoses and a justification for this reclassification. Finally, we indicate that the recognition of extensive diversity within the Oxyrrhis genus provides productive new avenues of research based on this important model organism.

  Culture Centre of Algae and Protozoa, Oban) and LB1133/2 (isolated from Essex, UK).

  for a comparison of cortical microtubular arrangements in O. marina and dinoflagellates). The longitudinal microtubules of O. marina are intact from pole to pole, and do not abut the transverse microtubules; this is in contrast to which the transverse microtubules abut the longitudinal at both the anterior and posterior ridges of the cingulum. Notably, O. marina does have a distinct

  editing, which is found in all other dinoflagellates

  marina and O. maritima (see diagnoses below). Following recommendations by[START_REF] Foissner | Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa), with emphasis on two contrasting environments, the Etosha region and the Namib Desert[END_REF], we have adopted the use of previously employed species names; thus we resurrect the synonymised specific epithet O. maritima to denote the second Oxyrrhis species.

Fig. 1 .

 1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. Illustrations of Oxyrrhis marina over the last 160 years: a. the original description

Fig. 3 .

 3 Fig. 3. Illustrations of the other three species described in the genus Oxyrrhis: a. O.

Fig. 4 .

 4 Fig. 4. Illustrations to indicate the extent and position of scales and mastigonemes on O.

Fig. 5 .

 5 Fig. 5. Schematic illustrations of a. Oxyrrhis marina and b. a generalised athecate

Fig. 6 .

 6 Fig. 6. Cladogram (redrawn from Lowe et al., 2010) of the four Oxyrrhis clades defined

  Table3. Criteria for the re-designation of species names in the genus Oxyrrhis.

Table 1 .

 1 Lowe et al. 2. Oxyrrhis marina taxonomy-phylogeny 30 A summary of studies (post 1950) providing morphological, cytological, ultrastructural, or molecular phylogenetic data concerning Oxyrrhis marina. The O. marina strain/isolate identity and the number of strains/isolates examined in each study are also noted.

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

ACKNOWLEDGEMENTS

The Authors would like to thank Emily Roberts, Michael Steinke, and Naoji Yubuki for constructive comments on the manuscript. We would also like to thank two anonymous reviewers for their suggestions.

FUNDING

This work was, in part, supported by a UK NERC grant (NE/F005237/1) awarded to PCW, CDL, and DJSM.

Study

Strain/Isolate Number of strains Phylogeny/genetic [START_REF] Lenaers | A molecular phylogeny of dinoflagellate protists (Pyrrhophyta) inferred from the sequence of 24S ribosomal-RNA divergent domain-D1 and domain-D8[END_REF] Dinoflagellate phylogeny Villefranche-sur-Mer (France) 1


Lisa Pettersson

Johan Rasmuson

Charlotta Andersson

Clas Ahlm

Magnus Evander
email: magnus.evander@climi.umu.se.

Hantavirus-specific IgA in saliva and viral antigen in the parotid gland in patients with hemorrhagic fever with renal syndrome

Keywords: Puumalavirus, HFRS, antibody, zoonosis, transmission Puumalavirus, HFRS, HCPS, antibody, zoonosis, transmission 22

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

has not been shown hitherto to be infectious [Hardestam et al., 2008b]. The reported 2 possibility of person-to-person transmission with the highly pathogenic ANDV (case-fatality 3 rate up to 40%) raise concerns whether hantaviruses could constitute a risk for further 4 epidemic spread among humans. Consequently, presence of virus in saliva during infection 5 and factors that could enhance or decrease transmission are therefore of utmost interest. secretions amounts to some 5-8g/day in adults [START_REF] Russel | Mucosal immune defense:Immunoglobulin A[END_REF]. In serum, IgA constitutes only 20 10-15% of the total amount of Ig and exists primarily as a monomer. The parotid gland IgA-21 to-IgG ratio is about 500 times increased compared to that in serum and 83-87% of salivary [START_REF] Hardestam | Sensitivity of Andes hantavirus to antiviral effect of 23 human saliva[END_REF] IgA in whole saliva is polymeric IgA [START_REF] Brandtzaeg | Do salivary antibodies reliably reflect both mucosal and systemic immunity?[END_REF]. The secretory IgA serves as an Of the 33 patients, 20 (61%) had PUUV RNA in their saliva and 27 (82%) in their plasma 3 (Table 1). The mean value of viral copy number in PUUV RNA positive samples was 33,091 4 copies/ml in saliva and 113,460 copies/ml in plasma, with a wide range between samples 5 (Table 1). Interestingly, PUUV RNA was detected in saliva (as long as 15 days) and plasma (as 6 long as 20 days) after disease onset (Table 1). As expected, in patients where saliva and 7 plasma were collected consecutively, the RNA-levels decreased with time (Table 1).

8

In patients where plasma and saliva were collected on the same day (for patients with 9 additional samples only one sample pair was evaluated), twelve of 19 sample pairs were 10 PUUV RNA positive in both saliva and plasma, three were negative in both, four were only 11 positive in plasma, and no sample was only positive in saliva (Table 1 andTable 2). There was 12 a significant association between presence of PUUV RNA in saliva and plasma samples, and 13 the RNA levels in plasma were significantly higher in RNA positive than in RNA negative 1) was collected only three days after disease onset and had high viral copy 2 number in the plasma. After two additional days, the patient was positive for IgG in serum. 3 The patient with serum negative for PUUV IgM (no. 28) was positive for PUUV RNA both in 4 plasma and saliva (Table 1).

5

PUUV-specific antibodies in saliva from acute phase 6 PUUV-specific IgA in the saliva was detected in eleven patients at the initial visit (Table 1) 7 and in one additional patient two days later (no. 12, Table 1). In total, twelve of 33 patients 8 with HFRS had salivary IgA and there was a statistically significant inverse association 9 (P=0.009, Mann-Whitney U test) between presence of salivary IgA antibodies and PUUV RNA 10 in the saliva samples (Fig. 1). Of the eleven patients with HFRS with salivary IgA antibodies in 11 their initial sample, seven were negative for PUUV RNA in the saliva (Table 1). In contrast, 16 12 of 20 patients with HFRS who were positive for PUUV RNA in their initial saliva sample had 13 no detectable PUUV-specific salivary IgA. Only five of 33 patients had both PUUV-specific IgA 14 and PUUV RNA in one of their saliva samples (Table 1). In two of these patients, consecutive 15 samples were collected. For patient 5, PUUV RNA in disappeared from the saliva four days 16 later, while patient 12 had no detectable salivary IgA at the initial visit, but IgA was present 17 in the patient's saliva two and four days later concomitant with a decrease and final 18 disappearance of PUUV RNA in the patient's saliva (Table 1). positive for IgG at this time. One patient (no. 13, Table 1) was positive for PUUV IgA in the 2 serum 32 months after the first sample, and two patients (no. 5 and 22, Table 1) were 3 weakly positive for IgA in their serum samples after 29 months and 28 months, respectively. antigen was detected in the capillary endothelium and within mononuclear cells (Fig. 2).

Long-term follow-up of PUUV-specific IgA antibodies in saliva and serum

3

Using real-time RT-PCR, 19 PUUV RNA copies/10,000 cells were demonstrated in the salivary 4 gland sample and 18,000 PUUV RNA copies/ml in the tracheal secretions. Unfortunately, no 5 saliva had been collected from this patient. Parotid samples from two non-hantavirus 6 patients were both negative for nucleocapsid antigen (data not shown). Escherichia coli as antigen in an ELISA [START_REF] Padula | Development and evaluation of a solid-phase enzyme immunoassay 23 based on Andes hantavirus recombinant nucleoprotein[END_REF]. In that report, ANDV-specific 1 salivary IgA was detected in six patients with HCPS up to a month after onset of symptoms 2 [START_REF] Padula | Development and evaluation of a solid-phase enzyme immunoassay 23 based on Andes hantavirus recombinant nucleoprotein[END_REF]. Similarly, the present study found one patient with HFRS who displayed 3 IgA antibodies in the saliva 32 days after disease onset. Furthermore, PUUV RNA was 4 detected in the saliva and plasma from patients with HFRS more than two weeks after 5 disease onset, a finding that is also similar to previous reports on PUUV RNA in plasma 6 [START_REF] Evander | Puumala 7 hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from 8 patients with hemorrhagic fever and renal syndrome[END_REF][START_REF] Saksida | Dobrava virus RNA load in patients who have 8 hemorrhagic fever with renal syndrome[END_REF]. A decreased nucleocapsid-specific serum IgA IgA was detected regardless whether PUUV-RNA was present or not. Complexes in the 1 saliva, including PUUV-specific IgA and PUUV particles, might cause a negative IFA result. In 2 the oral cavity, secretory IgA is known to exist in complex with salivary agglutinin (agglutinin 3 gp-340; [START_REF] Ligtenberg | Binding 19 of salivary agglutinin to IgA[END_REF] and mucin [START_REF] Biesbrock | Interaction of a salivary mucin-secretory immunoglobulin 6 A complex with mucosal pathogens[END_REF][START_REF] Wickstrom | Macromolecular organization of saliva: 12 identification of 'insoluble' MUC5B assemblies and non-mucin proteins in the gel phase[END_REF]

6 between 10 -

 610 Hantavirus genus are rodent borne, zoonotic viruses of the Bunyaviridae 2 family and belong to the group of viral haemorrhagic fevers. Typically, hantaviruses cause 3 haemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary 4 syndrome (HCPS) in the Americas. HFRS symptoms include fever, renal failure, and 5 hemorraghia and HCPS symptoms include fever and severe pulmonary oedema; however, 30% of HFRS patients caused by Puumala virus (PUUV) (genus Hantavirus and 7 family Bunyaviridae) also display symptoms in the lower respiratory tract [Kanerva et al., 8 1996; Linderholm et al., 1993; Linderholm et al., 1997]. The hantaviruses are negative 9 stranded RNA-viruses consisting of spherical, enveloped particles 90 to 120 nm in diameter 10 that contain two glycoproteins (Gn and Gc) and enclose three unique negative-stranded 11 RNAs (L, M, and S) associated with the RNA dependent RNA polymerase and the infection is the result of inhalation of aerosol containing virus shed in 15 rodent excreta (saliva, urine, and faeces). However, in South America several person-to-16 person transmissions with Andes virus (ANDV) have been noted; the highest risk of 17 contracting hantavirus by person-to-person transmission is attributed to very close contact 18 (sexual relations or intimate kissing) with a person infected with a hantavirus [Ferres et al., 19 2007; Martinez et al., 2005]. In a previous study, saliva from patients with HFRS was found to 20 contain PUUV RNA [Pettersson et al., 2008]. Similarly, rodent saliva contains infectious 21 hantavirus [Douron et al., 1984; Hardestam et al., 2008a; Padula et al., 2004; St Jeor, 2004]. 22 These observations indicate that human saliva aerosol or droplets could be a route of 23 -person transmission, yet human saliva from HFRS patients containing PUUV RNA 1

6 7

 6 Whole human saliva may inhibit the infectivity of several different viruses in vitro[Fox et al.,[START_REF] Meisel | Development of novel immunoglobulin G (IgG), IgA, and IgM enzyme immunoassays 10 based on recombinant Puumala and Dobrava hantavirus nucleocapsid proteins[END_REF] 1988;[START_REF] Hartshorn | Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have 4 broad anti-influenza activities and interactions with surfactant protein D that vary according 5 to donor source and sialylation[END_REF]]. In the Hantavirus genus, human saliva inhibited propagation in 9 cell culture for PUUV and Hantaan virus (HTNV), while ANDV was less sensitive to the saliva 10 antiviral effect[START_REF] Hardestam | Sensitivity of Andes hantavirus to antiviral effect of 23 human saliva[END_REF]. Several components of human saliva could be 11 responsible for this inhibition and mucin had some inhibitory effect on HTNV infection in 12 vitro [Hardestam et al., 2008b]. Furthermore, presence of hantavirus-specific salivary 13 antibodies in an infected individual could be important for protection against the disease 14 and transmission. In saliva, Immunoglobulin (Ig) A is the predominant immunoglobulin class: 15 it exists as polymeric IgA with a secretory component bound to the Ig molecules. This 16 secretory IgA has increased resistance against proteolytic degradation. Secretory IgA is 17 produced by plasma cells that are concentrated along mucous cell membrane surfaces and 18 the daily production of IgA is greater than that of any other Ig class. The output in most 19

  of defence on epithelial surfaces and the binding of secretory IgA to 1 pathogens can inhibit adherence of microorganisms to mucosal cells and neutralize viruses. 2 For instance, specific parotid and salivary IgA can neutralize Human immunodeficiency virus 3 type 1 (HIV-1) (family Retroviridae, subfamily Orthoretrovirinae, genus Lentivirus) [Devito et 4 al., 2002; Moja et al., 2000]. 5 Only one study has described salivary IgA antibodies against hantavirus where ANDV-specific 6 IgA was detected in saliva (six patients with acute HCPS) [Padula et al., 2000]. In serum, 7 PUUV-specific IgA was shown to have a neutralizing effect against PUUV [de Carvalho 8 Nicacio et al., 2000]. The present study investigated the presence and importance of salivary 9 IgA in relation to virus in saliva by analysing PUUV-specific IgA and viral RNA in saliva in 10 acutely ill patients with HFRS. In addition, viral RNA and antigen were examined in a salivary 11 gland of a fatal case with HFRS.

14 Patients and sample collection 15 In 21 [

 141521 part of a prospective study of patients with HFRS, saliva, plasma, and serum samples were 16 collected from 33 consecutive patients verified by PUUV specific IgM or real-time RT-PCR at 17 the Division of Infectious Diseases at Umeå University Hospital (Umeå, Sweden). The hospital 18 is situated in the endemic area in northern Sweden. Patients were added to the study from 19 January 2007 to February 2009. The patients were 26-82 years (mean 52 years); 21 were 20 female. The sample collection was performed during the acute phase as previously described Pettersson et al., 2008] and was random with no consideration to time of day or recent 22food intake. For some patients, additional samples were obtained later in the acute phase.

  2010. Twelve patients agreed to participate in the follow-up and samples 2 were collected in the same manner as previously described. Tissue from the parotid salivary 3 gland was sampled at autopsy three days post-mortem from a patient with HFRS who died 4 five days post onset of disease. 5 All experiments were performed in compliance with relevant laws and institutional 6 guidelines and in accordance with the ethical standards of the Declaration of Helsinki. The

7 project 11 ( 9 10 The

 711910 was approved by the Regional Ethics Review Board in Umeå and informed consent 8 was obtained from all patients. 9 Real-time RT-PCR 10 RNA from patient saliva, plasma, or tissue was extracted using a QIAamp® Viral RNA kit Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions. The real-time 12 RT-PCR was performed as previously described[START_REF] Evander | Puumala 7 hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from 8 patients with hemorrhagic fever and renal syndrome[END_REF]. Briefly, the RNA was 13 reverse-transcribed followed by a real-time PCR TaqMan® assay in triplets with PUUV-14 specific primers and probe from the S-segment. Taqman® RNAse P control reagents (Applied 15 Biosystems, Foster City, CA, USA) were used to determine cell numbers in tissue. The real-16 time PCR was performed using an ABI Prism 7900HT Sequence Detection System 2-specific IgM and IgG antibodies in serum from patients with HFRS, IFA was 20 performed as described previously[START_REF] Evander | Puumala 7 hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from 8 patients with hemorrhagic fever and renal syndrome[END_REF]. Briefly, the samples were diluted in 21 PBS (1:16 for IgM and 1:40 for IgG) and then applied onto spot-slide wells in a moist[START_REF] Hardestam | Sensitivity of Andes hantavirus to antiviral effect of 23 human saliva[END_REF] local strain PUUV Umeå/hu[START_REF] Johansson | Complete gene 10 sequence of a human Puumala hantavirus isolate, Puumala Umea/hu: sequence comparison 11 and characterisation of encoded gene products[END_REF] grown in VeroE6 cells 1 as antigen at 37°C over night for IgM analysis and 60 minutes at 20°C for IgG analysis. For IgA 2 analysis, the same antigen was used to detect PUUV-specific IgA antibodies in serum and 3 saliva. The slides were incubated over night with patient saliva (diluted 1:10) and 90 minutes 4 with serum (diluted 1:10 and 1:40) at 37°C and then PUUV-specific IgA antibodies were 5 detected by a polyclonal rabbit anti-human IgA-FITC (F0204, DAKO A/S, Glostrup, Denmark) 6 diluted 1:40 and incubated 60 minutes at 37°C. The salivary IgA results were confirmed by 7 detection of PUUV-specific salivary IgA antibodies using another detecting antibody -the 8 anti-human IgA-FITC (diluted 1:30) (AF010.M, Binding Site ltd, Birmingham, UK). Immunohistochemistry parotid salivary gland was examined for presence of viral antigen using PUUV 11 nucleocapsid protein specific monoclonal antibody (A1C5, Progen Biotechnik GmbH, 12 Heidelberg, Germany). Staining was performed on formalin-fixed paraffin-embedded 4µm-13 sections that were processed for immunohistochemistry using a biotin, streptavidin, and 14 peroxidase technique visualized with diaminobenzidine. Parotid samples from two non-15 hantavirus patients were used as negative controls. 16 Statistical analysis 17 For statistical calculations and graphs, the SPSS software (SPSS, Inc., Chicago, USA) was used. 18 Associations between parameters were evaluated using the Mann-Whitney U test. P 19 values<0.05 were considered statistically significant. RNA in saliva and plasma from acute phase 2

20 To

 20 study the long-term duration of PUUV-specific IgA, patients were invited for a follow-up 21 sampling between 17 months and 32 months after falling ill with HFRS. Twelve patients 22 a follow-up saliva and serum sample. All subjects were negative for IgM and 1

4 7 PUUV

 7 These three patients were all negative for IgM and positive for IgG at follow-up. The 5 remaining patients were negative for IgA in their sera. All patients were negative for IgA in 6 the saliva. No PCR was performed in the follow-up samples. IgA and RNA in serum and saliva in relation to airway symptoms, treatment, and 8 radiological findings 9 In addition to the typical symptoms and clinical signs of HFRS, 22 (67%) patients had 10 respiratory tract symptoms. Perhaps, PUUV RNA and PUUV-specific IgA originated from the 11 lungs through coughing and may be more abundant in individuals where lungs and airways 12 are affected. Seventeen of the 22 HFRS patients with respiratory tract symptoms had 13 dyspnea and 13 were coughing. Eight of the patients were treated with oxygen due to low 14 oxygen-saturation in the blood. A chest x-ray was performed on 12 patients; nine of these 15 were pathological (including infiltrates and/or pleural fluid) and three were normal. 16 However, no statistical significant associations (Mann-Whitney U test) were found between 17 presence of PUUV-specific IgA or PUUV RNA in their saliva or plasma and symptoms, 18 treatment, and radiological findings (data not shown). 19 Presence of PUUV antigen and RNA in a human salivary gland 20 To further investigate whether PUUV RNA present in the saliva could originate from the 21 salivary glands, a parotid salivary gland sample from a patient with HFRS was analysed for 22 RNA and PUUV antigen using a monoclonal antibody (A1C5) specific for 1 the PUUV nucleocapsid protein. Using immunohistochemistry, the PUUV nucleocapsid 2

7 8 DISCUSSION 9 Can 5 [

 7895 hantavirus in human saliva be infectious, and if not, why? In the studied patients with 10 HFRS, both PUUV RNA and nucleocapsid antigen were found in the saliva and salivary gland, 11 respectively. Presence of hantavirus in saliva could potentially promote transmission unless 12 salivary components inhibit this infectivity route. 13 Both PUUV RNA and nucleocapsid antigen were detected in a human parotid gland. The viral 14 antigen was demonstrated in the capillary endothelium and within mononuclear cells; 15 however, no staining was found within the secretory epithelium. The findings indicate that 16 virus found in saliva can be the result of shedding from the salivary gland. Presence of 17 hantavirus antigen in the human salivary glands has not previously been published, but 18 hantavirus antigen in rodent and mouse salivary glands has been demonstrated [Botten et 19 al., 2000; Compton et al., 2004; Lee et al., 1981]. Hantavirus is infectious in rodent saliva and 20 experimental studies with sigmodontine rodents hosting ANDV have investigated the 21 hypothesis that saliva is one of the sources of infection within reservoir populations [Padula 22 ., 2004]. Person-person transmission of ANDV has been suggested to be mediated by 1 close contact, such as having a sexual relations or engaging in intimate kissing with a person 2 infected with hantavirus [Ferres et al., 2007; Martinez et al., 2005]. One of the studies 3 suggests that the unique route of transmission of ANDV is by means of small-particle 4 infectious saliva or respiratory aerosols during the close contact between both persons Martinez et al., 2005] .For other hantaviruses, this type of transmission has not been 6 demonstrated and, except for ANDV, whole human saliva has an inhibitory effect on several 7 hantaviruses in vitro [Hardestam et al., 2009]. Hantavirus-specific antibodies could also play 8 a role in decreasing the possibility for transmission during an infection. 9 In the study, the presence of secretory IgA antibodies in the saliva of a patient with HFRS 10 was significantly inversely associated with the presence of PUUV RNA in saliva, suggesting 11 that the virus could be contagious before appearance of salivary IgA. Increased titers of IgG 12 antibodies in the serum of patients with HFRS coincide with the disappearance of PUUV RNA 13 in the plasma [Evander et al., 2007] and similar kinetics of the virus-antibody relationship 14 may occur in the human oral cavity. For other viruses, it has been shown that secretory IgA 15 protects against infection [Belec et al., 2003; Renegar et al., 2004]. Hantavirus-specific IgA in 16 saliva could potentially provide protection against person-to-person transmission. For HIV-1, 17 specific salivary IgA neutralises infection in vitro and mucosal IgA seems to be more efficient 18 than serum IgA [Moja et al., 2000], which could be explained by the polymeric structure of 19 the mucosal secretory IgA [Hocini et al., 1997]. In addition, the antibody function of 20 secretory IgA is most likely enhanced by the high level of cross-reacting activity detected in 21 human secretions [Ma et al., 1998]. Only one study has described detection of salivary IgA 22 antibodies in hantavirus disease; the study used ANDV nucleoprotein expressed in 23

7 response

 7 in convalescent and late-convalescent-phase sera has been observed in serum 8 from patients with HFRS [de Carvalho Nicacio et al., 2000; Elgh et al., 1998; Groen et al., 9 1994; Meisel et al., 2006; Padula et al., 2004], a finding also similar to the present study; 10 however, serum IgA could be detected in convalescent sera for up to ten years [de Carvalho 11 Nicacio et al., 2000]. The possibility of boosting with hantavirus during the follow-up period 12 cannot be excluded. In the present study, samples from 12 patients with HFRS more than 13 two years after disease were all negative for PUUV-specific IgA in their saliva, while three 14 patients were still IgA positive in sera.

15 16

 15 In the acute phase, all patients but one had serum IgA while the proportion of detectable IgA 17 in saliva was lower. PUUV-specific IgA directed both against hantavirus nucleocapsid and 18 glycoproteins was detected since the antigen in the IFA consisted of PUUV-infected cells. The 19 conjugate used to reveal IgA allowed recognition of all potential molecular forms of PUUV-20 specific IgA antibodies -e.g., monomeric, dimeric, and secretory IgA -but close to 90% of 21 salivary IgA in whole saliva is polymeric IgA[START_REF] Brandtzaeg | Do salivary antibodies reliably reflect both mucosal and systemic immunity?[END_REF]. In most samples, IgA was not 22 detectable in saliva until the PUUV-RNA was cleared, which is in contrast to serum, where 23

4 17 [

 17 and they inhibit infection by aggregation with virus [Habte et al., 2006; Hartshorn et al., 5 2006]. Further studies that optimize hantavirus IgA detection in salivary samples could 6 increase the sensitivity of IgA detection. In the present study, PUUV RNA was detected both 7 in saliva and plasma and virus RNA in saliva was associated with viremia in patients with 8 HFRS, indicating that analysis of oral fluid could be useful for molecular investigation during 9 outbreaks. Saliva is an attractive non-invasive sample for diagnostics and epidemiological 10 studies and oral fluid samples have been shown to be useful for many infectious agents such 11 as the early detection of HIV [Nugent et al., 2009]. Determination of specific viral nucleic 12 acids and antibodies in saliva samples makes this test even more useful for isolated 13 communities and it has been suggested as a sampling technique for some native populations 14 in South America who, due to cultural reasons, do not always accept venepuncture [Padula 15 et al., 2000]. The direct detection and identification of many viruses in saliva by PCR could 16 become a standard method and numerous studies on different viruses have been reported Corstjens and Malamud, 2008]. Detection of hantavirus-specific secretory IgA is non-18 invasive technique and has a promising future as a diagnostic tool. In addition, hantavirus-19 specific secretory IgA could provide insight into the dynamics of hantavirus infection in 20 relation to the host immune response. Here, PUUV-specific salivary IgA was present in saliva 21 of infected patients and appearance of salivary IgA was associated with the disappearance of 22 the virus.

1 2 Figure 1 .Figure 2 .

 212 Figure 1. Inverse association between presence of IgA antibodies and PUUV RNA in saliva

a

  IgA was diluted 1:10 and scored as negative (-), strongly positive (++) or weakly positive (+) by IF.b PUUV-specific PCR on patient 13 was only performed by conventional PCR and scored as positive (pos) or negative.Inverse association between presence of IgA antibodies and PUUV RNA in saliva samples (P = 0.009). Mean saliva PUUV RNA copy number was 30,960 copies/ml in patients without PUUVspecific salivary IgA and 2,046 copies/ml in patients with PUUV-specific salivary IgA. ° Outlier, * Extreme outlier 166x130mm (96 x 96 DPI) in the parotid salivary gland of an HFRS patient. Using PUUV nucleocapsid protein specific monoclonal antibody and immunohistochemistry technique, viral antigen was demonstrated within the capillary endothelium of the organ. Specific punctuate granular staining within the capillary endothelial walls are indicated. 215x159mm (300 x 300 DPI)

  IgM or IgG were found in the sera from all patients at first visit. The only 17 patient with a negative IgA serum (no. 27, Table1) was IgG and IgM positive in the serum18 and IgA negative in the saliva. Interestingly, this patient had been sick for six days and had[START_REF] Devito | Cross-clade HIV-1-specific neutralizing IgA in mucosal 21 and systemic compartments of HIV-1-exposed, persistently seronegative subjects[END_REF] an extremely high viral load in the serum, 1.38 x 10 6 copies/ml. Three patients were only 20 weakly positive for PUUV-specific IgA in serum (no. 20, 28 and 30, Table1). Notably, the only 21 patients with negative IgM and IgG in the serum were found in this group (no. 28 and 20 all three were IgA negative in the saliva. The sample with negative IgG (no.

		respectively) and
		F o r
		P
		e
		e r
			R
	14	saliva (P=0.010, Mann-Whitney U test).	e v i e
			w
	22
			9

15

PUUV-specific antibodies in serum from acute phase 16 PUUV-specific IgA, 1 20, Table

Table 1 .

 1 PUUV-specific IgA and RNA in HFRS patients.

		Day of			Day of
		saliva			plasma
		collection			collection
		(after			(after
	Patient	disease	PUUV IgA in	PUUV RNA	disease	PUUV RNA
	no.	onset)	saliva a	(copies/ml) in saliva	onset)	(copies/ml) in plasma
	1	7	++	0	6	81,330
	2	10	++	0	8	0
	3	12	++	0	12	5,027
	4	9	++	0	8	0
	5	6	++	8,810	8	43,013
		10	++	0	11	3,145
	6	25	++	0	27	0
		32	++	0	32	0
		52	-	0	52	0
	7	10	+	0	10	0
	8	7	+	0	7	9,721
		9	-	0	9	0
		11	-	0	11	0
	9	9	+	9,582	10	3,724
	10	5	+	2,589	5	36,231
	11	5	+	1,530	8	3,044
	12	7	-	31,284	7	73,291
		9	+	8,142	9	11,809
		11	+	0	11	5,142
	13	3	-	pos b	3	pos b
	14	6	-	43,095	6	26,756
	15	11	-	57,965	11	64,809
		15	-	14,090	15	0
	16	12	-	0	12	0
	17	5	-	23,130	7	26,842
	18	5	-	100,800	5	48,741
	19	9	-	15,195	9	110,454
		11	-	0	11	39,176
		13	-	0	13	17,517
		20	-	0	20	1,801
	20	3	-	0	3	149,903
		14	-	0	14	0
	21	8	-	83,100	8	20,516
	22	9	-	32,594	9	31,411
	23	17	-	0	16	0
	24	6	-	121,323	5	959,294
	25	2	-	66,994	2	117,562
	26	5	-	44,898	7	40,427
	27	6	-	17,516	5	1,381,413
	28	5	-	6,372	5	26,626
	29	7	-	3,745	7	189,233
	30	5	-	2,163	4	54,315
	31	6	-	0	6	1,952
	32	6	-	0	7	5,215
	33	2	-	0	3	41,271

ACKNOWLEDGEMENTS 2

Irene Eriksson is greatly acknowledged for her skilled technical assistance. This project was

3 supported by grants from the Kempe Foundation, The Swedish Heart-Lung Foundation, the 4 Swedish Society of Medical Microbiology, the County Councils of Northern Sweden, the 5 County Council of Västerbotten, and the Medical faculty of Umeå University.