Tridacna Ningaloo Penny

Willan

Philippe Borsa
email: philippe.borsa@ird.fr

Cécile Fauvelot

Serge Andréfouët

Tsun-Thai Chai

Hirofumi Kubo

Li-Lian Liu

On the validity of Noah's giant clam Tridacna noae (Röding, 1798) and its synonymy with Ningaloo

Keywords: Genetics, taxonomy, nomenclature, cryptic species, Tridacna maxima

, is an invalid name. We assessed the validity of resurrecting T. noae and designating a neotype for it against the rules of zoological nomenclature and found no flaw in these acts. We then compared the genetic and morphological characters used in the respective diagnoses of T. noae and the newly-described Tridacna ningaloo. No difference was apparent between T. ningaloo and T. noae except, possibly, in mantle ornamentation patterns. In particular, the holotype of T. ningaloo possesses a mitochondrial DNA haplotype identical to T. noae. Thus, the hypothesis that T. ningaloo is a species distinct from T. noae was not supported by clear morphological evidence and it was contradicted by the available genetic evidence.

Tridacna ningaloo should be regarded as a junior synonym of T. noae.

INTRODUCTION

Giant clams (genera Hippopus Lamarck, 1799 andTridacna Bruguière, 1797; Bivalvia: Cardiidae) are among the most remarkable inhabitants of coral reefs: they are large, conspicuous and often colourful; they have raised the attention of early naturalists and they have fascinated adventurers. The conservation of giant clams also raises concern, given their multiple commercial and cultural uses, and their vulnerability to fishing [START_REF] Rosewater | The family Tridacnidae in the Indo-Pacific[END_REF][START_REF] Newman | On the status of giant clams, relics of Tethys (Mollusca: Bivalvia: Tridacninae)[END_REF][START_REF] Bin Othman | The distribution and status of giant clams (family Tridacnidae)a short review[END_REF]. Updated taxonomy and distributions of giant clams are required to assist in their conservation [START_REF] Newman | On the status of giant clams, relics of Tethys (Mollusca: Bivalvia: Tridacninae)[END_REF][START_REF] Bin Othman | The distribution and status of giant clams (family Tridacnidae)a short review[END_REF][START_REF] Borsa | Distribution of Noah's giant clam, Tridacna noae[END_REF]. Giant clam taxonomy has seen a surge in new species descriptions since [START_REF] Rosewater | The family Tridacnidae in the Indo-Pacific[END_REF] revision of the group. Recently-described new species in the genus Tridacna include T. rosewateri [START_REF] Sirenko | Tridacna rosewateri sp. n. a new species of giant clam from Indian Ocean (Bivalvia: Tridacnidae)[END_REF], T. tevoroa Lucas, Ledua & Braley, 1990, and T. costata Roa-Quiaoit, Kochzius, Jantzen, Al-Zibdah & Richter, 2008[START_REF] Richter | Collapse of a new living species of giant clam in the Red Sea[END_REF]. The latter two species were subsequently shown to be junior synonyms of species described decades before, respectively, T. mbalavuana [START_REF] Ladd | Geology of Vitilevu, Fiji[END_REF]and T. squamosina Sturany, 1899[START_REF] Newman | On the status of giant clams, relics of Tethys (Mollusca: Bivalvia: Tridacninae)[END_REF][START_REF] Huber | Tridacna (Chametrachea) costata Roa-Quiaoit, Kochzius, Jantzen, Al-Zibdah & Richter from the Red Sea, a junior synonym of Tridacna squamosina Sturany, 1899 (Bivalvia, Tridacnidae)[END_REF]. Another species, Noah's giant clam T. noae [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF], has been recently resurrected from synonymy with the small giant clam, T. maxima [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF] after it was found to be reproductively isolated from the latter [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. The name T. noae was chosen upon comparing the shell characteristics of specimens from Japan and Taiwan with the historic drawings [START_REF] Chemnitz | Neues systematiches Conchylien-Cabinet, siebender Band[END_REF] plate 49, fig. 494) on which the initial description of T. noae was based [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. An additional giant clam species, T. ningaloo Penny & Willan, 2014 was published recently. In the same paper, Penny & Willan (2014: p. 202) suggested that T. noae might be an invalid species: "We are aware that some workers [START_REF] Kubo | On two sympatric species within Tridacna "maxima[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]) recognise an additional species from the tropical northwestern Pacific Ocean, but the characters by which this species is defined morphologically, as well as its taxonomy and the nomenclature, are too controversial for us to accept it as valid at present".

Tridacna noae under its present taxonomic definition [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] has a large Indo-Pacific distribution (Fig. 1), which includes Ningaloo Reef, Western Australia as indicated by mitochondrial DNA sequences [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF][START_REF] Borsa | Distribution of Noah's giant clam, Tridacna noae[END_REF]. Ningaloo Reef is also the type locality of T. ningaloo, which its authors [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] initially identified as a cryptic species distinct from T. maxima based on the very same set of sequences as [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF]. [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF] Tridacna sp. specimens were identified as T. noae by [START_REF] Borsa | Distribution of Noah's giant clam, Tridacna noae[END_REF]. There is no mitochondrial DNA evidence in [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF] samples from Ningaloo Reef (15 individuals labelled "Tridacna sp.") of material other than T. noae.

This paper addresses two issues of interest to giant clam taxonomists, conservation biologists, and reef ecologists. The first one concerns the validity of the name T. noae, i.e. whether this name should be kept to designate the cryptic species morphologically close to T. maxima that has initially been discovered in Japan and Taiwan [START_REF] Kubo | On two sympatric species within Tridacna "maxima[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. The second issue is whether T. ningaloo is taxonomically distinct from T. noae. We compared T. ningaloo with T. noae on the basis of their diagnoses in, respectively, [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] and [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. Diagnostic or partly-diagnostic characters include DNA sequences, shell morphology, and mantle ornamentation. Nucleotide sequences at the 16S ribosomal RNA (16S) and the cytochrome oxidase subunit 1 gene (CO1) loci used for the comparison of T. noae with other species in the genus Tridacna are listed in table 1 of [START_REF] Borsa | Distribution of Noah's giant clam, Tridacna noae[END_REF]. At the time of writing, T. ningaloo sequences, including those of the holotype, are not yet accessible from GenBank (http://www.ncbi.nlm.nih.gov/) or from related public sequence databases. However, we note from figure 5 of [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] that the concatenated (16S + CO1) nucleotide sequence of T. ningaloo's holotype is identical to the concatenated (16S + CO1) nucleotide sequence of individual no. ET918 [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF]. This individual, itself from Ningaloo Reef, is represented by GenBank sequences JX974878 and JX974908 at loci 16S and CO1, respectively. The complete mitochondrial DNA sequence of Acanthocardia tuberculata [START_REF] Linnaeus | Systema naturae per regna tria naturae, secundum classes, ordines, species, cum characteribus, differentiis, synonymis, locis. Tomus I, editio decima, reformata[END_REF] (Bivalvia: Cardiidae) (GenBank DQ632743), which is phylogenetically close to Tridacna spp. [START_REF] Plazzi | Towards a molecular phylogeny of mollusks: Bivalves' early evolution as revealed by mitochondrial genes[END_REF] was chosen as reference for numbering nucleotides at the CO1 locus. The sequences were aligned visually under BIOEDIT [START_REF] Hall | BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The variable nucleotide sites were then highlighted using MEGA 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF].

METHODS

For

RESULTS

Röding's (1798) mention of T. noae (under "Tridachnes … T. Noae. Die Vater Noahmuschel") and his explicit reference to figure no. 494 of Chemnitz ( 1784) satisfy the requirement of availability expressed in Article 12.1 of the Code. To further assess the validity of the name T. noae, we consulted all the articles and books cited in the References section of the present paper and searched for potential senior synonyms to T. noae. We are not aware of such species descriptions earlier than [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF]. If it were the case, the name associated with such a description would now be considered nomen oblitum and T. noae be maintained, because it has been used widely since it has been formally fixed by P.F. Röding [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF][START_REF] Mclean | A revision of the pelecypod family Tridacnidae[END_REF][START_REF] Kubo | On two sympatric species within Tridacna "maxima[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]Borsa et al., 2015, and references therein). The requirement of Article 23.1 of the Code is thus fulfilled. [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF] did not explicitly designate a holotype for T. noae and neither is there mention of the existence of one in [START_REF] Rosewater | The family Tridacnidae in the Indo-Pacific[END_REF] revision.

The designation of a neotype was justified because the original description of T. noae is insufficient to distinguish it with certainty from T. maxima [START_REF] Rosewater | The family Tridacnidae in the Indo-Pacific[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] explicitly stated: "Since no type material is extant and no types have been designated to date, a neotype of Tridacna noae is hereby designated" thus satisfying Condition 75.3.1 of the Code. They provided a list of characters allowing the partial (their table 4 and figure 6) or complete (their figures 2-5 and 7) distinction of T. noae from T. maxima and from all other extant Tridacna species (their table 3 and figures 2-5), as well as reference to previous work allowing the recognition of T. noae as an entity genetically and morphologically distinct, and reproductively isolated from T. maxima; this satisfies Condition 75.3.2 of the Code. Condition 75.3.3 of the Code was met by the provision of the precise length and height of the neotype, along with a series of pictures [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]; their figures 6G-6L) representing the two valves of its shell under different angles; we here add that the neotype of T. noae is numbered "13" in table 2 and figures 2, 3 of [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] and marked "13" with waterproof ink on the interior of its two valves [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]their figures 6H and 6K). Condition 75.3.4 of the Code was addressed through searching the relevant taxonomic literature [START_REF] Chemnitz | Neues systematiches Conchylien-Cabinet, siebender Band[END_REF][START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF][START_REF] Mclean | A revision of the pelecypod family Tridacnidae[END_REF][START_REF] Rosewater | The family Tridacnidae in the Indo-Pacific[END_REF]. The authors further wrote that "the shells of the [cryptic] species were consistent with the figure referred to by Röding for the species he named 'noae'", thus satisfying Condition 75.3.5 of the Code. There is no mention of a type locality by [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF] or any indication from Chemnitz (1784) on the geographical origin of the specimen he examined, making Condition 75.3.6 inapplicable in the present case. Last, [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] wrote that "the neotype specimen … is deposited in the National Museum of Natural Science, Taiwan, with catalogue number NMNS-6928-001", thus fulfilling Condition 75.3.7 of the Code. The type-locality of T. noae is the locality where the neotype was collected, which is Naliao, Taiwan [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF].

Table 1 presents the nucleotide sites of the partial CO1 gene sequence that distinguish T. noae from all other Tridacna species currently considered as valid with the exception of T. mbalavuana, T. rosewateri and T. squamosina, for which no CO1 gene sequences were available. Fifteen such sites were identified across a fragment 399 base-pairs (bp) long. Sequence GenBank JX974908 (T. ningaloo), which is 390 bp long and spans 14 of the 15 diagnostic nucleotides sites, unambiguously identifies it as T. noae (Table 1).

At locus 16S, T. noae also possesses nucleotide G at site 268 (numbered starting from the first nucleotide of GenBank KC456036, which is that of T. noae's neotype) whereas all other Tridacna spp. except, perhaps, T. rosewateri for which no sequence is yet available, have A at this site. T. ningaloo 16S sequence GenBank JX974878 does not span this site. In summary, at locus CO1 we found no nucleotide that would distinguish T. ningaloo from T. noae, as represented by the three sequences listed in Table 1; neither did we find diagnostic differences at locus 16S, where T. ningaloo is represented by sequence GenBank JX974878 and T. noae by homologous sequences GenBank KC456036, KC456040, KC456041 and DQ119339. Actually, the partial 16S sequence of T. ningaloo was identical to that of T. noae's neotype. Table 2 compares the morphological characters highlighted in the diagnostic features of T. noae relative to T. maxima [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]their table 4) with the homologous characters used in the diagnosis of T. ningaloo (see [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF]. No morphological character distinguished T. ningaloo from T. noae as redescribed by [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF], except uncertainty concerning mantle colour patterns. The pictures of live T. ningaloo presented along the original description of the species [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] have poor resolution. The authors of T. ningaloo, S.S. Penny and R.C. Willan, kindly allowed us to examine higher-resolution pictures of T. ningaloo's holotype and paratype. Neither specimen seemed to exhibit finely whitish-delineated ocellate spots on the mantle's margin, an important feature in T. noae's re-description [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. Nevertheless, such individuals with finely whitish-delineated ocellate spots are present in Coral Bay, which is part of the Ningaloo Reef complex (Fig. 2). Other characters mentioned in the diagnosis of T. ningaloo include the following: "posterior end of valves bluntly subtruncate; termination of interdigitating processes sharply triangular; angle of intersection of hinge plate and posterior ridge less than 120°; up to 8 vertical plications on interior of posterior margin of valve; hinge less than half length of shell with ligament terminating before end of valve; cardinal tooth rounded; anterior lateral teeth short; byssal notch deep; posterior pedal retractor muscle almost equal in size to adductor muscle" [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF]. Not one of these characters was used in the redescription of T. noae [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. However, the foregoing features are all present in either the drawing of Chemnitz's (1784: fig. 494) specimen subsequently referred to as T. noae [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF] or in the pictures of T. noae's neotype [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF].

DISCUSSION

Since it has been formally described by Röding (1798), T. noae has been used as a valid species name by a number of authors, until recently. The morphology of the shell being not fully diagnostic between T. maxima and T. noae [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF], T. noae could have been considered a nomen dubium (International Commission on Zoological Nomenclature, 1999: p. xxvii). This is no longer the case, because these doubts have been lifted by the fixation of a neotype. We here examined the circumstances under which [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] resurrected T. noae and we found no flaw in this nomenclatural act.

Defining species as separately evolving metapopulation lineages [START_REF] De Queiroz | Species concepts and species delimitation[END_REF], one expects to observe either reproductive isolation, or consistent genetic differences, or substantial morphological differences between sister species, at a degree depending on whether separation is complete or only incipient [START_REF] De Queiroz | Species concepts and species delimitation[END_REF]. There is no necessity to solely rely on morphological characters in the description or in the diagnosis of a species [START_REF] Cook | Need morphology always be required for new species descriptions?[END_REF]. Tridacna noae as redescribed recently [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] includes a genetic diagnosis. Large genetic differences between T. maxima and T. noae (17-26% nucleotide divergence at the CO1 locus; [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] point to millions of years of separate evolution since their last common ancestor. Unviable hybrid embryos indicate that the two species are reproductively isolated [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. Notwithstanding genetic evidence, [START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] have invoked controversy in the morphological definition of T. noae. We agree that shell morphological characters may not be sufficient for an unambiguous diagnosis of T. noae relative to T. maxima, but mantle ornamentation patterns so seem to be, at least in Japan and Taiwan [START_REF] Kubo | On two sympatric species within Tridacna "maxima[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF]. Above all, mitochondrial sequences provide a profusion of nucleotide substitutions allowing unambiguous diagnosis of T. noae relative to all other extant Tridacna species [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF].

Conversely, we found no difference among the genetic and morphological characters on which the diagnoses of T. noae and T. ningaloo are based, except, possibly, in mantle ornamentation patterns. In the diagnosis of T. ningaloo, no mention is made of the "oval patches with different colours bounded by white margins" [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] that are thought to be characteristic of T. noae [START_REF] Kubo | On two sympatric species within Tridacna "maxima[END_REF][START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF][START_REF] Borsa | Distribution of Noah's giant clam, Tridacna noae[END_REF]. However, giant clams with typical T. noae mantle patterns do occur at Ningaloo Reef.

Two alternative hypotheses are proposed and discussed in the following. The first hypothesis is that T. ningaloo is a species distinct from T. noae. It is not yet clear whether the apparent lack of thinly whitishcontoured, ocellated spots on the mantle's edge is a feature solely of the specimens chosen as type material, or of all individuals of T. ningaloo. It cannot be excluded that the apparent lack of such spots in the type material of T. ningaloo be the extreme of a continuum. Not only was there no genetic difference between T. noae and T. ningaloo based on the mitochondrial DNA marker, but the fact that the mitochondrial haplotype of some T. ningaloo specimens was identical to those sampled from T. noae would indicate very recent or current genetic exchange between the two species through introgressive hybridization. This in turn would imply that the two species cross-breed, in fact challenging the hypothesis of distinct biological species. They would have cross-bred to the point of erasing any genuine T. ningaloo mitochondrial haplotypes, as no mitochondrial sequence other than those characteristic of known Tridacna species including T. noae has been reported from Ningaloo Reef so far [START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF][START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF]. Thus, morphological evidence does not clearly separate T. ningaloo from T. noae, while the available genetic evidence contradicts the hypothesis of T. ningaloo being a species distinct from T. noae. In summary, based on the available evidence, this first hypothesis should be rejected. The second, alternative hypothesis is that the giant clam described as T. ningaloo is a local population of T. noae, implying that the two names are synonymous. The principles of priority and nomenclatural stability expressed in the Code then command that T. noae be the only name maintained. Table 1. Nucleotide substitutions at the cytochrome oxidase enzyme subunit 1 (CO1) locus that distinguish Tridacna noae [START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF]

-G C G G C C C G A G G C C C T. noae Taiwan KC456023 b C G C G G C C C G A G G C C C T. noae Taiwan DQ168140 C G C G G C C C G A G G C C C T. noae Molucca Sea KF446463 C G C G G C C C G A G G C C C T.

  examining the availability of the name T. noae and the validity of resurrecting it, we referred to the relevant articles of the International Code of Zoological Nomenclature, 4th edition (International Commission on Zoological Nomenclature, 1999), hereafter abbreviated as the Code. Relevant parts of the Code are Chapter 4 on criterias of availability, including Article 12 on names published before 1931, Chapter 6 on the validity of names and nomenclatural acts, including Article 23 on the principle of priority; and Chapter 16 on types in the species group, including Article 75 on neotypes and Article 76 on type locality.

Fig. 1 .

 1 Fig. 1. Geographic distributions of Tridacna noae (Röding, 1798): (A) after Su et al. (2014); (B) updated by Borsa et al. (2015); and T. ningaloo Penny & Willan, 2014: (C) after Penny & Willan (2014). Squares: localities of T. noae specimens sampled by Su etal. (2014); circles: T. noae records compiled by Borsa et al. (2015).

Fig. 2 .

 2 Fig. 2. Tridacna noae (Röding, 1798) on the reef in Coral Bay, Western Australia, 23°09'S 113°47'E, 14 August 2008 (photographed by: Tsun-Thai Chai).

  from other species in the genus Tridacna Bruguière, 1797. n.a. not acknowledged.

	Species	Sample locality	GenBank	Nucleotide site no. c
			accession no.
				1	2 2 2 2	2 2	2	3 3 3	4	4	4	5
				9	0 1 4 5	6 7	8	4 7 9	2	4	5	7
				5	7 3 0 8	4 9	2	8 5 0	0	7	6	1
	T. crocea	Indo-Malay region EU341379	T	A T A T T A T A T A C A T A
	T. derasa	n.a.	GQ166591	.	T .	.	A .	G .	T .	.	T G .	.
	T. gigas	Samar, Philippines KJ202113	.	T .	.	A .	.	G .	C .	.	G G .
	T. maxima	Taiwan	DQ155301	.	T .	.	C .	.	.	T .	.	.	.	.	.
	T. ningaloo	Ningaloo Reef	JX974908 a

Table 2 .

 2 numbering of nucleotide sites starts at the first nucleotide of the CO1 gene in the Cardiidae Acanthocardia tuberculata (GenBank DQ632743)[START_REF] Dreyer | The complete sequences and gene organisation of the mitochondrial genomes of the heterodont bivalves Acanthocardia tuberculata and Hiatella arctica and the first record for a putative ATPase subunit 8 gene in marine bivalves[END_REF]; the fragment considered in the comparison was comprised between nucleotide sites nos. 187 and 585 of the gene; dot nucleotide identical to homologous nucleotide in GenBank EU341379 (T. crocea); dash no data Diagnostic morphological characters listed for Tridacna noae[START_REF] Röding | Pars secunda continens conchylia sive testacea univalvia, bivalvia & multivalvia[END_REF] [START_REF] Su | Tridacna noae (Röding, 1798)a valid giant clam species separated from T. maxima (Röding, 1798) by morphological and genetic data[END_REF] and comparison with homologous characters listed for T. ningaloo[START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF] (from Penny & Willan, 2014).

	squamosa	Indo-Malay region EU346364	.	T .	.	A .	.	.	.	.	.	.	.	.	.

a sequence of Tridacna sp. individual ET918 of

[START_REF] Huelsken | A novel widespread cryptic species and phylogeographic patterns within several giant clam species (Cardiidae: Tridacna) from the Indo-Pacific Ocean[END_REF]

identical to that of T. ningaloo's holotype

[START_REF] Penny | Description of a new species of giant clam (Bivalvia: Tridacnidae) from Ningaloo Reef, Western Australia[END_REF]

; their figure

5

) b neotype

(Su et al., 2014) c

ACKNOWLEDGEMENTS

We are grateful to S.S. Penny and R.C. Willan for sharing some of their pictures of T. ningaloo and for exchanging viewpoints; and to N. Malchus and three anonymous reviewers for helpful comments on a previous version of the paper. Scans of old books and articles were obtained from the Animal Base database (http://www.animalbase.org/), the Biodiversity Heritage Library


Rim Touhami
email: rim.touhami@gmail.com

Patrice Buche
email: buche@supagro.inra.fr

Juliette Dibie
email: juliette.dibie@agroparistech.fr

Liliana Ibanescu
email: liliana.ibanescu@agroparistech.fr

Ontology Evolution for Experimental Data in Food

Keywords:

Throughout its life cycle, an ontology may change in order to adapt to domain changes or to new usages. This paper presents an ontology evolution activity [1] applied to an ontology dedicated to the annotation of experimental data in food [2], and a plug-in, DynarOnto, which assists ontology engineers for carrying out the ontology changes. Our evolution method is an a priori method which takes as input an ontology in a consistent state, implements the changes selected to be applied and manages all the consequences of those changes by producing an ontology in a consistent state.

Introduction

Ontologies are one of the fundamental layer of the Semantic Web and are designed to represent the knowledge from a domain in terms of concepts (or classes), relations between these concepts and instances of these concepts [START_REF] Guarino | What is an ontology[END_REF]. An ontology, defined as a formal, explicit specification of a shared conceptualisation [START_REF] Studer | Knowledge engineering: Principles and methods[END_REF] may change whenever the domain changes or when domain experts need to add or to restructure the knowledge. When an ontology is used as a system component (the knowledge backbone) of an advanced information system its evolution is a complex process and raise many challenges as, for example: the formal representation of ontology changes, the verification of ontology consistency after applying the ontology changes, and the propagation of those changes to the ontology related entities (e.g. underlying data sets) (see [START_REF] Zablith | Ontology evolution: a process-centric survey[END_REF] for a complete and detailed overview of the current research activities in ontology evolution).

In [START_REF] Buche | Fuzzy web data tables integration guided by an ontological and terminological resource[END_REF] we presented a complete system, called ONDINE (ONtology-based Data INtEgration), designed to extract experimental data from tables and store them into a data warehouse with the purpose of enriching incomplete local data sources and to allow afterword a flexible querying of the knowledge base. The backbone of ONDINE system is an ontology which was first designed for predictive microbiology in food [START_REF] Buche | Flexible querying of web data to simulate bacterial growth in food[END_REF]. Later a new version of the ontology was designed for the assessment of chemical risk in food. Experimental results of ONDINE system in those two domains were presented in [START_REF] Buche | Fuzzy web data tables integration guided by an ontological and terminological resource[END_REF]. When ONDINE system was used for the MAP'OPT project in the domain of food packaging, its ontology had to be adapted to this new domain, and we defined in [START_REF] Touhami | An Ontological and Terminological Resource for n-ary Relation Annotation in Web Data Tables[END_REF] an Ontological and Terminological Resource (OTR), called naRyQ, dedicated to represent an experiment involving a studied object, some control parameters and a result. During this project, ontology engineers had to manage naRyQ changes. Based on the methodological guidelines given in [START_REF] Palma | Ontology evolution[END_REF], we propose the workflow given in Figure 1 for carrying out the ontology evolution activity, then we combined, adapted and extended existing approaches in ontology evolution [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF][START_REF] Jaziri | Approach and tool to evolve ontology and maintain its coherence[END_REF][START_REF] Tissaoui | EvOnto -joint evolution of ontologies and semantic annotations[END_REF][START_REF] Haase | Consistent evolution of owl ontologies[END_REF][START_REF] Djedidi | Approche d'évolution d'ontologie guidée par des patrons de gestion de changement[END_REF][START_REF] Khattak | Change management in evolving web ontologies[END_REF], and finally we implemented a plug-in, called DynarOnto, to fit naRyQ evolution needs. Our paper is organized as follows. We first briefly recall the structure of the OTR dedicated to the annotation of experimental data in food and present our definition of its coherence in Section 2. In Section 3, the evolution process is detailed, and we present in Section 4 the implementation and evaluation of our plug-in designed to assist ontology engineering in the evolution activity.

naRyQ model and its coherence

In this section, we first recall naRyQ model presented in [START_REF] Touhami | An Ontological and Terminological Resource for n-ary Relation Annotation in Web Data Tables[END_REF] then we define the coherence constraints it must respect.

naRyQ model naRyQ (n-ary

Relations between Quantitative experimental data) is designed to represent experiments in order to annotate data tables representing scientific experiments results in a given domain (see [START_REF] Touhami | An Ontological and Terminological Resource for n-ary Relation Annotation in Web Data Tables[END_REF] for more details). As recommended by W3C [START_REF] Noy | Defining n-ary relations on the semantic web[END_REF] experiments which involve a studied object, several experimental parameters and a result are represented using n-ary relations without distinguished arguments. More precisely "pattern 1" is used and it consists in representing a n-ary relation thanks to a concept associated with its arguments via properties.

Example 1. Let us consider the experiment where the permeability, which is the experiment's result, of a given packaging, which is the studied object, is studied in a set of control parameters specified by the packaging thickness, the temperature and the differential partial pressure. This experiment with 6 arguments can be represented by a n-ary relation Permeability Relation as given in Figure 2. The conceptual component of naRyQ is composed of a core ontology to represent n-ary relations between experimental data and a domain ontology to represent specific concepts of a given application domain. Figure 3 gives an excerpt of naRyQ in the domain of risk in food packaging. The representation of n-ary relations between experimental data requires a particular focus on the management of quantities. In the up core ontology, generic concepts Relation Concept and Argument represent respectively n-ary relations and arguments. In the down core ontology, generic concepts Dimension, UM Concept, Unit Concept and Quantity allows the management of quantities and their associated units of measure. The sub-concepts of the generic concept Symbolic Concept represent the non numerical arguments of n-ary relations between experimental data. The domain ontology contains specific concepts of a given application domain. They appear in naRyQ as sub concepts of the generic concepts of the core ontology. All concepts are represented as OWL classes, hierarchically organized by the subsumption relation subClassOf and pairwise disjoints.

The terminological component of naRyQ contains the set of terms describing the studied domain and are used to annotate data tables. Sub concepts of the generic concepts Relation Concept, Symbolic Concept and Quantity, as well as instances of the generic concept Unit Concept, are all denoted by at least one term of the terminological component. Each of those sub concepts or instances are, in a given language, denoted by a preferred label and optionally by a set of alternative labels, which correspond to synonyms or abbreviations. Labels are associated with a concept or an instance thanks to SKOS labeling properties3 recommended by W3C. For instance, in Figure 3, English terms Ethylene vinyl alcohol and EVOH denote the symbolic concept Ethylene Vinyl Alcohol.

naRyQ CC-coherence

In [START_REF] Haase | Consistent evolution of owl ontologies[END_REF] the coherence of an ontology is classified in three categories: i) structural coherence which is related to constraints of the ontology's representation languages, ii) logical coherence where the semantic correctness of the ontology's entities are checked and iii) user-defined coherence which refers to specific user requirements and constraints related to the ontology's context of use. Inspired by [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF] we define a set of conditions that the ontology must respect for each category of coherence. These conditions are called Coherence Constraints, denoted by CC-constraints. An ontology is CC-coherent if it respects a set of defined CC-constraints. We briefly present below the CC-constraints for naRyQ.

Structural CC-coherence We have modeled naRyQ using a subset of OWL2-DL entities (i.e. classes, properties, constructors of classes, etc.) and axioms (see the technical report [START_REF] Touhami | Definition of naRyQ OTR CC-Constraints and kits of changes[END_REF] for more details).

To define structural coherence associated to the conceptual part of naRyQ, the constraint defined by the W3C group, which says that every OWL axiom must be well defined 4 was extended to associate it with each axiom and constructor used in the modeling of naRyQ: if an entity refers to an other entity, this latest must be defined in the OTR. We defined 15 structural CC-constraints, denoted by CCs. We present below two examples of CCs.

-CS1-Each anonymous class defined by a value restriction, owl:allValues-From or owl:someValueFrom links a pair property-class or property-data type. The property and the class used in the definition of the anonymous class must be defined in the OTR. -CS2-Each instance of skos:concept must have at least a preferred label.

Logical CC-coherence We defined 6 logical CC-constraints, denoted by CCl, which take into account the subset of OWL constructors and axioms used to model naRyQ. Those constraints are taken from the literature [START_REF] Djedidi | Approche d'évolution d'ontologie guidée par des patrons de gestion de changement[END_REF]. We present below two examples of CCl.

-CL1-A class can not be disjoint with its superclass.

-CL2-If two values restrictions owl:allValuesFrom which connect a pair property-concept are associated with the same concept c, then the concepts defining the restrictions cannot be disjoint.

User-defined CC-coherence The user-defined CC-constraints, denoted by CCu, correspond either to quality criteria modeling [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF] or to specific criteria corresponding to the modeled task. We defined 9 generic CCu which refer to quality criteria modeling and 20 new CCu which are specific to the annotation task, detailed in [START_REF] Touhami | Definition of naRyQ OTR CC-Constraints and kits of changes[END_REF]. We present below two examples of CCu.

-CU1-A n-ary relation has at least two arguments.

-CU2-Each quantity must be associated with its units of measurement (at least one) through the owl:allValuesFrom restriction and the property hasUnitConcept. The units of measurement are defined by enumeration using the owl:oneOf constructor.

Coherent evolution of naRyQ

As suggested in [START_REF] Palma | Ontology evolution[END_REF] our ontology evolution activity consists in applying changes to an ontology while preserving its CC-coherence, i.e. respecting all its CCconstraints presented in the previous section (i.e. structural, logical and userdefined constraints). Figure 1 presents the evolution process composed of three main steps. The first step consists in presenting all the possible changes for naRyQ evolution to the ontology engineer. From this list of changes, the ontology engineer chooses the ones to be applied. The second step consists in preserving the CC-coherence of naRyQ during its evolution. To do this, an additional set of changes is added automatically to the requested changes in order to maintain a priori (i.e. before the application of the requested changes) the CC-coherence of the OTR. In the third and last step, requested and additional changes are applied to the OTR. In the following, we present the two first steps.

Change representation

This first step consist in presenting to the ontology engineer all the possible changes for naRyQ. In order to generate all possible changes we first identified all entities and axioms used to model naRyQ in OWL2-DL and SKOS. We selected 55 changes from the literature and defined 26 new changes to take into account the specificity of naRyQ. The complete list is available in [START_REF] Touhami | Definition of naRyQ OTR CC-Constraints and kits of changes[END_REF]. Among these changes, some changes are not accessible to ontology engineer but are rather used in the kits of changes, presented in the following section.

Selection of a change that maintains CC-coherence

After applying a change, one or more CC-constraint may be violated (e.g. adding a new concept which represents a n-ary relation violates CU1 constraint presented in Section 2.2). Hence, a second step is necessary to restore the CCcoherence of the OTR while applying a change. To achieve this goal, we adapted the notions of kit of changes [START_REF] Jaziri | Approach and tool to evolve ontology and maintain its coherence[END_REF] and of strategy [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF] to our needs.

Kit of changes

To maintain a priori the CC-coherence of naRyQ during its evolution, we associated a kit of changes with each change that violates one or more CC-constraints and wich is accessible to ontology engineer. Its definition takes into account all the CC-constraints which can be violated by the requested changes. preconditions: a set of assertions which must be true in order to apply c to O; the change c; mandatory additional changes: a set of changes which are attached to c in order to correct the inconsistencies that may occur in O when c is applied to it; optional additional changes: a set of changes which can be applied in addition to mandatory additional changes. post-conditions: a set of assertions which must be true after the application of c to O.

Two examples of kits of changes are presented in [START_REF] Touhami | Definition of naRyQ OTR CC-Constraints and kits of changes[END_REF] and 15 kits from 63 were implemented. Definition 2. The addClass change allows a new class to be added to the OTR. The kit of changes associated with the addClass change is defined in the following: Because of the constraint "An ontology should not contain two concepts with the same URI", the precondition states that a new class newc can be added to the set of concepts C of naRyQ if its URI doesn't already exist in C. The requested change, which is the creation of the new class newc, is then applied to naRyQ. Unfortunatly, the application of this change violates several CC-constraints of the OTR. The application of a set of mandatory additional changes is then required:

1. The additional changes addSkosConcept and addPrefLabel are triggered to resolve the CC-incoherence linked to the terminological part of naRyQ due to the constraint "Each n-ary relation and each argument is associated with a terminological part".

The creation of a disjunction relationship between the new class and all its

sibling classes resolves a second CC-incoherence due to the contraint which requires that "n-ary relations and their arguments should be mutually disjoint". 3. If the new class newc is a n-ary relation, then at least two arguments must be associated with newc. Additional changes addAllValuesFromRestrictionToClass or addSomeValuesFromRestrictionToClass or addHasValueRestrictionToClass are applied. 4. If the new class newc is a n-ary relation, then exact (or greater than or equal to 1) cardinality restrictions must be associated with the mandatory arguments of newc to ensure their existence at the instance level. 5. If the new class newc is a quantity, then a dimension and a unit of measurement must be associated with it. 6. Adding a specialization relationship between newc and its superclass c resolves the CC-incoherence due to the constraint "Each concept must be connected by a relation of specialization to at least one other named concept.

Besides these mandatory additional changes, it is possible to apply other optional additional changes which are not necessary to maintain the CC-coherence of naRyQ but which may be requested by the ontology engineer. These optional additional changes may be for instance: adding other prefered and alternative labels to the new class, adding other arguments to the new class if it is a n-ary relation or other units of measurement if it is a quantity.

It is important to notice that each mandatory or optional additional change can in turn cause other CC-incoherences which can be resolved by triggering the required kits of changes. So each additional change can also call a kit of changes.

Evolution strategies The kit of changes defined in Definition 2 can be used when there is only one possible solution to solve an incoherence. But sometimes there can be several possible solutions to correct violated constraints. In this case, we have to use evolution strategies [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF] in order to represent the different alternatives.

Definition 3. Given a CC-coherent ontology O and a change c, if the application of c to O leads to several possible alternatives to maintain the CC-coherence of O, then we call evolution strategy the choice made between these alternatives.

There exist two types of kits of changes: those with evolution strategies and those without. The kit of changes associated with the change addClass, presented above, is an example of a kit without evolution strategies. Kits with evolution strategies can be considered as a variant of the first type since they need to fix the strategy (i.e. the choice) before applying the kit.

Example 2. In order to delete a concept, there are several solutions to deal with its sub concepts and there are also several solutions to deal with its terms:

-To deal with orphaned concepts (sub concepts of deleted concept), the possible solutions are i) delete them, 2) reconnect them to super concepts or 3) reconnect them to the root. -To deal with terms of deleted concept, the possible solutions are: 1) delete them or 2) reconnect them to super concepts.

4 Implementation and evaluation

Implementation

We implemented the ontology evolution activity presented above as a Protégé plug-in called DynarOnto. Figure 4 shows the different designed menus:

-The Relation Changes menu presents the changes that can be applied to a n-ary relation (e.g. add a n-ary relation, delete a n-ary relation, update the arguments of a n-ary relation, etc.). -The Argument Changes menu presents the changes that can be applied on arguments of a n-ary relation (i.e. Quantity and Symbolic Concept).

-Evolution Parameters menu helps the ontology engineer to define its evolution strategy that will be taken into account during the OTR evolution.

The screen shot presented in Figure 4 is the interface for the kit of changes associated with the change addClass presented in Definition 2. Let us consider that we want to add the new n-ary relation CO2Permeability Relation, which is a sub relation of the existing n-ary relation Permeability Relation. CO2Permeability Relation is defined by the following arguments: Packaging, Thickness, Temperature, Partial pressure difference, Relative humidity and CO2 permeability. Arguments presented in bold are specific to the sub nary relations. The other arguments are inherited from its super n-ary relation Permeability Relation (see Figure 2). To add this new n-ary relation, ontology engineer starts by clicking on Add relation of the Relation changes menu. Using the panel of the displayed interface, the ontology engineer enters the name of the new n-ary relation, CO2 permeability Relation. Then, he chooses Permeability Relation as his parent class in the hierarchy of n-ay relations of the OTR displayed by clicking on "Choose" button. By confirming the choose of the nary relation's parent, inherited arguments appears in panel 3 of the displayed interface. The ontology engineer can both specialize inherited arguments and add new ones by indicating their numbers in panel 2. To define the new n-ary relation CO2 permeability Relation, ontology engineer should add the new argument Relative humidity and specialize the inherited argument Permeability in CO2 permeability, using the panel 2. To associate terminology to the new n-ary relation, ontology engineer uses panel 4. Finally, when the ontology engineer clicks on "OK" button to validate the add of CO2 permeability Relation, DynarOnto plugin checks that the associated preconditions are verified (e.g. CO2 permeability Relation does not exist in the OTR). The interface facilitates the verification of some preconditions. For instance, when the ontology engineer decides to specialize an argument, the plugin displays only the hierarchy of more specific concepts. Once the preconditions are checked, the plugin applies the requested change and the set of additional mandatory changes, allowing violated CC-constraints to be resolved.

Evaluation of DynArOnto plugin

DynArOnto was evaluated in an incremental way by ten users, with different backgrounds.Three evaluation sessions were organized, DynArOnto interface being improved after each session. Users were asked to apply three changes (add, update n-ary relations and delete arguments). A questionnaire was created to collect their evaluations. Most participants affirm that using DynArOnto to manage the evolution is easier than using Protégé. It is also reflected in the time spent by users to apply the changes: the add relation change (resp. update/suppress change) was done with Protégé in an average of 42 minutes (resp. 11 minutes). Using DynArOnto, the duration was reduced by 20% (resp. 27%). Finally, Dy-nArOnto helped the users to manage the evolution of an OTR dedicated to the annotation of experimental data while guaranteeing its CC-coherence, which was not the case in Protégé where the users were not guided and make some errors (e.g. incorrect use of OWL restrictions used to link arguments to n-ary relations, forgetting to associate terms to concepts, etc.).

Related work

In [START_REF] Zablith | Ontology evolution: a process-centric survey[END_REF], a recent overview of the current research activities in ontology evolution, authors give first the different definitions of ontology evolution, then they present and discuss the various process models that were proposed for the ontology evolution tasks and afterword they propose a unified ontology evolution cycle thus providing a unique overview over several research fields. Our evolution method is defined as in the NeOn Glossary of ontology engineering tasks [START_REF] Palma | Ontology evolution[END_REF] which states that ontology evolution is "the activity of facilitating the modification of an ontology by preserving its consistency" and this is a narrower view than in [START_REF] Zablith | Ontology evolution: a process-centric survey[END_REF].

To maintain the coherence of an ontology during its evolution, the authors of [START_REF] Haase | Consistent evolution of owl ontologies[END_REF][START_REF] Djedidi | Approche d'évolution d'ontologie guidée par des patrons de gestion de changement[END_REF][START_REF] Khattak | Change management in evolving web ontologies[END_REF] have proposed a posteriori approaches. This type of approach allows the application of changes in order to evaluate the evolution impact on the ontology, then suggests how to repair inconsistencies in case of problems. To avoid backtracking after modification, resulting in a loss of time and resources, a priori approaches, where the coherence checking is made before the application of changes, have been proposed in the literature [START_REF] Jaziri | Approach and tool to evolve ontology and maintain its coherence[END_REF][START_REF] Tissaoui | EvOnto -joint evolution of ontologies and semantic annotations[END_REF][START_REF] Mahfoudh | Algebraic graph transformations for formalizing ontology changes and evolving ontologies[END_REF]. The work of Stojanovic [START_REF] Stojanovic | Methods and Tools for Ontology Evolution[END_REF] is the first to propose an ontology evolution process defined for KAON ontologies and using strategies for the task of managing changes. In [START_REF] Jaziri | Approach and tool to evolve ontology and maintain its coherence[END_REF] authors define kits of changes in order to manage the inconsistencies generated by each change. The authors of [START_REF] Tissaoui | EvOnto -joint evolution of ontologies and semantic annotations[END_REF] proposes a system of evolution of an OTR dedicated to the semantic annotation of text documents. Nevertheless their approach maintains the coherence of the OTR and the semantic annotations without defining explicitly the type of coherence which is considered. In [START_REF] Jaziri | Approach and tool to evolve ontology and maintain its coherence[END_REF], a preventive approach that manages the inconsistencies generated by each change is described. A set of rules that must be maintained during the evolution of an ontology is defined but, as in [START_REF] Tissaoui | EvOnto -joint evolution of ontologies and semantic annotations[END_REF], the authors don't define explicitly the type of coherence which is considered. More recently, the authors of [START_REF] Mahfoudh | Algebraic graph transformations for formalizing ontology changes and evolving ontologies[END_REF] proposed a framework based on graph rewriting rules that maintains a set of constraints. However, their approach takes into account only one way to manage the inconsistencies after applying changes (e.g. the remove of a class results automatically in removing all its instances). To the best of our knowledge, our preventive approach is the first one which is based on a clear definition of ontology coherence and simultaneously permits to manage evolution of an ontology involving quantitative data.

Conclusion

We have proposed in this paper an ontology evolution activity for an OTR dedicated to the annotation of experimental data which preserves its CC-coherence.

To do this, we first identified all the necessary constraints (i.e CCs, CCl and CCu) to be checked in order to avoid possible inconsistencies. Secondly, we identified all the required changes for the evolution of naRyQ. Then, we identified all the CC-incoherences that can occur after the application of each change. In order to solve these CC-incoherences, we defined a kit of changes for each change that violates one or more CCs-constraints. A kit of changes allows the CC-coherence of naRyQ to be preserved a priori by checking a set of preconditions, by applying a set of additional changes and/or by using evolution strategies. The originality of our method is to propose an evolution of an OTR containing inter dependent concepts to manage quantitative data. Further work is to explore how to propagate changes to all the ontology related artifacts: individuals, mapping, applications, metadata.

Fig. 1 .

 1 Fig. 1. The ontology evolution activity

Fig. 2 .

 2 Fig. 2. n-ary relation Permeability Relation.

Fig. 3 .

 3 Fig. 3. An excerpt of naRyQ in the domain of risk in food packaging.

Definition 1 .

 1 Given a CC-coherent ontology O and a change c. If the application of c to O doesn't maintain the CC-coherence of O, then a kit of changes is associated with c. It is composed of:

Fig. 4 .

 4 Fig. 4. Screen shot of the interface to add a new n-ary relation

Table 1 .

 1 Table 1 contains a subset of changes required for the evolution of naRyQ, where changes in bold are new ones compared to the literature. A subset of change operations for naRyQ

	Change	Element	Role
	addClass	NamedClass	add an OWL class to an ontology
	addSubClassOf	SubClass	add a subsumption link
	updateDomain	Domain	update the domain of a relation
	updateSomeValuesFromRestriction SomeValuesFrom	update an existential restriction
	addDataTypeRestriction	DataTypeRestriction	add a value range
	deleteFromAllDisjointClasse	DisjointClasses	delete a class of a set of disjoint
			classes
	updatePrefLabel	SkosPrefLabelAssertion update a preferred label of a
			SKOS concept

http://www.w3.org/TR/skos-reference/

http://www.w3.org/TR/owl-ref/#OWLDL

Acknowledgements

This work has been supported by the French Research Agency (ANR) in the framework of ALIA/MAP'OPT project, by the Carnot Institute 3BCAR in the framework of the IC2ACV project and by the Labex NUMEV.


Marc Le Vee

Elodie Jouan

Gregory Noel

Bruno Stieger

Olivier Fardel
email: olivier.fardel@univ-rennes1.fr

Polarized location of SLC and ABC drug transporters in monolayer

Keywords: Canalicular secretion, Drug transporter, Hepatocytes, Polarization, Primary culture

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

Introduction

Liver transporters, belonging either to the solute carrier (SLC) transporter family or to the ATP-binding cassette (ABC) transporter family, are now well-recognized as major actors of hepatic drug clearance [START_REF] Giacomini | Membrane transporters in drug development[END_REF]. Indeed, they are expressed either at the sinusoidal pole of hepatocytes, where they mediate uptake of drugs from blood into hepatocytes, i.e., the so-called phase 0 of the hepatic detoxifying system, or at the canalicular pole of hepatocytes, where they secrete drugs or drug metabolites into the bile, i.e., the socalled phase 3a of the hepatic detoxifying system [START_REF] Funk | The role of hepatic transporters in drug elimination[END_REF]. Additionally, some sinusoidal ABC transporters can secrete drug metabolites back into the blood, i.e., the so-called phase 3b, for a secondary renal elimination [START_REF] Pfeifer | Role of hepatic efflux transporters in regulating systemic and hepatocyte exposure to xenobiotics[END_REF].

Activity of hepatic transporters as well as their expression can be regulated by various drugs [START_REF] Jigorel | Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes[END_REF][START_REF] Klaassen | Xenobiotic, bile acid, and cholesterol transporters: function and regulation[END_REF], which can result in clinicallysignificant drug-drug interactions through altered hepatic elimination of the co-administrated drugs handled by the targeted transporters [START_REF] Konig | Transporters and drug-drug interactions: important determinants of drug disposition and effects[END_REF]. Other xenobiotics such as environmental pollutants and some physiological factors, including hormones and inflammatory cytokines, can also impair hepatic drug transporter expression and activity [START_REF] Fardel | Regulation of human hepatic drug transporter expression by pro-inflammatory cytokines[END_REF][START_REF] Fardel | Regulation of biliary drug efflux pump expression by hormones and xenobiotics[END_REF][START_REF] Klaassen | Regulation of hepatic transporters by xenobiotic receptors[END_REF]. Identifying putative interactions of drugs, chemical pollutants or endogenous substances with hepatic drug transporters and characterizing the cellular and molecular mechanisms involved in such interactions are therefore likely important issues that have to be addressed, notably during the development of new molecular entities in pharmaceutical companies, as recently recommended by drug regulatory agencies [START_REF] Giacomini | Transporters in drug development and clinical pharmacology[END_REF][START_REF] Prueksaritanont | Drug-drug interaction studies: regulatory guidance and an industry perspective[END_REF].

For performing such studies on hepatic drug transporter activity and expression, primary cultures of hepatocytes represent an in vitro choice model [START_REF] Brouwer | In vitro methods to support transporter evaluation in drug discovery and development[END_REF][START_REF] Ramboer | Primary hepatocyte cultures as prominent in vitro tools to study hepatic drug transporters[END_REF]. For rodent hepatocytes, such cultures are usually performed under a sandwich configuration, i.e., hepatocytes are plated on collagen-coated dishes and overlaid with collagen or matrigel [START_REF] Lecluyse | Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration[END_REF], which permits to recover a polarized status of hepatocytes and functional bile canaliculi networks [START_REF] Swift | Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity[END_REF]. By contrast, rodent hepatocytes cultivated in a conventional monolayer configuration, i.e., hepatocytes are plated on plastic or collagen-coated dishes, failed to exhibit bile canaliculi-like structures and are therefore not convenient for studying canalicular drug secretion [START_REF] Luttringer | Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes[END_REF][START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF]. In analogy to sandwich-cultured rodent hepatocytes, sandwich-cultured human hepatocytes are thought to represent the gold standard for in vitro investigating human hepatic transporters [START_REF] Bi | In vitro evaluation of hepatic transporter-mediated clinical drug-drug interactions: hepatocyte model optimization and retrospective investigation[END_REF][START_REF] De Bruyn | Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity[END_REF]. It is however noteworthy that monolayercultured human hepatocytes also display notable activity of various drug transporters [START_REF] Jigorel | Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes[END_REF][START_REF] Payen | Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes[END_REF] and that absolute quantification of sinusoidal and canalicular transporters through a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method revealed no major difference between sandwich-and monolayercultured human hepatocytes [START_REF] Schaefer | Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDPglucuronosyltransferases in cultured primary human hepatocytes[END_REF]. Moreover, primary human hepatocytes cultivated in a monolayer configuration retain regulatory ways of transporter expression and have been consequently used for analyzing the effects of drugs or physiological effectors such as cytokines or growth factors on drug transporter levels and activities [START_REF] Jigorel | Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes[END_REF][START_REF] Vee | Down-regulation of organic anion transporter expression in human hepatocytes exposed to the proinflammatory cytokine interleukin 1beta[END_REF]Le Vee et al., 2009a;[START_REF] Richert | Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes[END_REF]. Whether such monolayer-cultured human hepatocytes exhibit polarized expression of drug transporters, and therefore may closely mimic the in vivo situation, remains however yet largely unknown, but is rather important to determine in order to fully assess the relevance of monolayer-cultured human hepatocytes as a valuable in vitro model to investigate hepatic drug transport. The present study was therefore designed to get insights about this point, using a combined experimental approach, based on mRNA quantification, immunofluorescence labeling and canalicular transport assays, and very similar to that recently used for characterizing drug transporter location in highly differentiated human hepatoma HepaRG cells (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF].

Materials and methods

Chemicals

Rhodamine 123, verapamil and probenecid were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France), whereas carboxy-2,7-dichlorofluoresceine (CF) diacetate was provided by Invitrogen/Life Technologies (Villebon sur Yvette, France). [ 3 H(G)] taurocholic acid (sp. act. 1.19 Ci/mmol), [6,7-3 H(N)] estrone-3-sulfate (E3S) (sp. act. 57.3 Ci/mmol) and [1-14 C] tetra-ethylammonium (TEA) (sp. act. 2.4 mCi/mmol) were from Perkin-Elmer (Boston, MA). All other chemicals were commercial products of the highest purity available.

Cell culture

Human hepatocytes were obtained from adult donors undergoing hepatic resection for primary and secondary tumors or other pathologies, via the Biological Resource Center (University Hospital, Rennes, France). Cells were prepared by enzymatic dissociation of histologically-normal liver fragments [START_REF] Fardel | P-glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture[END_REF]. These freshly isolated hepatocytes were either immediately collected for further RNA isolation and analysis or were seeded on standard tissue-culture plastic plates (BD Biosciences, Le Pont de Claix, France) at a density of approximately 2 × 10 5 cells/cm 2 in Williams' E medium (Invitrogen, Cergy-Pontoise, France), supplemented with 10% fetal calf serum (Perbio Sciences, Brébieres, France), 5 g/ml bovine insulin (Sigma-Aldrich), 100 IU/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine (Invitrogen). After 24 h, this seeding medium was discarded, and hepatocytes were cultured in the fetal calf serum-containing Williams' E medium defined above and supplemented with 5 × 10 -5 M hydrocortisone hemisuccinate (Upjohn, Paris La Défense, France) and 2% dimethyl sulfoxide (DMSO), as reported previously [START_REF] Chouteau | A short N-proximal region in the large envelope protein harbors a determinant that contributes to the species specificity of human hepatitis B virus[END_REF]Le Vee et al., 2009b), knowing that DMSO is well-known to promote differentiation and survival of hepatocytes [START_REF] Isom | Maintenance of differentiated rat hepatocytes in primary culture[END_REF]. The culture medium was routinely renewed every 2 days. All experimental procedures complied with French laws and regulations and were approved by the National Ethics Committee. Hepatocytes usually form nearly-confluent monolayers of viable cells and were used for experiments after a 8-10 days culture period.

RNA isolation and analysis

Total RNA was isolated from freshly isolated and monolayer-cultured human hepatocytes using the TRIzol reagent (Invitrogen/Life Technologies). RNA (20 ng) was then subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR), using the fluorescent dye SYBR Green methodology and an ABI Prism 7300 detector (Applied Biosystem, Foster City, CA, USA), as previously described (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF].The gene primers used in the study were exactly as previously reported (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF][START_REF] Moreau | Drug transporter expression in human macrophages[END_REF]; their adequate efficiency was routinely checked using serially diluted RNA samples. Amplification curves of the PCR products were analyzed with the ABI Prism SDS software using the comparative cycle threshold (Ct) method. Relative quantification of the steady-state target mRNA levels for transporters was next calculated after normalization of the total amount of cDNA tested to an 18S RNA endogenous reference, knowing that the amount of 18S RNA in freshly isolated human and monolayer-cultured human hepatocytes was found to be constant, i.e., the Ct numbers determined from qPCR assays were similar in these cells (Fig. S1), and was arbitrarily set at 10 6 units in each PCR sample [START_REF] Moreau | Drug transporter expression in human macrophages[END_REF]. This finally allowed to get a relative value of expression for each transporter comparatively to the 18S RNA reference.

Light microscopy analysis

Light microscopy analysis of monolayer cultures of human hepatocytes was performed using an Axiovert microscope (Carl Zeiss, Le Pecq, France), as previously described (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF].

Immunolocalization studies

Immunofluorescence analyses were performed as previously reported (Le Vee et al., 2009b;[START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF]. Human hepatocytes, cultured on glass coverslips (Millicell EZ slides, Merck Millipore, Billerica, MA) for 8-10 days at the density of approximately 2 × 10 5 cells/cm 2 in the medium described above, were first fixed in ice-cold acetone for 10 min.

Cells were next incubated for 3 h with mouse monoclonal or rabbit polyclonal antibodies diluted to 1:50 (mouse antibodies) or 1:25 (rabbit antibodies) in phosphate-buffered saline supplemented with 4 % (weight/weight) bovine serum albumin. The mouse monoclonal antibodies were directed against organic cation transporter 1 (OCT1)/SLC22A1 (Abcam, Cambridge, UK), multidrug resistance gene 1 (MDR1)/ABCB1/P-glycoprotein (Alexis Corporation, Lausen, Switzerland), multidrug drug resistance-associated protein (MRP) 2/ABCC2 or MRP3/ABCC3 (Merck Millipore), whereas the rabbit polyclonal antibodies were raised against organic anion transporting polypeptide (OATP) 1B1/SLCO1B1 or OATP2B1/SLCO2B1 [START_REF] Huber | Characterization of two splice variants of human organic anion transporting polypeptide 3A1 isolated from human brain[END_REF]. Controls were performed in parallel with appropriate mouse or rabbit isotypic Ig controls. After washing, goat AlexaFluor 488-labeled secondary antibodies (Invitrogen/Life Technologies) were added for 1 h, and nuclei were subsequently stained with 4,6-diamidino-2-phenylindole. Immunofluorescence images were finally detected with a Zeiss Axioskop A1 microscope (Carl Zeiss) and a Nikon DS-2MBW camera (Nikon, Champigny-sur-Marne, France), using the NIS-Elements F3.2 software (Nikon).

Drug transport assays

Sinusoidal influx assay

Uptake transport activities due to the sinusoidal transporters sodium-taurocholate cotransporting polypeptide (NTCP/SLC10A1), OATPs and OCT1 were analyzed through measuring sodium-dependent-intracellular accumulation of the NTCP substrate taurocholate, probenecid-sensitive uptake of the OATP substrate E3S and verapamil-sensitive uptake of the OCT1 substrate TEA, using a defined transport assay medium consisting of 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 11 mM d-glucose, 10 mM HEPES, and 136 mM N-methyl glucamine (sodium-free buffer) or 136 mM NaCl (standard sodium-containing buffer), as previously described [START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF]. Briefly, cells were incubated at 37 °C for 10 min in the transport medium supplemented with 43.4 nM [ 3 H] taurocholate in the presence or absence of sodium, 3.4 nM [ 3 H] E3S in the presence or absence of the OATP inhibitor probenecid (2 mM) or 40 M [ 14 C] TEA in the presence or absence of the OCT1 inhibitor verapamil (50 M). After washing in phosphate-buffered saline, cells were lyzed and accumulation of radiolabeled substrates was determined through scintillation counting.

Taurocholate accumulation values in the presence of sodium minus accumulation values in the absence of sodium, E3S uptake values in the absence of probenecid minus uptake values in the presence of probenecid and TEA uptake values in the absence of verapamil minus uptake values in the presence of verapamil are thought to represent NTCP-, OATPs-and OCT1-related transport activities [START_REF] Jigorel | Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes[END_REF].

Canalicular secretion assay

Biliary secretion of taurocholate, CF and rhodamine 123 was determined through measurement of radiolabeled or fluorescent substrate accumulation into bile canaliculi-like structures, as previously reported [START_REF] Annaert | Pglycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes[END_REF][START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF]. Briefly, monolayer-cultured human hepatocytes were first incubated for 10 min at 37°C with the defined Ca 2+ -containing transport assay buffer described aboved or with the same buffer, except that 1.8 mM CaCl 2 was withdrawed and 100 µM EGTA was added; such an incubation in this Ca 2+ -free buffer is known to promote disruption of tight junctions and opening of bile canaliculi networks. Buffers were then removed and cultured human hepatocytes cells were further incubated for 10 min at 37°C in transport assay medium containing 43.4 nM [ 3 H] taurocholate, 3 µM CF diacetate or 5.25 µM rhodamine 123. After washing with ice-cold phosphate-buffered saline, accumulations of taurocholate, CF or rhodamine 123 into cells + bile canaliculi (Ca 2+ -containing conditions) and into cells only (Ca 2+ -free conditions) were determined by scintillation counting (for taurocholate) or spectrofluorimetry (for CF and rhodamine 123) using a SpectraMax Gemini SX spectrofluorometer (Molecular Devices, Sunnyvale, CA) (excitation and emission wavelengths were 485 and 535 nm, respectively).

Biliary excretion index (BEI) was finally calculated using the following equation (Liu et al., 1999a): 2.7 Statistical analysis. Quantitative data were analyzed using the two-sided Student's t-test.

The criterion of significance was p < 0.05.

Accumulation (Cells+Bile canaliculi) BEI= Accumulation (Cells+Bile canaliculi) -Accumulation (Cells) X 100

Results

Relative mRNA expressions of SLC and ABC drug transporters in monolayer-cultured human hepatocytes and freshly isolated human hepatocytes were first quantified by RT-qPCR. As shown in Fig. 1, expressions of the sinusoidal SLC transporters NTCP and OCT1

as well as those of the sinusoidal ABC efflux transporters MRP1 and MRP3 were similar and not statistically different in both cultured human hepatocytes and freshly isolated human hepatocytes. By contrast, mRNA expressions of the sinusoidal SLC transporters OATP1B1, OATP2B1, organic anion transporter 2 (OAT2/SLC22A7) and, in a more marked manner, OATP1B3 (SLCO1B3), were reduced in cultured human hepatocytes when compared to freshly isolated hepatocytes; the repression factors, i.e., the ratio mRNA levels in freshly isolated hepatocytes versus those in primary counterparts, range for these transporters from 1.8 (for OATP2B1) to 22.2 (for OATP1B3). Expressions of the sinusoidal efflux transporters MRP4 (ABCC4), MRP5 (ABCC5) and MRP6 (ABCC6) were found to be induced in monolayer-cultured human hepatocytes (Fig. 1), knowing that MRP4 and MRP5, in contrast to MRP6, remain rather poorly expressed in monolayer-cultured hepatocytes (expression < 1 arbitrary unit for MRP4 and MRP5 versus expression = 177.5 arbitrary units for MRP6). With respect to canalicular transporters (Fig. 2), MDR1 and MRP2 mRNA levels were up-regulated in cultured human hepatocytes when compared to freshly isolated hepatocytes, whereas those of bile salt export pump (BSEP/ABCB11) and breast cancer resistance protein (BCRP/ABCG2) were reduced and those of multidrug and toxin extrusion 1 transporter (MATE1/SLC47A1) unchanged.

To schematically categorize drug transporter expression in freshly isolated human hepatocytes and in monolayer-cultured counterparts, transporters were next classified according to their level of mRNA expression: low expression (<1 unit), moderate expression

(1-15 units), high expression (15-100 units) and very high expression (>100 units) (Table 1).

Most of drug transporters were ranged into the same expression level category in both freshly isolated human hepatocytes and monolayer-cultured counterparts (Table 1). In particular, OCT1 exhibited a very high expression, whereas MRP1, MRP4 and MRP5 were among the less expressed.

Light-microscopic analysis of hepatocyte cultures next demonstrated that monolayercultured human hepatocytes exhibited refringent bile canaliculi-like structures, allowing to discriminate sinusoidal-like and canalicular-like membranes (Fig. 3A). Immunofluorescence labeling then revealed that various sinusoidal hepatic transporters such as OATP1B1, OATP2B1, OCT1 and MRP3 were expressed at the sinusoidal pole of monolayer-cultured human hepatocytes cells (Fig. 3B). With respect to the canalicular ABC transporters MDR1/P-glycoprotein and MRP2, they were located to the rather wide bile canaliculi-like structures exhibited by cultured hepatocytes (Fig. 3B).

In order to determine whether sinusoidal transporters were active in monolayercultured human hepatocytes, activities of NTCP, OATPs and OCT1 were then determined through measuring intracellular accumulation of reference substrates. As shown in Fig. 4, cultured human hepatocytes exhibited sodium-dependent taurocholate accumulation, thus indicating the presence of NTCP activity in these cells. In the same way, probenecidinhibitable accumulation of the OATP substrate E3S and verapamil-inhibitable accumulation of the OCT1 substrate TEA were clearly detected in cultured human hepatocytes (Fig. 5), thus reflecting OATP and OCT1 activity in such cells.

To finally investigate whether bile canaliculi-like structures were functional in monolayer-cultured human hepatocytes, canalicular secretions of taurocholate, a substrate for BSEP [START_REF] Stieger | The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation[END_REF], of rhodamine 123, a substrate for MDR1/P-glycoprotein [START_REF] Drenou | Detection of P glycoprotein activity on normal and leukemic CD34+ cells[END_REF] and of CF, a substrate for MRP2 [START_REF] Ellis | Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley, Wistar and Mrp2-deficient Wistar (TR(-)) rats[END_REF][START_REF] Laupeze | Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood[END_REF] were analyzed and quantified using the BEI approach based on Ca 2+ withdrawal-mediated disruption of canaliculi (Liu et al., 1999b). Cultured human hepatocytes pre-incubated in Ca 2+ -free conditions exhibited decreased retention of taurocholate, rhodamine 123 and CF, comparatively to counterparts maintained in the presence of Ca 2+ (Fig. 5). This demonstrates that monolayer-cultured human hepatocytes secreted taurocholate, rhodamine 123 and CF in bile canaliculi-like structures, allowing to calculate BEI values (55.1 %, 22.1 % and 10.9 % for taurocholate, CF and rhodamine 123).

Discussion

The data reported in the present study demonstrate that human hepatocytes cultured in a standard monolayer configuration exhibit polarized expression of various sinusoidal and canalicular drug transporters, associated to detectable sinusoidal drug transporter activities and to the presence of functional bile canaliculi-like structures. Such data highlight the relative similarity to the in vivo situation of these human hepatocyte cultures and, consequently, their potential interest for hepatic drug transport studies.

This conclusion is also supported by the fact that most of sinusoidal and canalicular transporters were expressed at notable mRNA levels in monolayer-cultured human hepatocytes, even if, for some of them such as OATPs and OAT2, mRNA expression was rather decreased when compared to levels found in freshly isolated hepatocytes. The global expression of main hepatic drug transporters at the mRNA level, associated to detectable activities of NTCP, OATPs and OCT1, was moreover observed in human hepatocytes maintained in primary culture for 8-10 days, which fully agrees with the conclusion that monolayer-cultured human hepatocytes retain notable levels of drug transporter activity and protein expression with time in culture [START_REF] Jigorel | Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes[END_REF][START_REF] Schaefer | Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDPglucuronosyltransferases in cultured primary human hepatocytes[END_REF]. By contrast, drug transporter expression, especially that of sinusoidal SLC transporters, is rapidly altered during primary culture for rodent hepatocytes, whatever the culture conditions [START_REF] Luttringer | Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes[END_REF][START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF][START_REF] Tchaparian | Effect of culture time on the basal expression levels of drug transporters in sandwich-cultured primary rat hepatocytes[END_REF], which has been hypothesized to reflect the in vitro acquisition of a cholestatic phenotype [START_REF] Wilson | Multiparametric High Content Analysis for assessment of neurotoxicity in differentiated neuronal cell lines and human embryonic stem cell-derived neurons[END_REF]. NTCP expression, assessed by LC/MS-MS, as well as taurocholate and rosuvastatin uptake are thus reduced in sandwich-cultured rat hepatocytes with culture time, but are well maintained in human counterparts [START_REF] Kotani | Culture period-dependent changes in the uptake of transporter substrates in sandwich-cultured rat and human hepatocytes[END_REF][START_REF] Qiu | Absolute measurement of species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) and its modulation in cultured hepatocytes[END_REF].This highlights interspecies discrepancies with respect to expression of highly-differentiated hepatocyte markers such as drug transporters in primary hepatocytes. Similarly, expression of other liver-specific markers such as drug-metabolizing cytochromes P-450 has been shown to be better preserved in cultured human hepatocytes [START_REF] Morel | Expression of cytochrome P-450 enzymes in cultured human hepatocytes[END_REF], which is fully consistent with the conclusion that primary human hepatocytes are phenotypically more stable than rodent counterparts [START_REF] Guillouzo | Liver cell models in in vitro toxicology[END_REF].

It is however noteworthy that some differences between freshly isolated human hepatocytes and monolayer-cultured counterparts with respect to transporter mRNA expressions exist and have to be kept in mind when using primary human hepatocytes for drug transport studies. It is notably the case for the sinusoidal transporter OATP1B3, whose expression is markedly reduced in monolayer-cultured human hepatocytes. Uptake of OATP1B3 specific substrates such as some digitalis-like compounds [START_REF] Gozalpour | Interaction of Digitalis-Like Compounds with Liver Uptake Transporters NTCP, OATP1B1, and OATP1B3[END_REF] may therefore be under-estimated when using monolayer-cultured human hepatocytes for investigating kinetic features of hepatobiliary disposition of these compounds. The sinusoidal efflux orphan transporter MRP6, involved in the autosomal recessive metabolic disease pseudoxanthoma elasticum [START_REF] Pahlman | Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation[END_REF], is, by contrast, markedly up-regulated in monolayer-cultured human hepatocytes compared to freshly isolated counterparts. Transport of drugs putatively handled by MRP6 and that remain to be unambiguously identified, may consequently be over-estimated with the use of primary human hepatocytes. Other ABC transporters such as MDR1, MRP4 and MRP5 were also induced in monolayer-cultured human hepatocytes, even if they remained among the less expressed drug transporters (Fig.

3); these efflux transporters have similarly been found to be induced in primary mouse hepatocytes and in well-differentiated hepatoma HepaRG cells when compared to freshly isolated hepatocytes (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF][START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF]; such up-regulations can therefore be interpreted as a key hall-mark of hepatocytes when placed in an in vitro environment.

Primary human hepatocytes used in the present study were cultured in a monolayer configuration, i.e., they were seeded on plastic dishes without subsequent addition of a collagen or matrigel overlayer. Culturing hepatocytes in sandwich configuration, which is thought to represent the gold standard for drug transport studies with hepatocytes [START_REF] Bi | In vitro evaluation of hepatic transporter-mediated clinical drug-drug interactions: hepatocyte model optimization and retrospective investigation[END_REF][START_REF] Swift | Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity[END_REF], seems therefore to be not an absolute requirement for getting polarized hepatocytes, at least for human hepatocytes. This assertion is interestingly supported by the fact that calcium-sensitive efflux of taurocholate has been previously reported in collagen monolayer-cultured human hepatocytes [START_REF] Kostrubsky | Evaluation of hepatotoxic potential of drugs by inhibition of bile-acid transport in cultured primary human hepatocytes and intact rats[END_REF], which likely suggests the presence of functional bile canaliculi-like structures in such cells. Moreover, no relevant difference for sinusoidal and canalicular drug transporter protein levels between sandwich-and monolayer-cultured human hepatocytes has been recently demonstrated though LC/MS-MS-based absolute quantification of transporters [START_REF] Schaefer | Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDPglucuronosyltransferases in cultured primary human hepatocytes[END_REF]. Finally, highly-differentiated human hepatoma HepaRG cells exhibit polarized location of main drug transporters associated to taurocholate canalicular secretion, without being cultured in a sandwich configuration (Le [START_REF] Vee | Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells[END_REF][START_REF] Sharanek | Different dose-dependent mechanisms are involved in early cyclosporine a-induced cholestatic effects in hepaRG cells[END_REF]. Interestingly, a shared component between the culture medium used for our primary human hepatocytes and that used for HepaRG cells is DMSO, which is well known to favor hepatocyte survival and differentiation [START_REF] Choi | Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells[END_REF][START_REF] Isom | Maintenance of differentiated rat hepatocytes in primary culture[END_REF][START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF] and to increase Ntcp and Oatp activity in primary rat hepatocytes [START_REF] Jigorel | Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes[END_REF]. The way by which DMSO may promote polarized in vitro expression of drug transporters remains nevertheless to be determined. It can be hypothesized that DMSO may induce formation and secretion of extracellular matrix components in primary cultures of hepatocytes, which may mimic the addition of a collagen or matrigel overlay. To clarify the exact role played by DMSO, additional studies comparing cultured human hepatocytes maintained in the presence or absence of this solvent are however likely required. Otherwise, it is noteworthy that monolayer hepatocyte cultures used in the present study were nearly confluent. This parameter has probably to be taken into account owing to the well-established influence of cell density on differentiated functions of cultured hepatocytes [START_REF] Greuet | Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture[END_REF].

The canalicular transporters BSEP, MRP2 and MDR1/P-glycoprotein were demonstrated to be functional in our primary human hepatocyte cultures, as demonstrated by the secretion of taurocholate, CF and rhodamine 123 into bile canaliculi-like structures, thus allowing to calculate BEI values, in a way similar to that used for sandwich-cultured hepatocytes. The BEI values for taurocholate (BEI=55.1 %) and CF (BEI=22.1 %) in monolayer-cultured human hepatocytes were however substantially lower than those previously reported in sandwich-cultured human counterparts (BEI around 70-75 % and BEI=34.0%, for taurocholate and CF, respectively) [START_REF] Hoffmaster | P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide[END_REF][START_REF] Marion | Use of sandwich-cultured hepatocytes to evaluate impaired bile acid transport as a mechanism of drug-induced hepatotoxicity[END_REF], which may indicate higher activity of BSEP and MRP2 in hepatocytes maintained in a three-dimensional configuration. With respect to rhodamine 123, the BEI value found in our human hepatocyte cultures (BEI=10.9 %) was low, knowing however that rather low BEI values (BEI around 10-20 %) have also been reported for rhodamine 123 in sandwichcultured rat and mouse hepatocytes [START_REF] Annaert | Pglycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes[END_REF][START_REF] Noel | Functional expression and regulation of drug transporters in monolayer-and sandwich-cultured mouse hepatocytes[END_REF]. Additional studies are likely required to carefully and more extensively compare BEI values for drugs in monolayer-and sandwich-cultured human hepatocytes.

In summary, primary human hepatocytes maintained in a monolayer configuration were found to exhibit a polarized location of various major sinusoidal and canalicular drug transporters, associated with efficient canalicular secretion of ABC transporter substrates and detectable sinusoidal drug transporter activities. Monolayer-cultured human hepatocytes have otherwise been demonstrated to retain signaling ways involved in transporter regulation [START_REF] Jigorel | Differential regulation of sinusoidal and canalicular hepatic drug transporter expression by xenobiotics activating drug-sensing receptors in primary human hepatocytes[END_REF]. Taken together, these data indicate that primary human hepatocytes cultured in a monolayer dimension may likely represent a valuable in vitro model for studying activity and regulation of drug transporters.

Legends to figures

Fig. 1 .

 1 Fig. 1. Sinusoidal drug transporter mRNA expression in monolayer-cultured human

Fig. 2 .

 2 Fig. 2. Canalicular drug transporter mRNA expression in monolayer-cultured human

Fig. 3 .

 3 Fig. 3. Drug transporter localization in monolayer-cultured human hepatocytes.

  Fig.1