Mathieu Jacomy
email: mathieu.jacomy@gmail.com

Emilija Jokubauskaitė
email: e.jokubauskaite@uva.nl

Unblackboxing Gephi: How a user culture shapes its scientific instrument

Network visualization tools are blamed for being black boxes, but depending on who criticizes, blackboxing has one of two meanings. Blackboxing by inscrutability blames opacity (inaccessibility of the method) while blackboxing by embodiment blames transparency (invisibility of the mediation), thus we propose to drop the confusing dichotomy of opacity/transparency. We focus on Gephi, a network visualization tool popular in the social sciences and humanities, addressing its criticism as a black box, in the hope of proposing useful interventions. By analyzing empirical material from participant observation, interviews, and a publication review, we find that Gephi's blackboxing problem is by embodiment. We observe that some researchers circulate Gephi images without a proper interpretative context, a situation we refer to as "storyletting", or use methodological shortcuts in time-constrained situations, trading methodological awareness for efficiency. We acknowledge the existence of an epistemic subculture around Gephi, and we argue that its practices are only partially aligned with the instrument's design. We contend that Gephi's black box materializes in cultural artifacts such as online documentation and tutorials. We conclude that these artifacts are a valid place of intervention to try unblackboxing Gephi, and that researchers are natural actors of this critical technical practice.

Introduction

Technology is not neutral -to paraphrase [START_REF] Kranzberg | Technology and History:" Kranzberg's Laws[END_REF] first law of technology -and accordingly, digital tools and data are said to have transformed the production of knowledge in the social sciences and humanities [START_REF] Savage | The coming crisis of empirical sociology[END_REF][START_REF] Berry | The computational turn: Thinking about the digital humanities[END_REF][START_REF] Manovich | Software Takes Command[END_REF][START_REF] Ruppert | Reassembling social science methods: The challenge of digital devices[END_REF][START_REF] Meyer | Knowledge Machines: Digital Transformations of the Sciences and Humanities[END_REF][START_REF] Masson | Humanistic Data Research: An Encounter between Epistemic Traditions[END_REF]. However, digital tools and practices are too rarely given an active role in the narratives about big data, neither from enthusiasts (e.g. [START_REF] Lazer | Computational social science[END_REF] nor from critics (e.g. [START_REF] Boyd | Critical questions for big data: Provocations for a cultural, technological, and scholarly phenomenon[END_REF][START_REF] Ruppert | Reassembling social science methods: The challenge of digital devices[END_REF]. The discussion tends to be about technology in general rather than tools in particular (or instruments, as we refer to them). Most authors agree on the material-semiotic nature of big data issues, yet the level where materiality can be directly observed, the instrument level, is not addressed often. This is surprising considering that the idea that tools shape practices (see e.g. [START_REF] Wertsch | Voices of the Mind[END_REF] is common in Science and Technology Studies [START_REF] Callon | Writing and (re) writing devices as tools for managing complexity[END_REF] as well as in software development [START_REF] Kelty | Geeks and recursive publics: How the internet and free software make things public[END_REF]. The premise of this paper is to focus on a given scientific instrument (Gephi, an open-source software), and address its criticism (being a black box) by studying the practices of researchers, to contribute to better understand how to improve it. In short, we wonder what makes Gephi a black box; but as we will see, that research question comes with complications: what does it mean to be a black box, and where does the problem lie, materially?

In this article we focus on a specific network analysis and visualization tool called Gephi (see figure 1). Some academic authors associate Gephi to a black box, as a point of criticism [START_REF] Lemercier | Quantitative Methods in the Humanities: An Introduction[END_REF][START_REF] Wieringa | The Field Notes Plugin: Making Network Visualization in Gephi Accountable[END_REF] or not [START_REF] Bruns | Faster than the speed of print: Reconciling 'big data' social media analysis and academic scholarship[END_REF][START_REF] Paßmann | Unknowing Algorithms: On Transparency of Unopenable Black Boxes[END_REF]. Other authors make similar arguments but about a wider class of instruments, such as big data visualization [START_REF] Ruppert | The Politics of Method: Taming the New, Making Data Official[END_REF], computational instruments [START_REF] Masson | Humanistic Data Research: An Encounter between Epistemic Traditions[END_REF], or quantitative visualization [START_REF] Drucker | Humanistic Theory and Digital Scholarship[END_REF][START_REF] Drucker | Graphesis: Visual Forms of Knowledge Production[END_REF]. These authors cite each other, suggesting a relatively unified argument; but that assumption does not stand examination. Indeed, their arguments are on two distinct fronts, echoing different understandings of the notion of black box: either inability to access the method embedded in the instrument, that we call blackboxing by inscrutability, or invisibilization of the technology as a mediation, that we call blackboxing by embodiment. The coexistence of these two versions has caused confusion within the critical literature about digital instruments, and clarifying the situation is a necessary step towards addressing Gephi's criticism.

We study Gephi's blackboxing problem from different angles: as narrated by researchers in their publications (literature review), as explained by Gephi users in interviews, and through participant observation in teaching situations. From these complementary perspectives we account for different ways users engage with Gephi, we retrace the material-semiotic web of their interactions, and we explore how "different realities are being woven into being in different practices" [START_REF] Law | Material Semiotics[END_REF]. We observe that a specific subculture has emerged around Gephi, and that Gephi's powers of embodiment and inscrutability do not only root in its software, but also in the cultural artifacts of its user community (e.g., teaching material, online discussions). This leads us to conclude that the tool design might not be the right place where to intervene, and prompts us to reconsider who or what has enacted Gephi's blackboxing, and how to care for the Gephi users' community.

The case of Gephi

The instrument this research focuses on is Gephi [START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF], an open-source software tool for network analysis and visualization. It operates via a number of embedded algorithms (e.g. [START_REF] Blondel | Fast unfolding of communities in large networks[END_REF][START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF] and a graphical user interface (see figure 1). In practice, it allows users to visualize a relational data set (a network) and execute different analytical tasks, such as computing statistics or filtering data. It has been referenced in more than five thousand papers i across a variety of disciplines, inside and outside the social science and humanities. Its popularity within the SSH has led it to be discussed from that perspective [START_REF] Grandjean | Translating Networks: Assessing Correspondence Between Network Visualisation and Analytics[END_REF][START_REF] Wieringa | The Field Notes Plugin: Making Network Visualization in Gephi Accountable[END_REF], in addition to a more general discussion, in computer science and information design, on the kind of visualizations it provides [START_REF] Gibson | A survey of two-dimensional graph layout techniques for information visualisation[END_REF][START_REF] Krzywinski | Hive plots-rational approach to visualizing networks[END_REF][START_REF] Dunne | Readability metric feedback for aiding node-link visualization designers[END_REF][START_REF] Munzner | Visualization Analysis and Design[END_REF].

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Gephi's most popular node placement algorithm, Force Atlas 2, has become an increasingly important part of contemporary network analysis. Algorithms of this type (i.e., "force-driven") work by simulating "a physical system in order to spatialize a network. Nodes repulse each other like charged particles, while edges attract their nodes, like springs. These forces create a movement that converges to a balanced state" [START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF]. We must emphasize, however, that network visualizations created this way are "not deterministic, and the coordinates of each point do not reflect any specific variable" [START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF]. While popular, forcedriven placement algorithms have also been extensively criticized (for an overview, see [START_REF] Gibson | A survey of two-dimensional graph layout techniques for information visualisation[END_REF][START_REF] Dunne | Readability metric feedback for aiding node-link visualization designers[END_REF], notably for their "inherent unpredictability, inconsistency and lack of perceptual uniformity" [START_REF] Krzywinski | Hive plots-rational approach to visualizing networks[END_REF] and "brittleness" [START_REF] Munzner | Visualization Analysis and Design[END_REF]. Additionally, due to the many parameters and user interactions that need to be taken into consideration while using Gephi, it is notoriously complicated to document, communicate and reproduce how the network visualization came into being [START_REF] Wieringa | The Field Notes Plugin: Making Network Visualization in Gephi Accountable[END_REF].

Analytical perspectives

Our conceptual framework has been chosen to accommodate our two starting points: the criticism of data visualization tools in the SSH literature, and the empirical engagement with the Gephi user community. We aimed at remaining coherent with the voiced criticism while supporting an empirical engagement with Gephi as a device, its interactions with its users, and its community over internet. This led us to mobilize concepts such as epistemic culture (Knorr Cetina 1999) and technical mediation [START_REF] Simondon | Du mode d'existence des objets techniques[END_REF]. We will present this criticism first, and second our main concepts, as the latter have been picked to address the former.

The criticism of data visualization tools in the SSH literature

Within the social sciences and humanities (SSH) some authors criticize data visualization and its tools on the ground that they hide the conditions of their production and/or the layers of mediation they involve. As we briefly retrace in this subsection, this argument roots in science and technology studies (STS) through Haraway, Latour and Law (see figure 2).

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When Evelyn Ruppert and Stephan Scheel (2019) account for the politics of method involving big data, they argue that the visualization becomes "not only a vehicle for the data, but first and foremost for its claimed self-evidence" (emphasis added). Here they draw on Johanna [START_REF] Drucker | Humanities approaches to graphical display[END_REF]), Donna Haraway (1988) (both summarized below), and on John Law's idea that "practices enact realities" (2012). Similarly, Eef [START_REF] Masson | Humanistic Data Research: An Encounter between Epistemic Traditions[END_REF] writes that "computational research tools are often assigned such values as reliability and transparency" (emphasis added), drawing on [START_REF] Drucker | Humanistic Theory and Digital Scholarship[END_REF][START_REF] Drucker | Graphesis: Visual Forms of Knowledge Production[END_REF] and [START_REF] Rieder | Studying Facebook via Data Extraction: The Netvizz Application[END_REF]Röhle (2012, 2017).

Bernhard Rieder and Theo Röhle (2017) write: "[t]he problem, again, comes from the fact that tools such as Gephi have made network analysis accessible to broad audiences that happily produce network diagrams without having acquired robust understanding of the concepts and techniques the software mobilizes. This more often than not leads to a lack of awareness of the layers of mediation network analysis implies and thus to limited or essentialist readings of the produced outputs that miss its artificial, analytical character" (emphasis added). This argument is a refined version of a point from their 2012 paper.

Johanna Drucker writes that maps, charts and graphs "are a kind of intellectual Trojan horse, a vehicle through which assumptions about what constitutes information swarm with potent force. … they pass as unquestioned representations of 'what is.' This is the hallmark of realist models of knowledge and needs to be subjected to a radical critique to return the humanistic tenets of constructedness and interpretation to the fore" (emphasis added). This passage from her book Graphesis (2014) is also in her 2011 paper. In 2012 she also explores the same idea: "[g]raphs and charts reify statistical information. They give it a look of certainty. Only a naive viewer, unskilled and untrained in matters of statistics or critical thought, would accept an information visualization at face value" (emphasis added).

Finally, Donna Haraway writes in her influential paper Situated Knowledges (1988): "all seems not just mythically about the god trick of seeing everything from nowhere, but to have put the myth into ordinary practice. … There is no unmediated photograph or passive camera obscura in scientific accounts of bodies and machines; there are only highly specific visual possibilities, each with a wonderfully detailed, active, partial way of organizing worlds" (emphasis added). Through "situated knowledges," Haraway reclaims the necessity for all knowledge to be critically re-examinable, to state explicitly the epistemic ground where it stands.

Main concepts

Epistemic (sub-)culture. We make use of Knorr Cetina's concept of epistemic culture. "Epistemic cultures are cultures that create and warrant knowledge" (Knorr Cetina 1999, p. 26). In her own words, the notion of epistemic culture "disrupts the idea of the epistemic unity of the sciences and suggests that the sciences are in fact differentiated into cultures of knowledge that are characteristic of scientific fields or research areas, each reflecting a diverse array of practices and preferences coexisting under the blanket notion of science" (Knorr Cetina and Reichmann, 2015, p. 873). In the case of Gephi, as the scale is noticeably smaller, we argue an epistemic subculture.

Laboratory. Still following Knorr Cetina (1999), we situate the SSH use of Gephi in its own laboratory. For Knorr Cetina, and for us, the laboratory is not necessarily a physical place, but wherever takes place the action of working with the inscriptions produced by inscription devices (see below), as opposed to studying objects in nature. The laboratory is "the locus of mechanisms and processes which can be taken to account for the success of science" (Knorr Cetina 1992, p. 116). Gephi's laboratory consists of digital and physical spaces where users share their work and seek help and advice, notably Twitter, a dedicated Facebook group ii , and blogs iii . Some official teaching material does exist (website iv , wiki v ) but most of it has been authored by the community, including three books. vi Gephi is also part of a few regular cursus, for instance the Digital Methods Initiative's Winter and Summer schools vii .

Inscription device. Latour and Woolgar (1986) introduce the notion of inscription device as "any item of apparatus or particular configuration of such items which can transform a material substance into a figure or diagram which is directly usable" (p. 51). We analyze Gephi as a scientific instrument but more precisely as an inscription device.

Black box.

As an inscription device, Gephi is said to be a black box. That concept is problematic because it has at least two distinct meanings. We will return to that point in the next subsection, after we have introduced a few necessary concepts.

(Technical) mediation. In line with the conceptual framework mobilized to criticize Gephi and data visualization, we draw on Gilbert Simondon's thinking of the technical object (1958), and more recently on Madeleine [START_REF] Akrich | Les formes de la médiation technique[END_REF] and Bruno [START_REF] Latour | On Technical Mediation[END_REF]. For [START_REF] Latour | On Technical Mediation[END_REF], the technical mediation refers to the fact that technical objects "act, displace goals, and contribute to their redefinition." An important feature of that framework is its flat ontology: objects act on people and symmetrically people act on objects. The mediation allows us inquiring into Gephi's material-semiotic ability to reorganize the goals of its users, and vice-versa.

Embodiment.

To analyze the material-semiotic effects of Gephi we draw on Maurice Merleau-Ponty's (1962) notion of "embodiment," that he applies specifically to mediational objects (see also [START_REF] Heidegger | Trans. John Macquarrie and Edward Robinson[END_REF]. As he argues, one stops perceiving the mediation as an object, and instead perceives through it as if it were part of one's body. His classic example is the blind man's stick, but one could also think of eyeglasses or the computer mouse. This phenomenological approach is widespread in the field of Human-Computer Interaction (HCI) with concepts such as "embodied interaction" [START_REF] Dourish | Where the action is: the foundations of embodied interaction[END_REF] and as a foundation of Activity Theory [START_REF] Kaptelinin | Activity theory in HCI: Fundamentals and reflections[END_REF].

Critical technical practice.

As participants to the Gephi development team and community, we also draw on the tradition of critical technical practice [START_REF] Agre | Toward a Critical Technical Practice: Lessons Learned in Trying to Reform AI[END_REF]; see also [START_REF] Van Es | The datafied society. Studying culture through data[END_REF], also popular in the field of HCI, "in which technology development is not simply an end in itself, but also becomes a means to reflectively explore the assumptions and attitudes that underpin ideas about technology and humanity" [START_REF] Dourish | Reflective HCI: Towards a critical technical practice[END_REF](Dourish et al., , p. 1727)).

Storyletting (and not storytelling) is a notion we propose further in this text to refer to the act of sharing or circulating an image (a network visualization) without providing the context necessary to its interpretation. One is then letting the image speak for itself (hence the name), which suggests its "self-evidence" [START_REF] Ruppert | The Politics of Method: Taming the New, Making Data Official[END_REF] and fails to consist of "situated knowledges" [START_REF] Haraway | Situated knowledges: The science question in feminism and the privilege of partial perspective[END_REF].

Two versions of blackboxing: by embodiment / by inscrutability

We find two distinct versions of the black box in the literature. Here we present their differences and give them distinct names for clarity. We call the first "by embodiment" because it grounds into phenomenology of perception, and the second "by inscrutability" because it points to a scrutiny problem.

Latour argues that blackboxing is "the way scientific and technical work is made invisible by its own success. When a machine runs efficiently, when a matter of fact is settled, one need focus only on its inputs and outputs and not on its internal complexity. Thus, paradoxically, the more science and technology succeed, the more opaque and obscure they become." (1999, p. 304). It corresponds to what [START_REF] Paßmann | Unknowing Algorithms: On Transparency of Unopenable Black Boxes[END_REF] call practical transparency, when the user "does not have to be bothered with the underlying machinery or software" as Star et al. put it (2003, p. 242). We call this version blackboxing by embodiment, drawing on Maurice Merleau-Ponty's (1962) notion of "embodiment" (see also [START_REF] Heidegger | Trans. John Macquarrie and Edward Robinson[END_REF], typical of mediational objects. Embodiment is not a problem by itself; on the contrary, it is required for a mediation to function [START_REF] Latour | On Technical Mediation[END_REF]). Yet by invisibilizing the mediational object, it allows it to escape scrutiny. It acts against what [START_REF] Haraway | Situated knowledges: The science question in feminism and the privilege of partial perspective[END_REF] calls "situated knowledges," where the knower's relation to the known must be re-examinable. The invisibilization of the mediation is the main problem pointed to by blackboxing by embodiment.

The alternative, that we call blackboxing by inscrutability, addresses the impossibility to know the method, as in the colloquial expression "opening the black box." This understanding is typically present in algorithm accountability and explainable AI (overview in [START_REF] Wieringa | What to account for when accounting for algorithms: A systematic literature review on algorithmic accountability[END_REF] where black boxes are defined as "opaque decision systems" [START_REF] Guidotti | A survey of methods for explaining black box models[END_REF]. Latour attributes this version to cyberneticians, who use it "whenever a piece of machinery or a set of commands is too complex ... That is, no matter how controversial their history, how complex their inner workings, how large the commercial or academic networks that hold them in place, only their input and output count." (2000, p. 681) Such black boxes are inscrutable by design.

Blackboxing by inscrutability is one of the wider-known points of tool criticism in academia, especially in the social sciences. Rieder and Röhle characterize it as the "practical [im]possibility to access the most obvious layer of functional specification, a tool's source code" (2017, p. 113), hindering "our ability to understand the method, to see how it works, which assumptions it is built on, to reproduce and criticise it" (2017, p. 112). This blackboxing opposes what Paßmann and Boersma (2017) call formalized transparency, the ability to access methodological information.

The coexistence of these distinct understandings of blackboxing has brought confusion to the debate. First problem: when a critique mentions blackboxing, we do not always know what it refers to, notably at the intersection of STS (where blackboxing by embodiment is prioritized) and Computer Science (where blackboxing by inscrutability prevails), and in HCI. Second problem: the opacity/transparency dichotomy often used to clarify the notion of blackboxing is confusing on its own. In the metaphor, transparency opposes blackness; yet counter-intuitively, transparency is a defining feature of blackboxing by embodiment. Therefore, demarcation attempts that rely on the notion of transparency sound confusing. For instance, Paßmann and Boersma (2017) demarcate practical transparency from formalized transparency. The former aligns with blackboxing by embodiment, while the latter opposes blackboxing by inscrutability. Their demarcation is productive on a conceptual level, yet it is awkward to articulate with blackboxing because the metaphors clash on an intuitive level. Third problem: these blackboxings call for different countermeasures. One must avoid addressing the wrong kind of blackboxing to improve Gephi. The distinction matters to tool makers and users. Indeed, as we will see, Gephi is mainly blackboxed by embodiment: its methodological ground is relatively accessible, yet problems arise from its mediational properties.

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Now equipped with these distinct concepts of blackboxing, we can articulate our general argument. We contend that Gephi's is a black box by embodiment. The layers of mediation it involves are made invisible by its own success, but that success partially depends on practices invented in the laboratory and institutionalized by its subculture as cultural artifacts. We argue that the apparent self-evidence of network visualizations partially roots in Gephi's ability, as a mediation, to displace the goals of its users: by facilitating the production of images that can easily circulate but not easily be understood, it incentivizes storyletting (images are let free to tell their own story). These knowledges therefore lack the situatedness required to reexamine the interpretative process.

Methodology

This paper is a collaboration around the material collected by one of the authors, Emilija Jokubauskaite, more extensively presented in her master's thesis (2018). It contrasts the voice of users with Mathieu Jacomy's designer and developer perspective on Gephi, to unpack the different dimensions of blackboxing.

We engaged in participant observation in a dozen of Gephi workshops over the last decade, often as trainers, and sometimes as trainees. We conducted and inductively analyzed semistructured interviews with 6 Gephi users of varying levels of experience, who were chosen due to having conducted work that utilizes it for academic purposes within media and communication studies. The interviews consisted of open-ended questions aimed at understanding how researchers communicate their usage of the tool and how they represent the method. Parts of each interview followed an elicitation interviewing method [START_REF] Johnson | Elicitation techniques for interviewing[END_REF]: the interviewees were asked to use Gephi to briefly interact with a data sample (either familiar or new to them) prior to the interview and take notes on their use of the tool. During the interview, the tools' graphical user interface was open, and the interviewees narrated their methodological steps of using it, followed by a discussion of the decision-making process, questions about starting to use the tool and overall experience, as well as reflections on Gephi use in the academic field in general and other topics.

To triangulate the information gathered from the interviews with the methodologies presented in the documentation, we also analyzed 17 published academic papers. The papers were chosen through a systematic sampling, including recent papers from 2009 up to 2018, filtering 50-100 papers published each year for those of most relevance to the area of social media research in media and communication studies.

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Gephi's blackboxing by embodiment Here we present Gephi's blackboxing problem as painted in our empirical material. Our analysis diagnoses an issue of blackboxing by embodiment: researchers have issues accounting for what Gephi performs, not because they fail to scrutinize it, but because they do not understand, or are not aware of, the layers of mediation involved.

Scholars do not fully acknowledge Gephi's methodological dimension

One of the interviewed researchers reflected on the need to understand how the specific settings of Gephi function. In their eyes, the level of understanding should be at least "to the extent that they can explain what is happening ..., because it is also a part of your job as a researcher to actually communicate this stuff". They add that on the other hand, "if you know a lot about this stuff, you get into a potential problem that you communicate too much about the method ..., whereas what you want to say is actually something about the data."

Here we account for how academic publications frame the methodology where Gephi is involved. Our publication review shows that authors rarely make explicit which method they use to analyze networks. Some of these publications do not present the settings employed to apply the algorithms of Gephi [e.g.,8,12,13,14,15]; as the vignette above shows, the authors may just consider it out of scope. In other papers, the explanation of methodological decisions draws on the settings of the tool [e.g., 16, 18] but their effects on the analysis are not discussed.

While some authors do elaborate on the method [e.g., 19, 20], their rationale does not directly relate to Gephi. An illustration of that situation can be found in the publication of Barnett and Benefield [19]. In their article, they expose and discuss the measures of in-degree centrality and betweenness centrality. Gephi is providing these statistical metrics but is just presented to illustrate the findings. Similarly, Bonini and Celli et al.'s articles [9,15] present the method of network analysis in the methodology section, but do not mention Gephi until a visualization is presented later in the text, somewhat separating the tool from the method. Additionally, as [START_REF] Bruns | Faster than the speed of print: Reconciling 'big data' social media analysis and academic scholarship[END_REF] writes, the referees for articles do not usually request such methodological information: "treatment of tools such as Gephi as black boxes whose interior operations can be ignored is not limited to scholarly authors alone", referring here to what we call blackboxing by embodiment.

To better understand the rationale behind the methodological decisions with Gephi, we turn to the interview data. In them, we observe, the users explain their methodological decisions from the viewpoint of the tool, for example stating that they used Force Atlas 2, a layout algorithm, "because it is actually optimized for Gephi" [6]; "because basically it was the one that worked best" [5] or because it "is the most convenient … to use" [1]. These examples suggest that the Gephi users may be paying more attention to finding the settings that allow extracting the "correct" or "best" network visualization, rather than rationalizing their practice based on how the method operates. For many scholars, Gephi is put to use before they fully grasp its methodological effects, which also points to blackboxing by embodiment.

Gephi is rarely perceived as inscrutable by its users

The interviewees reflect on the level of scrutiny offered by the instrument. For example, some of them state that "it gives you a very direct feedback" [1] or that they don't necessarily know what it does, but it "gives a reasoning behind it after you run it" [2]. They also report on specifically trying out some functionalities of the tool to gain an understanding of them [1]. Moreover, researchers say that "you always have sort of a clear methodological thing, that you can reference and people can look at it after" [2]. They appreciate that the tool provides them with references to academic literature sources, that contextualize the various metrics and functions one can apply with the tool. Gephi is not perceived as an inscrutable black box; but of course, inscrutability is not the only way to be blackboxed.

Users do not necessarily understand how Gephi works

Discussing their use of Gephi, one of the interviewees reflects about using one of the different layout algorithms by saying that it "is sort of you use [it] and then try your best, which feels sort of unmethodological or unscholarly, because I feel like the advice I have gotten is just 'try them all and see what fits', which is just like rolling a dice. Feels sort of a bit random." Another interviewee reflected on how she learned to use Force Atlas 2. She said that another student explained it to her via text messaging by telling her to "put gravity on 10 and enable the 'prevent overlap' setting", however they were not able to provide rationale for it and when the interviewee asked them why, they replied "nevermind, just do it". Some users reflect on often using the tool without making the decisions consciously [1,2,4] and not being aware of what and why they should question in the process of implementing the tooluse in their research [2,3]. They articulate that the instrument is "complicated and there's a lot of things that you have no idea what is happening and what is going on" [3]. Moreover, one of the interviewees also argued that the interface does not necessarily help the user understand what is happening method-wise [4]. These observations can be complemented by Bruns' ( 2013) argument that even though there is quite a lot of information about the technical aspects behind various algorithms in Gephi, understanding them to a needed level is often out of the skillset of humanities and social sciences scholars. Not everyone has the knowledge necessary to grasp what Gephi performs. Likewise, "beginners" are of particular concern for Lemercier and Zalc, in supplemental material viii to their book Quantitative Methods in the Humanities: An Introduction (2019). They warn against software that "presents some analysis tools like 'black boxes'" and comment that "contrary to many, we are no fans of Gephi, for this type of reasons; … we do not find it well-suited to beginners." The mediation performed by Gephi is not visible or understandable to all users at all moments. Rieder and Röhle (2017, p. 118) also highlight the interplay between the tool and its public: "The problem … comes from the fact that tools such as Gephi have made network analysis accessible to broad audiences that happily produce network diagrams without having acquired robust understanding of the concepts and techniques the software mobilizes. This more often than not leads to a lack of awareness of the layers of mediation network analysis implies." For them, the problem is at the same time about visibility ("awareness") and "understanding" of the method. But as we have seen, Gephi is not perceived as inscrutable; most users are aware that it involves algorithms described in academic papers, and gradually improve their understanding of the "layers of mediation" involved [START_REF] Rieder | Digital methods: From challenges to Bildung[END_REF]. Interviewed researchers say, for example, that "through the time of working with the actual tool you … get to know what it … represents. So, it gives you more insight into what it is … that you are working with" [1]. Moreover, several of the interviewees express a wish to do more related research to learn more about the tool [2,4]. Gephi is perceived as an entry point to academic literature. However, this knowledge is not required to use it. This situation is in line with Gephi's original design intent (documented in e.g., [START_REF] Jacomy | ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software[END_REF]. It was initially developed for the needs of the e-Diasporas Atlas project [START_REF] Diminescu | e-Diasporas Atlas[END_REF], a collaborative exploration of the web of diasporas (on the apparatus, see [START_REF] Diminescu | Digital Diasporas Atlas Exploration and Cartography of Diasporas in Digital Networks[END_REF] on the project, see [START_REF] Diminescu | Diasporas on the Web[END_REF]. The participating scholars were specialists of migrations inexperienced in network analysis. Gephi was largely designed for the persona of the scholar-going-digital: someone trained to the social sciences in an analog setting, and willing to extend their skills to digital methods. It was designed to be functional for a user aware of the existence of an underlying methodology, but without the ability to understand it. The design intent assumed that users would be willing to acquire the necessary knowledge, thus it was not rendered necessary to using the instrument in practice.

Our empirical material rules out a major problem of blackboxing by inscrutability, and leaves us with the logical conclusion, substantiated by several observations, that Gephi suffers from blackboxing by embodiment. Yet it is also clear that the visibility and understandability of Gephi's layers of mediation also depend on who is using it, and in which context. Which begs the question: to whom, and in which situation, is Gephi a black box?

How the black box is materialized in the artifacts of Gephi's epistemic subculture Enacting self-evidence through "storyletting"

In a research seminar at the IT University of Copenhagen, a few PhD students have the occasion to present their work in 10 minutes and one of them has a slide with a network visualization. It shows a colorful screenshot of a decently sized Gephi network on a black background. It is hard to read because many labels overlap but it seems to have a clustered structure. The student just comments: "We also do these kinds of fancy visualizations" and immediately moves on to the next slide without providing any explanation of what we are supposed to see, or how it was useful to them.

One does not have to find a network visualization useful, but then why showcase it without providing the context necessary to its interpretation? We gave this situation a name: storyletting, the act of circulating an image without an explanation, letting it tell its own story. Indeed, a Gephi screenshot is prone to tell stories such as: my data is complex and massive; my research is computational and quantitative; my skillset includes advanced analysis techniques… Visualization plays a clear role in the politics of method [START_REF] Ruppert | The Politics of Method: Taming the New, Making Data Official[END_REF][START_REF] Savage | The coming crisis of empirical sociology[END_REF]. Naming the practice allows us to refer to it without assuming malice, or even intent; Gephi images may circulate context-free for many reasons. As we have seen, most academic papers featuring Gephi do not provide any discussion or justification of the methodological decisions related to it [e.g., 8, 12, 13, 14, 15, 17, 21] (see also [START_REF] Bruns | Faster than the speed of print: Reconciling 'big data' social media analysis and academic scholarship[END_REF]. Several researchers presented Gephi as "only one tool they used" [4; see also 13, 14] or "just a data visualization" tool [3; also in 10, 12, 14, 15, 17, 19, 20, 21, 22, 24], and some interviewees [2,4] reflect that scrutinizing Gephi is not necessary because network analysis is deemed peripheral to their research. Yet regardless of the justification and intent, the act of storyletting signifies that the visualization does not require explanation, that it is "self-evident" [START_REF] Ruppert | The Politics of Method: Taming the New, Making Data Official[END_REF]. As critiques have argued, this absence of context prevents us from re-examining our relation, as knowing subjects, to the known object (the network), failing to situate the knowledges we produce and/or circulate [START_REF] Haraway | Situated knowledges: The science question in feminism and the privilege of partial perspective[END_REF].

Brooke Foucault Welles and Isabel Meirelles (2014) account for the circulation of the network visualization from Lada Adamic and Natalie Glance's (2005) influential paper Divided they Blog. Adamic and Glance had situated their claims, yet other authors stripped the visualization of its context when they circulated it, transforming it into a (claimed!) self-evident illustration of the polarization of US politics. As [START_REF] Latour | Visualisation and Cognition: Drawing Things Together[END_REF] argues, the materiality of visualization may matter more than its semiotics, notably through its ability to circulate while maintaining its consistency (what he calls "immutable mobiles"). Situatedness is about more than the production process of a visualization. To understand Gephi's blackboxing by embodiment, we need to widen the focus and look at the practices with it, in the context of its "laboratory" [START_REF] Knorr Cetina | Epistemic cultures: How the sciences make knowledge[END_REF].

Shortcut practices within Gephi's laboratory

When asked about what situation they were in when they were first learning to use Gephi, one of the interviewees reflects on participating in the fast-paced data sprints of the Digital Methods Initiative (presented in [START_REF] Berry | The data sprint approach: exploring the field of Digital Humanities through Amazon's application programming interface[END_REF]: "Well, usually it's in our Winter or Summer Schools. There is a project and suddenly there is a network, I need to open it and do things. And then it's more like, OK, learn what you need to do [in] this specific project to solve this kind of issue you have. So, it's more like learn how to do a specific thing with the software, related to the project and less about how to use all the specific features and all the settings." As we observed many times, in the heat of a data sprint the use of scientific instruments gets less reflexive and more pragmatic. Gephi is often used in a time-restricted environment. Interviewees explain that they learned how to use the tool during data sprints [1], short introductory classes [4] or project-based university courses [4,6] and other project-to-project cases. That is, while the researchers often arrive at the use of Gephi without former knowledge of network analysis, their educational path from the beginning can be seen as largely focused on producing findings in short-time projects. They often learn from step-by-step tutorials and "speed courses" [1] or "best practices" that increase productivity when using the tool. Following that, the researchers report being encouraged to "just use it" [3] or instructed by their peers to take some methodological steps without providing further information on why and what the implications might be [1,4]. Consequently, some researchers refer to their use of Gephi as automatic, for instance "if I want a specific end result, I know how to get there, but it's very automatic, I don't really understand why this is what I do, it's just that I go through the moves, because that's what you have to do." This situation is typical of a know-how teaching, where reproducing the actions of the trainer or tutorial is the first step of the learning process, but there is more to it. This researcher accepts to trade methodological awareness for efficiency. Shortcuts are problematic but ordinary, and Gephi is relatively aligned with such utilitarian uses. It can be mobilized purposefully to produce a visualization, and once this goal is met, it will offer no resistance to the repurposing of that image, for instance as a dubious marketing asset in some slides. This situation matches Rieder and Röhle's (2017) observation of "broad audiences that happily produce network diagrams without having acquired robust understanding of the concepts and techniques the software mobilizes." With a nuance, however: it does not only point at the users' lack of "digital bildung" [START_REF] Rieder | Digital methods: From challenges to Bildung[END_REF]) but also at Gephi's lack of resistance to the most utilitarian practices.

Gephi's design only resists one kind of blackboxing Gephi's design addressed blackboxing, but in the sense of inscrutability, not embodiment. Gephi's proxy paper [START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF] is too short to discuss its design, but another paper developed by the same team [START_REF] Jacomy | ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software[END_REF] offers some elements. The authors state: "Our goal was to provide some network analysis methods to social scientists, that would not require learning graph theory." This commoditization goal implies blackboxing by embodiment: concealing complexity to offer more practicality. When the authors ix explain why their algorithm displays its process in real time they emphasize the importance of embodiment, but when they criticize black boxes, they limit the notion to inscrutability: "Visualizing the 'live' spatialization is a key feature of Gephi. It provides a very intuitive understanding of the layout process and its settings. … Social scientists cannot use black boxes, because any processing has to be evaluated in the perspective of the methodology." Gephi's design aims at resisting blackboxing by inscrutability but does not address blackboxing by embodiment.

Should we then conclude that Gephi's blackboxing problem roots in its design? The situation is not so simple, at least because its design implementation differs from the stated intent. First, many aspects of the instrument are unpolished if not unfinished, and most users find it frustrating at times. Second, Gephi's interface occasionally exercises intentional resistance. As an emblematic illustration, let us look at how the Force Atlas 2 algorithm [START_REF] Jacomy | ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software[END_REF], commonly construed as Gephi's default layout, is contextualized in the software. It features as a mere item in a list that you must unfold to see; it is not the first item of the list; it is not selected by default; it is not highlighted (see figure 3). The rationale behind this design choice was to resist blackboxing by inscrutability: forcing users to acknowledge the existence of multiple algorithmic alternatives, none if which is intrinsically better. But notice that by imposing friction (more clicks and choices) it also resists blackboxing by embodiment. What makes it the default algorithm is not inscribed in the software, but in cultural artifacts: the Force Atlas 2 paper presents it as Gephi's "default layout algorithm"; teaching material presents it as the go-to solution; its popularity x in other papers reinforces its status of standard within the Gephi subculture. In this case, the practices have counteracted the design and institutionalized a default option despite its absence from the material semiotics of the instrument.

[INSERT_FIGURE_3] [START_REF] Latour | On Technical Mediation[END_REF] highlights the symmetry of technical mediation: the tool shifts the goals of its users and, symmetrically, users shift the goals of the tool. We have observed both sides of it but in different situations. First, Gephi yields to utilitarian pressure in time-restricted situations where users are the most willing to trade methodological awareness for efficiency. In this case, users shift Gephi's goals, cutting through intentionally featured resistances with popular and/or heuristic recipes. Second, by facilitating the production of images (outputs or screenshots), it produces an "epistemic surplus" [START_REF] Rieder | Digital methods: From challenges to Bildung[END_REF] susceptible to get repurposed through storyletting. Here it is Gephi that shifts the goals of users.

Conclusion

We have seen that SSH scholars criticize data visualization in general, and network visualization in particular, including Gephi, for being a "black box." This argument blames network maps for appearing self-evident, which leads to forgetting that they come with methodological commitments. By analyzing empirical material from participant observation, interviews, and a publication review, we endeavored to diagnose Gephi's blackboxing problem. We clarified that blackboxing may refer to two different things, and we advocated for dropping the confusing opacity/transparency dichotomy. We called blackboxing by inscrutability the problem of limited or nonexistent access to relevant information about the method performed, and blackboxing by embodiment the problem of invisibilization of the mediation. Each problem calling for a different solution, we assessed each possibility. We observed that Gephi is not perceived as inscrutable, and that many scholars put it to use before they fully grasped its methodological effects. This led us to conclude that Gephi is primarily a black box by embodiment. We observed that some users circulated network images produced by Gephi without providing any interpretative context, a situation we refer to as storyletting. Letting such images telling their own narrative, allows them to get received as self-evident by the audience, as if they did not need to be explained, which contributes to invisibilizing the layers of mediation involved. We also observed that Gephi is often used in time-constrained situations where users are willing to trade methodological awareness for efficiency. On this ground, we argued that Gephi's blackboxing happens for two reasons: because of utilitarian practices in its epistemic subculture, and because it facilitates storyletting by generating an epistemic surplus: network visualizations that cannot be understood outside of the laboratory.

Ultimately, what makes Gephi a black box? Gephi's design philosophy explicitly acknowledged blackboxing as an issue, but it conceived it as a matter of opacity (by inscrutability) to fight with transparency. It therefore missed that transparency could also lead to blackboxing (by embodiment). The same approach is common in algorithm accountability, but as the case of Gephi shows, it fails to address a criticism about the concept of technical mediation, rooted in STS and phenomenology of perception. Indeed, the context of the laboratory is necessary to understand how Gephi gets blackboxed. The time-constrained environment contributes to it; the cultural artifacts of the community, like tutorials promoting a utilitarian use, contribute to it; and of course, Gephi's design choices contributes to it. Users shape their instrument through interactions and inscriptions that extend far beyond its source code. Their repurposing of the tool can gradually get institutionalized through the artifacts of its epistemic culture, and contribute to reframing it. This makes it clear that it takes more than software design to succeed in unblackboxing Gephi. Intervening through critical technical practice requires taking into account the multiplicity of the situations where blackboxing arises, which leads to two major questions. First, how can instrument design contribute or be adapted to caring for a largely selfdetermined epistemic subculture? Second, how to resolve the inherent tension between usability and embodiment? As [START_REF] Latour | On Technical Mediation[END_REF] reminds us, blackboxing is necessary for a mediation to succeed, but it is also reversible. The black box can be opened and embodied, although not at the same time. That point may be worth reminding to SSH scholars: through the place they give to their apparatus in the knowledge they produce, they too can engage in critical technical practice, and contribute to unblackboxing their digital instruments.


Handi Dahmana

Nadia Amanzougaghene

Bernard Davoust

Thomas Normand

Olivier Carette

Jean-Paul Demoncheaux

Baptiste Mulot

Bernard Fabrizy

Pierre Scandola

Makhlouf Chik

Florence Fenollar

Oleg Mediannikov

Great diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species

Keywords: Piroplasmosis, Equidae, PCR assays, Sub-Saharan Africa, France

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

Introduction

Apicomplexa protists are stealth invaders, they can escape the immune response in host cells while using them as a source of nutrients [START_REF] Striepen | Building the perfect parasite: Cell division in apicomplexa[END_REF]. Almost all apicomplexan are parasites, including multiple pathogenic species, both for humans and animals like malaria, toxoplasmosis, cryptosporidiosis, and piroplasmosis.

The parasites belonging to the apicomplexan order Piroplasmida include three genera namely: Babesia, Theileria, and Cytauxzoon (Megan E. Schreeg et al., 2016). Equine piroplasmosis is an infectious tick-borne disease caused by the hemoprotozoan parasites Theileria equi and Babesia caballi [START_REF] Wise | Review of equine piroplasmosis[END_REF]. These piroplasms affect equid species, including horses, donkeys, mules and zebras [START_REF] Wise | Review of equine piroplasmosis[END_REF]. T. equi was initially named Piroplasma equi by Laveran in 1901, but after the discovery of schizogony in horse lymphocytes, which is known for Theileria but not for Babesia, this parasite was reclassified by Mehlhorn and Schein as T. equi [START_REF] Uilenberg | Babesia-A historical overview[END_REF]. In 1912, B. caballi, formerly named Piroplasma caballi, was first identified as another parasite infecting equids and different from T. equi and was assigned to the genus Babesia [START_REF] Nuttall | On the Occurrence of two Species of Parasites in Equine "Piroplasmosis" or "Biliary Fever[END_REF].

Equine piroplasmosis caused by B. caballi and T. equi is endemic in tropical and subtropical zones; the latter is most prevalent [START_REF] Friedhoff | An account on equine babesioses[END_REF]. Southern Europe and Africa are highly endemic [START_REF] Wise | Equine Piroplasmosis[END_REF]. Moreover, a high genetic diversity of these parasites has been observed in Tunisia [START_REF] M'ghirbi | Prevalence and genetic diversity of piroplasm species in horses and ticks from Tunisia[END_REF] and Jordan [START_REF] Qablan | Infections by Babesia caballi and Theileria equi in Jordanian equids: epidemiology and genetic diversity[END_REF].

Equine piroplasmosis has serious impacts on horses' health. Agricultural production is strongly affected with high cost of control rules and impact on the carriage of merchandises and international commerce(World Organization for Animal Health. Biological Standards Commission., 2012). In the United States, with a population of about 9.2 million horses, the direct economic impact of the equine industry is about 39 billion dollars per year. This industry also supports approximately 1.4 million full-time jobs (The American Horse Council, 2005). It took 25 years and $23 million for Southern California to eradicate equine piroplasmosis [START_REF] Usda | Equine Piroplasmosis and the[END_REF].

For a long time, T. equi and B. caballi were considered specific to their hosts. However, both parasites were recently identified in clinically healthy dromedaries by PCR, in Jordan [START_REF] Qablan | Quest for the piroplasms in camels: Identification of Theileria equi and Babesia caballi in Jordanian dromedaries by PCR[END_REF]. Both were also detected in a dog in Croatia in 2009 [START_REF] Beck | Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia[END_REF].

In France, in 2010, Fritz et al. analyzed 166 dogs;31 were infected by T. equi and one by B. caballi [START_REF] Fritz | A PCR study of piroplasms in 166 dogs and 111 horses in France (March 2006 to March 2008)[END_REF]. Finally, Babesia canis, Babesia rossi and Babesia capreoli were recently reported to infect also horses [START_REF] Fritz | A PCR study of piroplasms in 166 dogs and 111 horses in France (March 2006 to March 2008)[END_REF][START_REF] Zanet | Horses infected by Piroplasms different from Babesia caballi and Theileria equi: species identification and risk factors analysis in Italy[END_REF].

Piroplasmosis is a typical zoonotic vector-borne infection [START_REF] Schnittger | Babesia: A world emerging[END_REF]. However, T. equi and B. caballi are not considered to infect humans [START_REF] Maslin | Babésioses[END_REF]. Ticks and iatrogenic blood transfers are efficient modes of transmission in equids [START_REF] Ueti | Ability of the vector tick Boophilus microplus to acquire and transmit Babesia equi following feeding on chronically infected horses with low-level parasitemia[END_REF]. More than 21 tick species, mainly hard ticks, are associated with the transmission of these parasites. Co-infections are frequently reported in Equidae, often associated with coinfestation by tick species of the genera Dermacentor, Hyalomma and Rhipicephalus, [START_REF] Tamzali | Equine piroplasmosis: An updated review[END_REF]. Babesia and Theileria exhibit different ecological relationships with their vectors and hosts. The reservoirs of Babesia include chronically infected animals and ticks [START_REF] Yabsley | Natural history of Zoonotic Babesia: Role of wildlife reservoirs[END_REF]. Infected ticks are able to transovarial and transstadial transmission of B. caballi from female ticks to its offspring. In contrast, Theileria are only transmitted transtadially [START_REF] Ueti | Ability of the vector tick Boophilus microplus to acquire and transmit Babesia equi following feeding on chronically infected horses with low-level parasitemia[END_REF]. Besides, Equidae are the primary reservoirs of Theileria.

Clinical signs of equine piroplasmosis are similar for both parasites [START_REF] Tamzali | Equine piroplasmosis: An updated review[END_REF].They occur after transmission within 10 to 30 days for B. caballi and 12 to 19 days for T. equi [START_REF] De Waal | Equine piroplasmosis: A review[END_REF]. In endemic areas, most of infected horses are asymptomatic carriers with low level of parasitaemia. In case of concurrent disease or stress, they may develop clinical equine piroplasmosis [START_REF] Allsopp | Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals[END_REF]. Acute forms of equine piroplasmosis can include fever (over 40°C), sweating, congested mucous membranes, limb and supraorbital edema, icterus, anorexia, tachypnoea, tachycardia, anemia, and occasionally petechiae or ecchymoses [START_REF] Tamzali | Equine piroplasmosis: An updated review[END_REF]. Death may occur in severe cases.

In this study, our aim was to develop sensitive and specific molecular tools able to potentially detect all piroplasm species and to analyze their phylogeny, in order to perform an epidemiological study on Equidae from sub-Saharan Africa and France.

Materials and methods

Study area and samples collection

In total, 548 blood samples and 97 hard ticks were collected from Equidae (horses and donkeys). Blood samples were collected in four countries from sub-Saharan Africa (Chad, Senegal, Djibouti, and Democratic Republic of the Congo [DR Congo] and from France (Marseille and Corsica) (Figure 1). Hard ticks have only been collected in Chad. All equines were apparently healthy at the time of sampling (Table S1).

In Chad, we had two periods of sampling of saddle horses belonging to the Chadian National Guard (GNT). Ninety-six male horses (from 4 to15-year-old) were sampled in 2012 and 60 in 2016 (59 male and one female). The infestation of horses by ticks is noticeable during wet periods especially, with 97 ticks collected in March 2016. In Senegal, an epidemiological survey, conducted from 2011 to 2014, allowed the inclusion of 126 horses and 54 donkeys from north-west villages. In Djibouti, in the Decan Reserve, 5 horses and 11 Somali wild donkeys (Equus asinus somalicus) were sampled in March 2010. In DR Congo, a screening survey performed in August 2012 at the Equestrian Club in Kinshasa covered 48 sport and leisure horses (34 males and 14 females from 5 to 33year-old). In the city of Marseille (France), a study was conducted in February 2015on 51 horses (30 males and 21 females from 7 to 35-year-old). Finally, in Corsica (French island in the Mediterranean Sea), 98 horses (59 males and 39 females from 1 to 29-year-old) were sampled in September 2014, in 13 equestrian centers.

The blood sampling was done aseptically using an ad-hoc device (BD Vacutainer® system; Becton Dickinson, Franklin Lakes, New Jersey, USA) and a single use needle (20G -0,9X40 mm) at the jugular vein. Vacuum tubes with EDTA K3 were used to collect the blood.

Then, specimens were stored and transported at +4°C. On arrival at the laboratory, specimens were frozen at -80°C.

The 97 ticks were collected manually from Chadian horses in 2016 and stored in 70% ethanol until they were identified under a binocular microscope. We used the available taxonomic keys and morphometric table to classify the ticks by family, genus and species (Walker et al., 2003). Each tick was washed three times in distilled water and stored at -20°C until DNA extraction.

DNA extraction

EZ1 DNA Kits (Qiagen, Courtaboeuf, France) have been used for the DNA extraction.

The DNA was extracted from 200 µl of blood and from half of a tick. DNA extracts were then stored at -20°C until PCR analysis.

Piroplasmida specific-PCR tools design

In order to detect piroplasms DNA, we designed primers and probe targeting the most conserved regions of encoding ribosomal RNA genes (28S, 18S, and 5.8S), as well as primers targeting mitochondrial gene (cox1). House-keeping genes that are typically constitutive genes, required to maintain basal cell function and that are reliable to study piroplasms phylogeny (Megan E Schreeg et al., 2016).

First, a real time PCR assay for the screening of all piroplasms targeting a conserved region of the 5.8S gene was developed. Then, for species identification, we designed three conventional PCR assays targeting 969-bp,750-bp, and 480-720-bp sequences from the 18S, 28S, and cox1 genes, respectively. In order to detect an eventual co-infection by the two equine piroplasmosis agents, PCR assays for specific amplification of B. caballi were developed targeting a 2,930-bp sequence of the 28S gene.

All primers and probe for real time PCR and conventional PCR assays were designed using free web Primer3 software, version 4.0 (http://frodo.wi.mit.edu/primer3/); their sequences were listed in Table 1. The specificity and sensitivity of all PCR assays were tested in silico using primer-BLAST (NCBI, USA) and were validated using a panel of DNA extracts from several species of piroplasms, arthropods and bacteria, as well as from human, donkey, horse, cattle, mouse and dogs (Table S2).

Piroplasmid DNA detection, PCR amplification and phylogenetic analysis

The analytical sensitivity of the 5.8S real time PCR was assessed using 10-fold dilution from horse infected blood, which harbored 1.64 E+5 parasites per ml (100ul of blood extracted and eluted in 50ul using EZ1 DNA Kits (Qiagen, Courtaboeuf, France).

The initial screening was performed using the 5.8S-based real time PCR assay. Then, the identification of Piroplasmida species in real time PCR-positive samples was based on the amplification of a 969-bp fragment of 18S rRNA gene. Finally, the 28S rRNA and cox1 genes were amplified from selected samples representing different genetic variants based on the 18S rRNA sequence analysis.

Reaction mix for the 5.8S real-time PCR contained 5 μl of the DNA template, 10 μl of Eurogentec Takyon™ Mix (Eurogentec, Liège, Belgium), 0.5 μl (20 μM) of each reverse and forward primers, 0.5 μl (5 μM) of the FAM-labeled probe (Table 1) and3 Conditions for conventional PCR were one incubation step at 95°C for 15 minutes, 40 cycles of one minute at 95°C, 30 seconds annealing at a different hybridization temperature for each PCR assay and one minute at 72°C followed by a final extension for five minutes at 72°C (Table 1). Negative and positive controls were included in each molecular assay. The success of amplification was confirmed by electrophoresis on a 1.5% agarose gel. The purification of PCR products was performed using NucleoFast 96 PCR plates (Macherey-Nagel, Hoerdt, France) according to the manufacturer's instructions.

Co-infections were confirmed by amplifying different species from the same DNA after cloning using PGEM easy vector system II A1380 (Promega, Charbonnières-les-Bains, France). The amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). The obtained sequences were assembled and edited using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia). Then, sequences were compared with those available in the GenBank database by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Our sequences obtained from positive samples in addition to sequences of validated piroplasms species already available in Genbank were aligned using Bioedit software version 7.0.5.3 (ClustalW multiple alignment) (Tom Hall, 2011). For taxonomic analyses, the Maximum-likelihood phylogenetic trees constructed using MEGA software version 7.0.21 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]with 100 bootstrap replications and species position confirmed using Topali software version V2.5 (2.5.13.04.03) [START_REF] Milne | TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops[END_REF]. More specific information found in front of figures.

Statistical analysis

The Epi Info version 7 program (http://www.cdc.gov/epiinfo/index.html) was used to compare prevalence. A difference was statistically significant when p-values were <0.05.

Results

Sensitivity and specificity of PCR assays

The 5.8S real time PCR, as well as the standard 18S RNA-based PCR assay, allowed the amplification of the following piroplasmids: T. equi, B. caballi, Theileria ovis, Theileria annulata, Theileria buffeli, Babesia canis, and Babesia vogeli, all are already identified and sequenced in our laboratory (Table S2). All 18S amplicons from controls were sequenced.

The obtained sequences allowed an accurate identification of all controls. None of the DNA extracts from negative controls were amplified.

The real time PCR is able to detect the presence of parasites with an efficiency of 121.5%, slope: 2.895, Y.int: 41.44 and an almost perfect coefficient higher than 0.98. The limit of detection was fixed at 1.64E+2 parasites/ml in blood which means 0.82~1 copy per reaction (Supp. Table S5)To insure the reproducibility of the results, samples from the DRCongo (n=48) were tested simultaneously by 5.8S real time PCR and 18S PCR tools. Both gave the same result (21 positive on 48 samples). None of them missed positive samples; 5.8S real time PCR tool was then used for screening in all our samples.

Results of the PCR tools on the samples

In total,548 blood samples of Equidae were tested for the presence of piroplasms' DNA using the real time PCR assay. 29.56% were found positive, including 54 samples from France and 108 from sub-Saharan Africa. The overall results on blood samples are presented in Table 2.

In France, real time PCR screening detected 15/51 (29.4%) positive horses in Marseille, and 39/98 (39.8%) positive horses from Corsica. There is no statistically significant difference in the prevalence of piroplasm between these two studied sites (Table 3).

In sub-Saharan Africa, we found 23/180 (12.8%) positive Equidae from Senegal and 21/48 (43.75%) positive horses from DR Congo. Only one horse of 16 (6.25%) Equidae from Djibouti was tested positive. All 11 wild Somali donkeys were found negative. In Chad, the prevalence was significantly higher in 2016 (43/59, 72.9%) than in 2012 (20/96, 20.8%), p<0.0001(Table S3).

Considering the species of Equidae we studied, the prevalence of piroplasm was significantly higher in horses (160/484, 33.06%) than in donkeys (2/64, 3.12%), p<0.0001.

The infection was also observed more often in females (46/124, 37,09%) than in males (116/424, 27.36%), p<0.20. More detailed data are given in Table 3.

Of the 97 ticks collected in Chad (2016), only 9 (8 female and one male ticks) were positive (9.27%). Interestingly, all positives ticks were Rhipicephalus decoloratus, which were collected from two horses (coded MGM04 and MGM10) that were also found positive for piroplasms.

Piroplasms species identification

To identify piroplasms species, all positive samples were subjected to standard PCR (targeting 18S rRNA gene) coupled with sequencing. Sequencing results showed the existence of a large genetic diversity (Table 4) allowing us to identify four piroplasms species: T. equi, B. caballi and two potential new species; provisionally named here Theileria sp. "Africa" and Theileria sp. "Europa". The highest prevalence was observed for Theileria sp. "Africa" (51.85%, 84/162) followed by T. equi (28.39%, 46/162), Theileria sp. "Europa" (19.14%, 31/162), and B. caballi (0.62%, 1/162). Only one horse from Senegal was positive for B. caballi.

We selected some samples corresponding to each 18S genetic variant to sequence using 28S and cox1 genes (Table 1). All sequences were submitted to the Genbank and their accession numbers were obtained (Table S3). A horse from Senegal was co-infected by T. equi and B. caballi which is confirmed by amplifying the two species from the same DNA after cloning.

Phylogenetical analysis

The phylogenetic trees constructed on the base of 18S, 28S and cox1 genes allowed the position of the identified species to be visualized (Figures 2,3,and 4,respectively). Both Theileria sp. "Africa" and Theileria sp. "Europa" formed separate clades with samples of different geographical origins grouped together with a good bootstrap support (Figures 2, 3, and4).

In a second time, we compared sequences of three genes of Theileria spp. identified in the present study with T. equi sequences of WA strain and USDA strains available in the Genbank (Table S4). We discovered that the differences among T. equi and the two potential new species varied between 4 to 14%.

Discussion

To the best of our knowledge, all published PCR-based assays are able to amplify either only one species or a group of piroplasms [START_REF] Qurollo | Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA[END_REF][START_REF] Salem | Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis[END_REF]. Herein, we have developed a real time PCR assay targeting the 5.8S gene able to detect well-known equine pathogens (T. equi and B. caballi), well-known mammal pathogens as well as potential new species. The 5.8S gene is a conservative gene easy to amplify by PCR using one pair of universal primers [START_REF] Gou | A DNA barcode for Piroplasmea[END_REF]. For the design of our 5.8S real time PCR assay, highly divergent regions flanked by highly conserved regions among piroplasms have been targeted from 32 aligned sequences of Theileria spp. and Babesia spp. The designed real time PCR assay exhibited a good sensitivity. As expected in silico, the tool allowed an accurate in vitro detection of all known piroplasms, as well as those not yet described. Besides, none of the DNA extracts from the control group were amplified with this molecular tool confirming its specificity. The cox1 gene is one of the most used markers for population genetics and phylogeographic studies [START_REF] Derycke | Exploring the Use of Cytochrome Oxidase c Subunit 1 (COI) for DNA Barcoding of Free-Living Marine Nematodes[END_REF]. The 18S and 28S genes, generally considered to be highly conserved, are actually composed of a mixture of conserved and divergent regions [START_REF] Gou | A DNA barcode for Piroplasmea[END_REF]. For the design of the 28S and cox1 PCR assays, 35 and 34 sequences were respectively aligned. The molecular phylogeny of Babesia and Theileria is usually based on 18S sequences [START_REF] Gou | A DNA barcode for Piroplasmea[END_REF]. For the design of our 18S PCR assay, the combination of primers F1 and R4 showed high specificity.

Overall, we analyzed 548 blood specimens from apparently healthy Equidae sampling in four sub-Saharan African countries (DR Congo, Djibouti, Chad, and Senegal) and France (Marseille and Corsica). The overall prevalence of piroplasms detected in blood samples was 29.6% (162/548). Only one case of B. caballi was diagnosed in Senegal, in a horse. All the other samples were positive for Theleiria spp. Thus, the overall prevalence of Theleiria spp. was28.4%. Besides, two new potential Theleiria species were detected, Theileria sp.

"Europa" and Theileria sp. "Africa". Theileria sp. "Europa" exhibited a prevalence of 5.7% (31/548). Theileria sp."Africa" was more commonly observed with a prevalence of 15.3% (84/548). This higher prevalence observed in Chadian horses in 2016 compared to 2012 can be explained by the inclusion of another sampling site such as Amguifel (Table S1). Previous studies reported PCR-negative samples while indirect fluorescent antibody (IFA) tests were positive. One of the hypotheses was the existence of variants that have not yet been identified and therefore cannot be detected by the available PCR tools [START_REF] Bhoora | Development and evaluation of realtime PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa[END_REF]. Indeed, we have identified here different genotypes that can easily be omitted by conventional PCR tools (Table 4).

Ticks from the genera Dermacentor, Hyalomma and Rhipicephalus are natural vectors of Babesia and Theileria [START_REF] Tamzali | Equine piroplasmosis: An updated review[END_REF]. The 97 ticks studied from Chad belonged to the two following genera: Hyalomma (4.1%, 4/97) and Rhipicephalus (95.9%, 93/97). Nine ticks (9.3%, 9/97) identified as Rh. decoloratus were found positive for Theileria sp. "Africa".

Besides, these ticks were found on two horses which were also positive for Theileria sp. "Africa". More studies on these ticks must confirm or disprove their capacity to transmit piroplasms.

Our study does not claim to be representative because of the reduced number of Equidae tested. Furthermore, our sampling was carried out in an opportunistic manner but it gives a picture on the epidemiological situation in the studied areas. The horses tested are certainly protected by a pre-immunization while latent infection persists. Tick infestations are regularly observed in areas where our study has been conducted and sporadic clinical cases of piroplasmosis are reported by veterinarians. It is therefore important to limit the infestation by ticks with external deworming measures to control the diseases they transmit. Asymptomatic infection is very persistent (this is known for T. equi) and tick infestation (especially nymphs) may pass unnoticed.

The sole case of B. caballi was identified in a horse from Senegal, which was coinfected with T. equi. We should notify that the sampling time plays a critical role in the detection of circulating parasites. Indeed, infections by B. caballi are self-limiting. Horses are generally able to eliminate the infection within one to three years naturally or after a sterilizing treatment [START_REF] Friedhoff | An account on equine babesioses[END_REF].

T. equi and B. caballi are the parasites most frequently involved in equine piroplasmosis. However, recent studies have reported the existence of other piroplasms species in horses. Indeed, B. canis has recently been reported in horses in France and Italy and B. capreoli in Italy [START_REF] Fritz | A PCR study of piroplasms in 166 dogs and 111 horses in France (March 2006 to March 2008)[END_REF][START_REF] Zanet | Horses infected by Piroplasms different from Babesia caballi and Theileria equi: species identification and risk factors analysis in Italy[END_REF]. The analysis of three different genes of the two-potential new Theileria genotypes clearly showed a distant position between these species and the others. These data strongly support the fact that they differ from other recognized species, including T. equi (Table S4). New species are isolated from equines and described and validated using phylogenetic analyses based on both 18S rDNA sequences and 223 nuclear-encoded protein-coding genes, extensive genome-wide differences and with predicted protein divergence with T. equi [START_REF] Knowles | Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria : implications for apicomplexan parasite surveillance[END_REF]. Finally, the pathogenicity of these potentially new species remains unknown.

Conclusion

Overall, sensitive and specific PCR assays have been developed to potentially identify all piroplasm species and to study their phylogeny. Four horse-infecting piroplasms, including two potentially new species, were identified in Equidae from sub-Saharan Africa and France.

However, few epidemiological investigations on equine piroplasmosis are conducted. Our study provides a better understanding of the situation. Further studies, covering larger geographical areas and larger number of samples, are nevertheless required to improve exploration of the health status of Equidae. These studies will assess more specifically the actual prevalence of equine piroplasmosis and its impacts on equine populations. Moreover, the establishment of high rigorous prophylaxis plans, allowing both surveillance and control of equine piroplasmosis should be proposed. The involvement of public authorities in the detection of these diseases remains essential to contain them, or at least to reduce their spread.

  .5μl of distilled water DNAse and RNAse free, for a final volume of 20 μl. The real time PCR amplification was carried out in a CFX96 Real-Time system (Bio-Rad Laboratories, Foster City, CA, USA) using the following thermal profile: Incubation at 50°C for two minutes for UDG action (eliminating PCR amplicons contaminant), then activation step at 95°C for three minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing-extension at 60°C for 30 seconds. Conventional PCR amplifications were performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). PCR reactions contained 5 μl of the DNA template, 25 μl of Amplitaq-Gold STAR™ Mix (Eurogentec, Liège, Belgium), 10 μM (1 μl) of each primer, and 18 μl of distilled water DNAse and RNAse free.

Figure 1 .

 1 Figure 1. Map of countries where samples were collected in sub-Saharan Africa and Europe.

Figure 2 .

 2 Figure 2. Maximum-likelihood phylogenetic tree of piroplasms, including our new species

Figure 3 .

 3 Figure 3. Maximum-likelihood phylogenetic tree of piroplasms, including our new species

Figure 4 .

 4 Figure 4. Maximum-likelihood phylogenetic tree of piroplasms, including species identified in the present study based on partial 480-bp cox1 gene.

Fig 1 Figure 2 JX177671

 12 Fig 1

Table 1 .

 1 Oligonucleotide sequences of primers and probe used for qPCRs and conventionalPCRs in this study.

	Targets	Targeted	Name	Primers (5'-3') and probe	Annealing
		gene			temperature
	Piroplasmida 5.8S	5.8S-F5	AYYKTYAGCGRTGGATGTC	60°C
			5.8S-R	TCGCAGRAGTCTKCAAGTC
			5.8S-S	FAM-TTYGCTGCGTCCTTCATCGTTGT-MGB
		18S	piro18S-F1	GCGAATGGCTCATTAIAACA	58°C
			piro18S-F4	CACATCTAAGGAAGGCAGCA
			piro18S-F3	GTAGGGTATTGGCCTACCG*
			piro18S-R3	AGGACTACGACGGTATCTGA*
		28S	28S-F2	CACCCCYGKCCGTACDGAT	58°C
			28S-R2	CCTCAYTGAGYTYGCCTTRGGAC
	B. caballi	28S	28Scab-F4	GCAGAAAAGAAAATAACCATG	62°C
			28Scab-R3	GCGTTCAGTCATTATCCAACGG
			28Scab-F6	GCTTTAGCACGGTTGCTAGGA*
			28Scab-F7	GGCATAGCAGTCCGGYYTTCGG*
			28Scab-F8	TGTCCCTATCTGCCATCTAG*
			28Scab-R6	TCGCCCCTATACCCGGATTTG*
			28Scab-R7	CCGAARRCCGGACTGCTATGCC*
		cox1	piro-F	AGGAAGTGGWACWGGITGGA	57°C
			piro-R	GATGISCCCAIACIARACAWCC
	Theileria sp.	cox1	383-F3	GAAGTGGWACHGGDTGGAC	57°C
	"Europe"		900-R1	CCHGADGTATACATATGRTG
	Theileria sp.	cox1	COI-F	GTGAYGTTGTTTTTCCAAG	57°C
	"Africa" &		COI-R	CCWGTTGTACCTCCAAYDAC
	Theileria equi
	*Primers used for sequencing only

Table 2 .

 2 Overall prevalence of piroplasms in blood and tick samples.

		Blood	Ticks
	Country	Number of positive / Number of tested samples (%)
	France (Marseille)	15/51 (29.4)	NA
	France (Corsica)	39/98 (39.8)	NA
	Senegal	23/180 (12.8)	NA
	DR Congo	21/48 (43.75)	NA
	Chad (2012)	20/96 (20.8)	NA
	Chad (2016)	43/59 (72.9)	9/97 (9.27)
	Djibouti	1/16 (6.25)	NA
	Total	162/548 (29.6)	9/97 (9.27)
	NA: Not available

Acknowledgements

This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03, the Région Provence Alpes Côte d'Azur and European funding FEDER PRIMI.

The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

We thank Masse Sambou, Maxence Aubadie ( †), Raphaël Tine, Mamadou Diara, Pierre Verhaeghe, Bertrand Lafrance, Mustapha Dahmani, Younes Laidoudi and Rima Saad-Eddine for their assistance during the field and laboratory work. We also want to thank the command of National and Nomadic Guard (GNNT) of Chad, the French Armed Forces Medical Service, in particular Jean-Lou Marié and the « Association Pour la Recherche en Infectiologie» (APRI) for their financial support.