Alexandre Lodie

The Conciliation Of Transparency Measures with the Processing of Possibly Sensitive Data by the Administration According to the French Administrative Judge*

9 of the GDPR.

Introduction

The free flow of data has become a central concern for the European market, as emphasised by the European Data Protection Supervisor (EDPS), Wojciech Wiewiorowski, who stated during the G7 DPA Roundtable in September 2022 that the "free flow of data is not only necessary to our digital economies and societies but even a precondition for a world placed under the auspices of cooperation and multilateralism". 1 This issue is related to the Big Data phenomenon which designates the inflation of data available online be they "generated from online transactions, emails, videos, audios, images, click streams, logs, posts, search queries, health records, social networking interactions, science data, sensors and mobile phone". 2 Public administration and public bodies are no exception when it comes to processing and storing data. As such, they must be considered as a data-sharing stakeholder, hence the proposal on the European level of the Data Governance Act which aims "to address the barriers to a well-functioning data-driven economy and to create a Union-wide governance framework for data access and use, in particular regarding the re-use of certain types of data held by the public sector". 3 Besides, States and public administration are encouraged to publish their data to improve the transparency of the public life and decision-making processes, it is what some States call "open data" policies. 4 However, such a wide data disclosure might sometimes run contrary to individuals' data protection. This issue is all the more critical when sensitive data related to health, cultural or ethnic origin, political opinions, sexual orientation are at stake. 5 The French Highest Administrative Court (Conseil d'État) released in 2021 a decision on the conciliation between the publication of administrative documents on the one hand and the protection of individuals' data, including sensitive data, on the other. 6 In this case, a public agent had been nominated as Inspector of Finance by virtue of a decree bearing on the access of disabled persons to state functions. 7 The appointment order, mentioning the said decree, was consequently published on the website of the Ministry of Economy and Finance. The agent considered that such a publication constituted a violation of his private life and did not comply with the GDPR. He asked French administrative Courts to delete his name and date of birth from the appointment order. In front of their refusal, the case was brought to the Conseil d'État, which is the French Highest Administrative Court.

From this background the Conseil d'État had to settle several issues such as whether the publication of said appointment order constituted data processing by automated means subject to the GDPR. Then the Conseil d'État had to determine whether such processing could be seen as "data processing concerning health" as regards Article 9 of the GDPR. This decision thus questions in a broader manner what can be considered as data processing by automated means, what sensitive data really are and how to conciliate the duty of the administration to publish administrative documents on the one hand with individuals' right to data protection on the other.

A broad conception of "data processing" in accordance with the ECJ view

One of the arguments put forward by the claimant was that the publication of the appointment order infringed European data protection law, including the GDPR. The problem was that the Appellate Court dismissed the application of the GDPR to the case since it claimed that "neither the publication by computerised means of a decree appointing public servants containing only the names of the persons concerned and an indication of the legal basis for their appointment, nor the decision refusing to put an end to this publication, could be regarded as relating to the processing of personal data by electronic means". 8 In other words, the Appellate Court considered that the publication of the appointment order did not constitute data processing, within the meaning of the GDPR.

The Conseil d'État however disapproved such a view, and repealed this decision by stating that "in rejecting the application of these rules, when the mere publication of personal data on a website is sufficient to make them applicable, the administrative court of appeal made an error of law". 9 The main issue was to consider whether the online publication of information regarding an individual could be considered as data processing.

By answering in the affirmative, the Conseil d'État seems to agree with the definition of data processing acknowledged by the European Court of Justice (ECJ). Indeed, in the Lindqvist decision, the ECJ considered that the publication of information related to an individual on a web page constituted data processing according to Article 3 of the GDPR. 10 More specifically the Court concluded that "the act of referring, on an internet page, to various persons and identifying them by name or by other means, for instance by giving their telephone number or information regarding their working conditions and hobbies, constitutes the processing of personal data wholly or partly by automatic means". 11 According to some scholars, following Article 3 of the GDPR, "any operation on the data constitutes processing, especially as the list is merely illustrative" [START_REF] Castets-Renard | La protection des données personnelles dans les relations internes à l'Union européenne[END_REF] which makes such a definition "particularly broad". 13 By concluding that the publication of an appointment order in this scenario was data processing subject to the GDPR the Council of State has therefore followed in the ECJ footsteps.

Another case law on the European stage tackled a similar issue. As a matter of fact, the ECJ, in its Google Spain decision released in 2014, used the same reasoning and concluded that "it is not contested that the data found, indexed and stored by search engines and made available to their users include information relating to identified or identifiable natural persons and thus 'personal data'". 14 It should logically be acknowledged that "when Google does this on its own page, it is itself carrying out such processing since it collects, extracts, records, indexes and makes available the personal data of third-party sites". [START_REF] Aubert | Chronique de jurisprudence de la CJUE: CJUE 13 Mai 2014[END_REF] Therefore, it can be deduced from ECJ case law that the mere publication of information related to an individual on a website or on search engines web pages constitute data processing subject to the GDPR.

To summarise, the French Conseil d'État, in its decision, adopts a similar approach to that of the ECJ, by considering that the publication of a civil servant's appointment order on the Ministry of Economy and Finance's website constitutes data processing by automated means which is regulated by the GDPR.

A restrictive view of what constitutes processing of "special categories of data"

The claimant argued that the publication of the appointment order, mentioning the decree on the access of disabled persons to state functions violated his right to privacy. The processing of data related to the health status of a data subject is not only data processing subject to the GDPR, but also processing of "special categories of data". However, the Conseil d'État surprisingly considered in this case that the administration did not process data concerning health, which questions what can be considered as processing of "special categories of data" revealing sensitive characteristics.

A narrow interpretation of health data

Once the Conseil d'État acknowledged that the GDPR applied, it had to determine whether such processingwhich was indirectly revealing16 the health status of the data subjectconstituted processing concerning health data. 17 Health data is a category of data which is defined pretty broadly by EU data protection law as "personal data related to the physical or mental health of a natural person, including the provision of health care services, which reveal information about his or her health status". 18 For instance, in the Lindqvist case cited previously the ECJ claimed that "the expression data concerning health […] must be given a wide interpretation so as to include information concerning all aspects, both physical and mental, of the health of an individual" 19 and that "reference to the fact that an individual has injured her foot and is on half-time on medical grounds constitutes personal data concerning health". 20 A semantic clarification must be made as a preamble: Article 9 makes a distinction between "processing of personal data revealing racial or ethnic origin, political opinions […]" 21 on the one hand and "data concerning health", 22 on the other. This distinction suggests that while an indirect connection between the processing and the nature of the data is enough to consider certain processing as processing of categories of data, the connection must be direct when considering health data. However, this interpretation has been rejected by the ECJ which considered that "personal data concerning health should include all data pertaining to the health status of a data subject which "reveal" information relating to the past, current or future physical or mental health status of the data subject". 23 Interestingly, the Conseil d'État does not reach the same conclusion at all, as it claims that "although the posting of such information online indirectly reveals that the persons recruited in this capacity suffer from a disability, it does not directly provide any information on the nature or seriousness of this disability and cannot therefore be regarded as processing data relating to the health of the persons concerned". 24 The degree of seriousness of a disability or an injury or the specificity of a disability are not relevant criteria as regards Article 9 of the GDPR, so such a statement by the Court can be a bit surprising. The Court's reasoning does not seem to be consistent, in the sense that it acknowledges that the processing involved indirectly reveals that the data subject suffers from a disability, but at the same time, it refuses to draw the right conclusion from this statement and to consider such processing as processing of "special categories of data" while the latter encompasses processing of data "pertaining to the health status" 25 of a data subject.

It is worth recalling that it is not the first time that the Conseil d'État makes the connection between the seriousness or nature of an injury or disability and the qualification of data as "data concerning health". For instance, in a decision which dates back from 2014, the Court stated that "the mention of the permanent incapacity rate or the disability rate of the "spouse or partner" and of the staff member's dependents is not data "relating to health" […] since it is not even alleged that it would provide information on the nature of the disability". 26 The Court concluded in the same manner in relation to a file on education facilities which contained also information relating to care facilities where pupils can be enrolled. 27 From a broader perspective this decision questions how to determine when data processing must be considered as processing bearing on "special categories of data".

3.2.A decision in conflict with the European view on what constitutes "special categories of data"

As mentioned previously the Conseil d'État adopted a quite narrow definition of what "health data" are while this category refers to "special categories" of data which benefit from additional protections under the GDPR. 28 Very few case law are related to the specific issue of which data can be considered as "indirectly revealing" sensitive characteristics and thus as "special categories" of data. Furthermore, none of themto our knowledgerefers to data revealing the health status of a data subject. However, it is worth analysing what the ECJ and data protection authorities throughout the EU have stated as regards data revealing sensitive characteristics, although such case law do not bear on health data.

The ECJ got referred lately for a preliminary ruling, in a case involving transparency measures required by the administration on the one hand and sensitive data protection issues on the other. The question that the Court had to settle was thus pretty similar to the Conseil d'État's, although not bearing on health data but on other sensitive data provided for by Article 9 of the GDPR.

In this case, the director of a Lithuanian public body had to release a declaration of interest containing several personal information about him and his partner, which was likely to reveal at least some sensitive characteristics, such as his sexual orientation. 29 Said declaration of interest was intended to be published, as required by Lithuanian Law. The ECJ was therefore asked to determine whether such data processing could be considered as processing sensitive data.

The ECJ concluded that "the publication, on the website of the public authority responsible for collecting and checking the content of declarations of private interests, of personal data that are liable to disclose indirectly the sexual orientation of a natural person constitutes processing of special categories of personal data". 30 Even though the processing was not directly related to the sexual orientation of the data subjects the ECJ considered that it was processing of special categories of data.

The ECJ, in its decision, does not make any relationship between the nature or specificity of sensitive characteristics and the nature of the data processing as "sensitive", lying in the scope of Article 9 of the GDPR. It is also possible to consider that the ECJ, in the abovementioned Lithuanian use case31 reached this conclusion because the information contained in the declaration of interest was revealing a specific sexual orientation, but such an interpretation would be far-fetched since the Court does not say anything on this specific point.

The decision of the Conseil d'État was released one year earlier, so it could not foresee what the ECJ would have stated in such a situation. However, one could have expected the Conseil d'État to follow a similar reasoning to that of the ECJ even though the nature of the data concerned was not exactly the same.

In any case, the view expressed by the Conseil d'État is likely to limit strongly the scope of Article 9 of the GDPR and the protection of individuals' sensitive characteristics. Such a restrictive view is all the more surprising that, in any case, the administration could have relied upon the "substantial public interest" 32 exception. In other words, even if the Conseil d'État had acknowledged that the online publication of an appointment order containing sensitive information constituted processing of "special categories of data", French administration would have possibly been able to carry out such processing since the publication of civil servants' appointment orders is mandatory under French Law and possibly serves a substantial public interest. The judges interestingly mention the exception of substantial public interest by citing the GDPR, without further explanation. It suggests that the administration could possibly rely on this exception.

To summarise, the Conseil d'État adopts a restrictive view of what constitutes processing of data concerning health, claiming that the sensitive nature of the data (and consequent processing) depends on the specificity and seriousness of the disability.

It is worth noting that the Norwegian DPA also had to address a similar issue and adopted a view which can be considered as the opposite of the Conseil d'État's reasoning on whether data need to be precise to be considered as data of a sensitive nature.

In this case the data protection authority had to determine whether a dating app for LGBTQI+ people processes data of a sensitive nature by revealing data subjects' sexual orientation. The Norwegian Datatilsynet claimed that "being a Grindr user strongly indicates, and appears in most cases to accurately reflect, that the data subject belongs to a sexual minority. […] As established above, the wording of Article 9 does not require a revealing of a particular "sexual orientation", and the purpose behind Article 9 discourages a narrow interpretation". 33 Even though this use case does not deal with data concerning health, it bears on the interpretation of what processing of "special categories of data" actually is. According to the Norwegian DPA, Article 9 of the GDPR must be interpreted in a broad manner, which indicates that the processing does not need to reveal a specific sexual orientation to be considered as processing of sensitive data. 34 In particular the company argued for its defense that everyone can subscribe to its services and not only LGBTQI+ people, so that the fact of being a Grindr user is not an indication of the user's specific sexual orientation. Despite this argument, the DPA concluded that the use of Grindr was a strong indication of one's sexual orientation, which means that it is reasonable to think that a Grindr's user belongs to a sexual minority. 35 In this case the sensitive characteristic is deduced and not specific, but the Datatilsynet still claimed that this was sufficient to consider the processing as processing of "special categories of data".

The debate on how specific data must be to qualify their processing as processing of sensitive data remains open. For instance, the EDPB claims, considering the relation between video devices and sensitive data, that "video footage showing a data subject wearing glasses 32 See Article 9 of the GDPR. 33 See Datatilsynet, 13 December 2021, 20/02136-18, Administrative fine -Grindr LLC. 34 Ibidem. 35 Ibidem.

or using a wheel chair are not per se considered to be special categories of personal data". 36 However, the processing of a video stream showing individuals who suffer from a disability would logically be seen as data concerning their health status. On the other hand, it would be impossible to acknowledge such an extreme view since almost every data processing would be qualified as processing of sensitive data.

Whereas the Conseil d'État stated that the seriousness and the nature of an injury were decisive factors to consider whether data are of sensitive nature, the Norwegian DPA claimed that a piece of information does not need to reveal a specific sexual orientation to be qualified as "sensitive". From this background it can be concluded that it can be hard to set the bar as whether data reveal sensitive features and thus, whether their processing should be considered as prohibited according to Article 9 of the GDPR.

Eventually, the processing was deemed unlawful by the Conseil d'État on other grounds such as non-compliance with proportionality and data minimisation principles.

The unlawfulness of such processing under necessity, proportionality and data minimisation principles

Even though the Conseil d'État concluded that the publication of an appointment order mentioning a decree on the access of disabled persons to state functions could not be seen as processing data concerning health, it eventually claims that such processing was unlawful because of the way the processing was carried out. In particular, judges consider that "the permanent display of these personal data on the Ministry's website exceeds what is necessary in view of the purposes of the processing in question, which are to guarantee the rights of third parties and respect for the principle of equal access to public employment as set out in Article 6 of the 1789 Declaration of Human and Citizens' rights". [START_REF]Conseil d'État, 10ème -9ème chambres réunies[END_REF] The proposed solution was to delete the legal basis of the appointment order once the latter's period of appeal expired. 38 This dictum is meant to strike a balance between data subjects' data protection rights and privacy on the one hand and the administration's duty to publish appointment orders on the other. This view is in line with the ECJ's case law. Indeed, in the Lithuanian case cited above [START_REF] See | [END_REF] the ECJ considered that "it must be found that the online publication of the majority of the personal data contained in the declaration of private interests of any head of an establishment receiving public funds, such as that at issue in the main proceedings, does not meet the requirements of a proper balance". 40 In other words, the existence of a legal requirement concerning the data processing and the narrow definition of the concept of "data concerning health" would have implied that the individuals' protection was reduced, but these factors are not sufficient to consider that data processing is lawful. Indeed, the Conseil d'État concludes in this case that although the administration did not carry out processing of sensitive data, it was not necessary to keep data online after the period of appeal was over.

Conclusion

In view of the above, it is worth noting that there is a pressing need to determine what constitutes processing of special categories of data, pursuant to Article 9 of the GDPR. There is indeed a wide array of data processing which can indirectly reveal some sensitive characteristics of a data subject. The question as to whether these processing must be considered as processing of sensitive data is largely debated, as the decision of French Conseil d'État illustrates. Indeed, the Conseil d'État seems to consider that only processing revealing the seriousness or the nature of a disability must be considered as processing of data concerning health. This view can be problematic since it runs contrary to the way the ECJ interprets the processing of health data and more broadly, to the way some DPAs interpret the processing of data likely to reveal sensitive characteristics. 41 This question is very critical since the development of machine learning technology enables the inference of sensitive characteristics from data which are not inherently sensitive. [START_REF]Article 29 Data Protection Working Party, Advice paper on special categories of data ("sensitive data[END_REF] One of the most relevant criteria to consider whether processing of personal data can be considered as unlawful processing of sensitive data is the purpose criterion. In other words, while the processing of data likely to reveal in an indirect fashion some sensitive data can be deemed lawful, the processing of said data with the intent to reveal sensitive characteristics should be deemed unlawful in whatever circumstances. For instance, the Information Commissioner's Office (ICO) claimed in the Cambridge Analytica use case that "since CA used the information collected to make predictions about data subjects' political affiliations and opinions, it is clear that the data should be considered sensitive personal data". [START_REF] Ico | Investigation into the use of data analytics in political campaigns[END_REF] The link is thus drawn between the intent (purpose) and the qualification of processing as revealing sensitive data. Further research should be undertaken on the topic of data processing indirectly revealing sensitive data through inferences.

Proposal for a Regulation of the European Parliament and of the Council on European data governance (Data Governance Act), COM (2020) 767 final, 25 November 2020.

See Commission Nationale de l'Informatique et des Liberté (CNIL), Publication en ligne et réutilisation des données publiques (« open data »), available at: www.cnil.fr/fr/publication-en-ligne-et-reutilisation-desdonnees-publiques-open-data.

 5 See Article 9 of the Regulation (EU) 2016/679 on the protection of natural persons with regard to the processing of personal data and the free movement of such data (General Data Protection Regulation

).6 Conseil d'État, 10ème -9ème chambres réunies, 10 June 2021, no.

431875.7 See Decree no. 95-979 of 25 August 1995, on the recruitment of disabled workers in the civil service, in application of article 27

of law no. 84-16 of 11 January 1984 on statutory provisions relating to the civil service. 8 Conseil d'État, 10ème -9ème chambres réunies, 10 June 2021, no. 431875 (Our translation)

S. Wachter and B. Mittelstadt, A Right to Reasonable Inferences: Re-Thinking Data Protection Law in the Age of Big Data and AI, in Columbia Business Law Review, no. 2, 2019, 563.

Conseil d'État, 10ème / 9ème sous-sections réunies, 28 March 2014, no. 36104 (Our translation).

Conseil d'État, 10ème et 9ème sous-sections réunies, 19 July 2010, no. 334014.

See Article 9 of the GDPR.

ECJ, Grand Chamber, 1 August 2022, case C-184/20, OT v Vyriausioji tarnybinės etikos komisija.

Ibidem.

Ibidem.

EDPB, Guidelines 3/2019 on processing of personal data through video devices, 29 January 2020, 17, available at:

by Inria action-exploratoire DATA4US. 1


Silvie Huijben

Sarah Reece

Marianna Szucs

Villa Manon

Berthomieu Arnaud

Rivero Ana
email: ana.rivero@cnrs.fr

Recommender

Keywords: drug-resistance, fitness costs, within-host dynamics, virulence, ACTs, artemisinin derivatives, kelch-13

Drug resistance is a major issue in the control of malaria. Mutations linked to drug resistance often target key metabolic pathways and are therefore expected to be associated with biological costs. The spread of drug resistance depends on the balance between the benefits that these mutations provide in the drug-treated host and the costs they incur in the untreated host. The latter may therefore be expressed both in the vertebrate host and in the vector. Research on the costs of drug resistance focusses on interactions with vertebrate host, yet whether they are also expressed in the vector has been overlooked. In this study, we aim to identify the costs and benefits of resistance against artesunate (AS), one of the main artemisinin derivatives used in malaria-endemic countries. For this purpose, we compared different AS-selected lines of the avian malaria parasite Plasmodium relictum to their ancestral (unselected) counterpart. We tested their within host dynamics and virulence both in the vertebrate host and in its natural vector, the mosquito Culex quinquefasciatus. The within-host dynamics of the AS-selected lines in the treated birds was consistent with the phenotype of resistance described in human P. falciparum malaria: a clearance delay during the treatment followed by a recrudescence once the treatment was interrupted. In the absence of treatment, however, we found no significant costs of resistance in the bird. The results of the two experiments to establish the infectivity of the lines to mosquitoes point towards a decreased infectivity of the drugselected lines as compared to the ancestral, reference one. We discuss the potential implication of these results on the spread of artesunate resistance in the field.

Introduction

Malaria is a life-threatening disease caused by Plasmodium parasites which are transmitted to people through the bites of infected mosquitoes. According to the latest World Health Organization Malaria Report, in 2020 there were an estimated 240 million episodes of malaria which caused over 500 000 deaths, most of them among African children (WHO 2021).

Currently, four major antimalarial drug classes exist for the treatment of malaria: quinolines, antifolates, atovaquone and artemisinin derivatives. The widespread use of these synthetic anti-malarials has saved thousands of lives, but has also exerted an intense selection pressure for the evolution of resistance in the parasite. Drug resistance in malaria has become a major human health issue because Plasmodium parasites have evolved resistance to all classes of anti-malarials that have gone into widespread use. The rapid evolution of drug resistance in Plasmodium highlights the urgent need to understand the selective pressures under which drug resistant mutations emerge and spread in the population [START_REF] Read | Evolutionary biology and the avoidance of antimicrobial resistance[END_REF][START_REF] Huijben | The fitness of drug-resistant malaria parasites in a rodent model: multiplicity of infection[END_REF].

The fate of mutations responsible for resistance is expected to be governed by the balance between the benefits accrued in the drug-treated hosts and the costs suffered in the untreated hosts. The benefits of resistance are obvious: drug resistant parasites show an improved ability to survive the drug treatment. The costs of resistance, on the other hand, arise from the metabolic costs of detoxification or the reduced biochemical efficiency associated with target site mutations and are expected to become manifest in the absence of the drugs [START_REF] Sirawaraporn | Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase[END_REF][START_REF] Hastings | The impact of antimalarial drug resistance mutations on parasite fitness, and its implications for the evolution of resistance[END_REF]. For this reason, these costs can be expressed both in the vertebrate host, but also in the vector. Current views about the impact of drug resistance on parasite fitness are, however, almost entirely based on data obtained from vertebrate hosts. In humans, costs of resistance have been inferred by the decrease in the frequency of drugresistance alleles when drug use is stopped or discontinued [START_REF] Laufer | Withdrawing antimalarial drugs: Impact on parasite resistance and implications for malaria treatment policies[END_REF][START_REF] Ord | Seasonal carriage of pfcrt and pfmdr1 alleles in Gambian Plasmodium falciparum imply reduced fitness of chloroquine-resistant parasites[END_REF][START_REF] Babiker | Seasonal fluctuation of drug-resistant malari parasites: a sign of fitness cost[END_REF]). The largest contributions to our understanding of the costs of drug resistance in the vertebrate host have, however, come from work carried out in animal models. In vivo competition experiments using laboratory selected drug resistant strains of the rodent malaria parasite Plasmodium chabaudi have shown that drug resistant strains are competitively suppressed by the sensitive strains in the absence of drug treatment [START_REF] Crawley | Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections[END_REF][START_REF] Wargo | Competitive release and facilitation of drugresistant parasites after therapeutic chemotherapy in a rodent malaria model[END_REF][START_REF] Huijben | Chemotherapy, within host ecology and the fitness of drug resistant malaria parasites[END_REF].

The costs of resistance in the mosquito vector have, in contrast, either been ignored entirely or given only cursory attention [START_REF] Koella | Costs and benefits of resistance against antimalarial drugs[END_REF]). Yet, the passage through a mosquito is an essential step in the life cycle of Plasmodium. Within mosquitoes the parasites reproduce sexually, differentiate, proliferate and migrate to the salivary glands to ensure transmission to the next host. Costs of drug resistance may be expressed at any one of these stages, profoundly impacting the probability of transmission of drug resistant strains. These costs may be expressed in two different ways: by altering the ability of the parasite to infect the mosquito vector (infectivity), or by altering the effects that the parasite has on the fitness of the mosquito vector (virulence).

Here, we investigate the costs and benefits of resistance to artesunate (one of the main artemisinin derivatives) both in the vertebrate host and in the vector. Artemisinin derivatives are the most potent and effective anti-malarial to date. The precise mode of action of artemisinin derivatives is still controversial, but several lines of evidence indicate that they exert their anti-malarial action by perturbing redox homeostasis and haematin detoxification in the parasite [START_REF] O'neill | The molecular mechanism of action of artemisinin-the debate continues[END_REF]. To prevent the evolution of resistance, the World Health Organisation recommends the use of the so-called artemisinin combination therapies (or ACTs) which combine the highly toxic, but short lived, artemisinin derivatives with a partner drug with a long half-life such as sulfadoxine-pyrimethamine, amodiaquine, or lumefantrine (WHO 2021). Despite the hopes raised by the implementation of ACTs, artemisinin resistance is already present in several countries of the Greater Mekong Subregion (WHO 2021), and worryingly, the de novo emergence of artemisinin resistance has been recently detected in Subsaharan Africa [START_REF] Uwimana | Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda[END_REF]. Phenotypically, artemisinin resistant parasites are characterized by (i) a delay in parasite clearance (resistant parasites take an extra 24-48 to be cleared from the blood) followed by (ii) a recrudescence of the parasite at the end of the standard 3-day therapeutic course [START_REF] Dondorp | Artemisinin resistance in Plasmodium falciparum malaria[END_REF]. This phenotype has been shown to be associated to several mutations in the Kelch 13 (K13) propeller domain of the parasite [START_REF] Straimer | K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[END_REF]. Despite the ongoing spread of artemisinin resistance across malaria-endemic areas we still lack a clear picture about the costs and benefits associated to these mutations (but see [START_REF] Nair | Fitness costs and the rapid spread of kelch13-C580Y substitutions conferring artemisinin resistance[END_REF][START_REF] Tirrell | Pairwise growth competitions identify relative fitness relationships among artemisinin resistant Plasmodium falciparum field isolates[END_REF].

Selecting for drug resistance in rodent and avian malaria has proven to be a straightforward procedure that provides a powerful tool for testing the effects of a particular drug resistance mutation on parasite fitness in vivo [START_REF] Greenberg | Mixed lethal strains of Plasmodium gallinaceum: drug-sensitive, transferable (SP) × drug-resistant, non-transferable (BI)[END_REF][START_REF] Beaudoin | Persistence of pyrimethamine resistance in the exoerythrocytic stages of Plasmodium gallinaceum[END_REF][START_REF] De Roode | Virulence and competitive ability in genetically diverse malaria infections[END_REF][START_REF] Walliker | Fitness of drug-resistant malaria parasites[END_REF][START_REF] Wargo | Competitive release and facilitation of drugresistant parasites after therapeutic chemotherapy in a rodent malaria model[END_REF][START_REF] Huijben | The fitness of drug-resistant malaria parasites in a rodent model: multiplicity of infection[END_REF]). Here we select for artesunate (AS, a widely-used artemisinin derivative) resistance in Plasmodium relictum (the most widespread species of avian malaria in the wild) by submitting it to gradually increasing doses of the drug. We then compare the within host dynamics and virulence of the drug-selected lines to the reference line both in the presence and in the absence of an AS treatment in the vertebrate host. We expect that: i) in treated hosts (birds), artesunate-selected lines will exhibit a similar phenotype to human malaria, i.e. delayed clearance followed by recrudescence, ii) in untreated hosts (birds) and vectors (mosquitoes), artesunate-selected parasites suffer higher fitness costs in comparison to their unselected counterparts.

Methods

Mosquito and parasite protocols

Plasmodium relictum (lineage SGS1) is the aetiological agent of the most prevalent form of avian malaria in Europe. The biology of this parasite species is similar to that of human malaria, both in the vertebrate host and in the mosquito. Avian malaria has historically played a key role in human malaria research, and in particular in the development and testing of the first anti-malarials [START_REF] Hewitt | Bird Malaria[END_REF][START_REF] Rivero | Evolutionary Ecology of Avian Malaria: Past to Present[END_REF]. Our parasite lineage was isolated from blue tits (Parus caeruleous) collected in the Montpellier area (France) in October 2016 and subsequently passaged to naïve canaries (Serinus canaria) by intraperitoneal injection. Since then, it has been maintained by carrying out regular passages between our stock canaries through intraperitoneal injections with the occasional passage through the mosquito (one passage through mosquito every c.a. 15 bird to bird passages, on average).

All experiments were carried out using a laboratory line of Culex quinquefasciatus (SLAB strain). Vectors in the Culex pipiens complex (Cx pipiens and Cx quinquefasciatus) are the principal natural vector of Plasmodium relictum in the wild. The larvae in all the experiments were reared at a constant density per tray (n=300 larvae) following previously published laboratory protocols (Vézilier et al 2010). Larval trays (n=20) were placed individually inside an "emergence cage" (40 cm x 28 cm x 31 cm) and emerged adults were allowed to feed ad libitum on a 10% glucose water solution.

AS-selected parasite lines were obtained by treating infected birds with increasing concentrations of artesunate (Sigma A3731). Birds were infected by injecting them intra-peritoneally with 100 µl of infected blood. The artesunate treatment was initiated 12 days later, to coincide with the peak of the acute phase of the parasite's infection. Artesunate was diluted in bicarbonate (50 mg/Kg) and administered through intra-peritoneal injections carried out twice daily (9am and 6pm) for four consecutive days. Five or six days later, 100 µl of blood from the treated birds was taken via a wing puncture, transferred to new canaries, and the treatment was thus repeated until a total of 5 passages were carried out. Three AS-selected lines were obtained in this way. Line AS1 was obtained by treating birds with 16 mg/kg artesunate for 3 passages followed by 32 mg/kg for a further 2 passages. Lines AS2 and AS3 were obtained by treating birds with 8 mg/kg for 3 passages followed by 16 mg for 2 passages. The reference line was maintained in parallel by injecting birds with bicarbonate following the same protocol. Experiments were initiated immediately after the 5 th passage.

Experiment 1. Phenotypic characterization of drug resistance in the bird

Artesunate-resistance is expected to be beneficial for the parasite in the presence of the drug, but to incur fitness costs when the drug is absent. In this experiment, we infected birds with either the reference line or one of the drug-selected lines (AS1, AS2, AS3). Half of the birds where then treated with a standard dose of artesunate, while the other half were given a sham (bicarbonate) injection. For each bird we quantified parasitaemia (proportion of red blood cells infected) through time and parasite virulence (bird weight loss and anaemia).

The detailed experimental protocol was as follows (Figure 1). Birds were inoculated with each of the parasite lines (reference, AS1, AS2 and AS3) by means of an intra-peritoneal injection (6 birds per line, all birds in these experiments were 1-2 years old). Each bird received between 70 000 to 90 000 parasites, except for the birds injected with the AS3 line, which received 30 000 parasites because the parasitaemia in the donor birds were too low. Bird weight and red blood cell count (RBC: number of red blood cells per ml of blood, Beckman Coulter Counter, Series Z1) were measured immediately before the infection and every two days thereafter. Bird parasitaemia (% red blood cells infected) was monitored every two days from the second day after the inoculation (henceforth "day 2 pbi": post-bird inoculation) onwards, using thin blood smears. On day 12 pbi, to coincide with the acute phase of the parasite infection, half of the birds were injected with 50 µl of 16mg/kg AS (dissolved in 50mg/kg bicarbonate) while the other half were injected with an equivalent amount of bicarbonate (50mg/kg). The injections took place twice a day (9 am and 6 pm) for 4 consecutive days (days 12-15 pbi). During this period, parasitaemia and virulence measurements were taken 24h after each injection (days 13-16 pbi). Bird parasitaemia, weight and RBC were thereafter followed every two days for 16 days after the end of the treatment (days 18-30 pbi, Figure 1). To account for the variance in parasitaemia between birds on the day immediately preceding the treatment (day 12 pbi, see Supplementary Table ST1), the parasitaemia on day 13 pbi onwards was calculated as a fold-increase (or decrease) in the number of parasites using the parasitaemia at 12 pbi as the baseline (px/pb, where px is the parasitaemia at a given day, and pb the baseline parasitaemia at day 12 pbi).

Experiment 2. Costs of drug resistance in the vector: parasite infectivity and virulence

The aim of this experiment was to establish whether AS-selected and reference lines differ in their infectivity and virulence to the mosquito. For this purpose, mosquitoes were allowed to blood feed on birds infected with the drug-selected and reference parasite lines. Mosquitoes fed on uninfected birds were used as controls. A subsample of the mosquitoes was used to quantify parasite density, while the rest were followed for several days to measure their fecundity and longevity. Since having too many mosquitoes in the cage risk compromising the wellbeing of the bird, and given that AS3 did not have the expected phenotype of resistance (delayed clearance followed by recrudescence), this experiment was done using only AS1 and AS2.

The detailed experimental protocol is as follows (Figure 1). Six naïve birds were infected with 100 000 parasites of either the reference, AS1 or AS2 lines (2 birds per line). Two further birds were used as uninfected controls. On day 12 pbi, birds were introduced individually into cages containing 150 12-day old female mosquitoes. The following morning, unfed females were counted and discarded from the study and all cages were provided with ad libitum sugar in the form of a 10% sugar solution. To quantify fecundity, three days after the blood meal (day 3 pmi, post-mosquito infection), a tray of water was placed in each to allow females to lay eggs. The trays were checked daily for the presence of eggs for 3 consecutive days (days 3-6 pmi). Culex pipiens females lay eggs in a single raft containing up to 250 tightly-packed eggs. This facilitates the estimation of the number of laying females and the eggs laid per female. The egg laying date was recorded and the egg rafts were photographed using a binocular microscope equipped with a numeric camera. The eggs counted using the Mesurim Pro freeware (Academie d'Amiens, France). Survival was assessed daily by counting dead individuals lying at the bottom of each cage until all females died.

To quantify parasitaemia, on day 8 and 9 pmi, 20 mosquitoes were haphazardly sampled from each cage and immediately dissected under a binocular microscope to extract the oocyst-infected midguts. Midguts were stained with a 5% mercurochrome solution to assess infection rate (oocysts present/absent) and oocyst burden (number of oocysts) under a phase contrast microscope.

Experiment 3. Costs of drug resistance in the vector: parasite dynamics

The aim of this experiment was to explore whether the results obtained in Experiment 2 may be explained by a difference in the within-vector parasite dynamics between AS-selected and reference lines. For this purpose, mosquitoes were allowed to blood feed on birds infected with the drug-selected and reference parasite lines (as above). Mosquitoes were dissected at regular intervals and the number of oocysts and sporozoites were compared between the AS-selected and the unselected reference lines.

The detailed experimental protocol is as follows (see Figure 1). Six naïve birds were infected with 100 000 parasites of either the reference, AS1 or AS2 lines (2 birds per line). As in the previous experiment, on day 12 pbi, birds were introduced individually into cages containing 150 12-day old female mosquitoes. The following morning, unfed females were counted and discarded from the study and all cages were provided with ad libitum sugar for the reminder of the experiment. Starting on day 4 post blood meal and every 2 days thereafter (4-20 dpmi), 20 mosquitoes were haphazardly sampled from each cage and immediately dissected under a binocular microscope to extract the oocyst-infected midguts and count the oocysts (as above). The mosquito thorax, containing the sporozoite-infected salivary glands, was then severed and frozen at -20 o C for the subsequent quantification of parasites via qPCR. Sporozoite quantification was calculated as a ratio between mosquito DNA (ace2 gene) and Plasmodium relictum DNA (cytb) as described previously [START_REF] Zélé | Wolbachia increases susceptibility to Plasmodium infection in a natural system[END_REF].

Ethics statement

Bird manipulations were carried out in strict accordance with the "National Charter on the Ethics of Animal Experimentation" of the French Government. Experiments were approved by the Ethical Committee for Animal Experimentation established by the authors' institution (CNRS) under the auspices of the French Ministry of Education and Research (permit number CEEA-LR-1051).

Statistical analyses

Analyses were carried out using the R statistical package (v3.4.4). The general procedure to build models was as follows: line (reference, AS1, AS2, AS3), sampling day, treatment (AS treated or shamtreated, Experiment 1 only) and parasitaemia were introduced into the model as fixed explanatory variables. Bird ID and the qPCR plates used to quantify sporozoites in the head-thorax fraction of the mosquito (Experiment 3 only) were fitted as random effects. Maximal models, including all higher order interactions, were simplified by sequentially eliminating non-significant terms and interactions to establish a minimal model. The significance of the explanatory variables was established using a likelihood ratio test (LRT) which is approximately distributed as a chi-square distribution [START_REF] Bolker | Ecological models and data in R[END_REF]) and using p = 0.05 as a cut-off p-value.

Mosquito survival (Experiment 2) was analyzed using Cox proportional hazards mixed effect models (coxme). Proportion data (oocyst and sporozoite prevalence) were analyzed using mixed linear models and a binomial distribution. Count data (eggs per raft) were analyzed using mixed linear models and a Poisson distribution. Response variables that were highly overdispersed (e.g. oocyst burden) were analyzed using mixed negative binomial models (glmmTMB). A posteriori contrasts were carried out by aggregating factor levels together and by testing the fit of the simplified model using a LRT [START_REF] Crawley | Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections[END_REF]. Statistical analyses are summarised in Supplementary Table ST2. Two additional standard measurements of survival were obtained in each cage: the median survival (the time at which 50% of the population is still alive) and the proportion of mosquitoes that survived till day 14 (the time at which sporozoites peak in all lines, see below).

Results

Experiment 1. Phenotypic characterization of artesunate resistance in bird

We infected birds with either the reference line or one of the drug-selected parasite lines (AS1, AS2, AS3). Twelve days later, half of the birds were then treated with AS, while the other half were given a control (bicarbonate) injection (Figure 1). For each bird, we quantified parasite density dynamics (proportion of infected red blood cell through time) and parasite virulence (bird weight loss and anaemia) before, during and after the AS treatment.

We started by analysing whether there were differences in parasitaemia between lines before the AStreatment (days 2-12 pbi). We fitted a full model that included day, line and treatment (and bird as a random factor to control for temporal pseudo-replication). There was no significant 3-way interaction and no significant differences in parasitaemia between birds infected with the different lines, or subsequently allocated to the treated or control treatments (although variance between birds was high, Supplementary Figures SF1A-D). This model only detected differences in parasitaemia between days (model 1, χ 2 = 33.34, p < 0.0001).

During the treatment (days 13-16 pbi), the parasitaemia of treated and untreated birds followed very different courses. Treatment with AS had a very significant effect on the parasitaemia, and the effect was dependent on both the day and the parasite line (model 2, χ 2 = 12.088, p = 0.0071, Figure 2A). To obtain a more accurate picture of the nature of this interaction, subsequent analyses were done separately for AStreated and reference birds. Treatment with AS resulted in an overall decrease in parasitaemia in all lines (see Supplementary Table ST3). On the first day after the treatment (day 13 pbi) the reduction in parasitaemia was greater in the reference line (-93.6% on average) than in either the selected AS1 (-59.8%), AS2 (-57.8) or AS3 (-69,6%) lines. This effect largely disappears in subsequent days (Supplementary Table ST3). The statistical analyses revealed a significant interaction between day and line (model 3, χ 2 = 20.563, p= 0.0147). In untreated birds, there was a significant difference between the lines (model 4, χ 2 = 9.1288, p= 0.0276) due to AS2 having a significantly higher parasitaemia than the other lines. This effect was independent, albeit marginally, of the sampling time (model 4, χ 2 = 7.7845, p= 0.0507, Figure 2B).

After the AS treatment, parasitaemia in the treated birds showed a different pattern between the lines (model 5, χ 2 = 8.8851, p= 0.0308, Figure 2A). Lines AS1 and AS2 had a significantly higher parasitaemia than the reference line. In untreated birds, the parasitaemia decreased gradually with time (model 8, χ 2 = 54.449, p < 0.01) and was independent of the parasite line (model 6, line: χ 2 = 7.9084, p= 0.0479, Figure 2B). Virulence was quantified as a change in weight and a change in RBC counts (anaemia) with respect to the weight and RBC counts on the first day of the experiment. Weight changes following the infection were significant and dependent both on the parasite line, on the treatment group (AS-treated or untreated), and on the timing with respect to the treatment (before, during or after, model 7, c 2 = 34.51, p = 0.001). In untreated birds, there was a weight difference between the lines, with birds infected with AS1 and AS2 (but not AS3) having a significant lower weight than those infected with the reference line throughout the experiment (model 9, c 2 = 26.56, p = 0.0002, Figure SF2B). In treated birds, however, the weight difference disappeared during the treatment, only to reappear after the treatment ended (model 8, c 2 = 29.28, p < 0.0001, Supplementary Figure SF2A). RBC counts were significantly lower during the treatment than before or after the treatment (model 10, c 2 = 54.55, p < 0.0001), but were independent of the line and were similar in AS-treated and control birds (Supplementary Figures SF3A andSF3B).

Experiment 2. Costs of drug resistance in the vector: parasite infectivity and virulence

Mosquitoes were allowed to blood feed either on uninfected birds or on birds infected with drugselected and reference parasite lines. A subsample of the mosquitoes fed on the infected birds was used to quantify oocyst density, while the rest were followed for several days to quantify their fecundity and longevity.

The overall prevalence of infection was low (29 ± 4% of female mosquitoes had at least 1 oocyst in their midgut) and independent of the parasite lines (model 11, χ 2 = 0.6017, p= 0.7402, Figure 3A). Oocyst burden (number of oocysts in infected females) was, however, significantly different between the lines (model 12, χ 2 = 8.0512, p= 0.0179); mosquitoes infected with the reference line had a significantly higher number of oocysts (mean ± s.e: 70 ± 22) than mosquitoes infected with the AS1 (mean ± s.e: 13 ± 5) and AS2 (mean ± s.e: 25 ± 11) lines (Figure 3B). There were also significant differences in mosquito fecundity (number of eggs per raft) between lines (model 14, χ 2 = 11.1, p= 0.0112). Post-hoc analysis showed that mosquitoes infected with the AS2 line laid significantly fewer eggs (mean ± s.e: 126 ± 4) than uninfected mosquitoes (142 ± 5) and mosquitoes infected with the refence (142 ± 4) and AS1 (148 ± 4) lines (χ 2 = 6.5741, p= 0.0104, Figure 4). There were no significant differences in longevity between infected and uninfected mosquitoes, or between mosquitoes infected with the different lines (model 13, χ 2 = 3.7758, p= 0.2867). The median longevity (the time at which 50% of the mosquitoes in each treatment is still alive) and the proportion of mosquitoes that survived till day 14 (peak sporozoite production) are given in Supplementary Table ST4.

Experiment 3 -Costs of drug resistance in the vector: parasite dynamics

This experiment was carried out to investigate whether the differences in oocyst burden between selected and reference lines may be due to a difference in the dynamics of development of the parasite within mosquitoes. Figure 5 shows the cumulative increase in oocysts in mosquitoes having fed in birds infected with each of the lines (reference or AS-selected). No mosquitoes in any of the lines were infected on day 4 so this time point was taken off the analyses. Mosquitoes infected with the reference line showed a faster cumulative increase in oocysts than mosquitoes infected with the AS-selected ones. As a result, by days 8-9 (corresponding to the dissection time in Experiment 2), mosquitoes fed on birds infected with the reference line had between 60-70% of the cumulative total number of oocysts, while mosquitoes fed from 3 out of the 4 birds infected with AS-selected lines had 0-16% of the cumulative total. Mosquitoes fed from one of the (AS1-infected) bird, however, showed an unusually high number of oocysts on the first dissection day. The mean oocyst prevalence (% mosquitoes containing at least 1 oocyst) averaged over the whole experiment was higher in reference (67.92 ± 6.47%, mean ± s.e.) and AS1 lines (53.65 ± 7.88%) than in the AS2 line (36.36 ± 10.49%). The same pattern was found with oocyst burden, which was higher in the reference (32.80 ± 11.95) and AS1 lines (18.90 ± 11.33) than in the AS2 line (7.75 ± 4.83). When day was fitted to the model the effect of line, however, disappeared for both oocyst prevalence (model 15, χ 2 = 5.3164, p= 0.070 and burden (model 16, χ 2 = 0.2313, p= 0.8908; Figure 6A and6B).

The mean sporozoite prevalence, averaged over the whole experiment, was roughly 2-times higher in the reference (28.30 ± 6.24%) and AS1 lines (27.90 ± 6.92%) than in the AS2 line (13.63 ± 7.48%). Sporozoite burden, on the other hand, was 4-5 times higher in the reference line (0.0126 ± 0.0067) than in the ASselected lines (AS1: 0.0026 ± 0.0009, AS2: 0.0034 ± 0.0029). As above, when day was fitted to the model the effect of line on sporozoite prevalence and burden disappeared (model 17, χ 2 = 0.1788, p= 0.9145, and model 18, dχ 2 = 1.9214, p= 0.3826, respectively Figure 6C and6D).

Discussion

The two telltale signs of P. falciparum resistance to artemisinin derivatives in the field are a delay in the clearance of parasites during ACT treatment followed by a recrudescence once the treatment is interrupted (Douglas et al. 2021). Artemisinin derivatives are remarkably efficient at clearing susceptible parasites from the blood (typically within 24-48 hours in P. falciparum). A slower clearance of parasites after the treatment, typically taking an extra 24-48 hours, is interpreted as evidence of resistance to the artemisinin-derivative component [START_REF] Witkowski | Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in Western Cambodia[END_REF][START_REF] Mok | Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance[END_REF]. This delayed clearance phenotype is a far cry from the standard definition of resistance (survival to a drug treatment), which is why artesunate resistance is commonly referred to as a 'partial resistance' (Uhlemann et al. 2010;[START_REF] Menard | Antimalarial drug resistance: a threat to malaria elimination[END_REF]. Partial artemisinin resistance rarely leads to ACT failure, but may select for drug resistance to the partner drug, as larger parasite populations remain in the blood stream for longer periods of time [START_REF] Fairhurst | Understanding artemisinin-resistant malaria: what a difference a year makes[END_REF]. In our study, the within-host dynamics of the drug-selected lines in the treated birds was consistent with the phenotype of resistance on P. falciparum in the field. AS-selected lines took longer to respond to the treatment than their reference line counterparts. In the first 24h after the beginning of the treatment the parasitaemia of the reference line dropped by over 90%, while that of the AS-selected lines only dropped by between 60% (AS1, AS2) and 70% (AS3). By the following day, there was no discernible difference between the parasitaemia of the reference and drug selected lines, (Figure 2A). There are no significant differences in the pre-treatment dynamics between the lines that may explain this significant difference in the parasitaemias in the first 24h between the treatments. (Supplementary Figure SF1). In keeping with previous studies on P. falciparum parasites, two of our drug-selected lines (AS1, AS2) also had a pronounced recrudescence after the treatment was interrupted, producing on average more than 20 times as many parasites in the two weeks following drug treatment than did the reference line (Figure 2A). One potential explanation of the different dynamic of AS3, may be found in the lower infective dose given to the birds (see above).

Drug resistance mutations are known to disrupt the parasite's metabolism, generating fitness costs. In drug-treated hosts these costs are largely compensated by the benefits conferred by the resistance. In untreated hosts, however, the magnitude of these costs will determine whether these mutations will persist and spread in the population. Current views about the impact of drug resistance on parasite fitness are almost entirely based on data obtained from the vertebrate host. In humans, costs of resistance have been inferred by the decrease in drug-resistance alleles when drug use is stopped or discontinued [START_REF] Laufer | Withdrawing antimalarial drugs: Impact on parasite resistance and implications for malaria treatment policies[END_REF] and from the decrease in the frequency of drug resistant parasites during the dry season, when there is no transmission and drug use is dramatically reduced . Direct experimental evidence of the costs of artemisinin resistance has been obtained by setting up in vitro competition experiments between drug-resistant and reference Plasmodium strains [START_REF] Hott | Fitness of artemisinin-resistant Plasmodium falciparum in vitro[END_REF][START_REF] Nair | Fitness costs and the rapid spread of kelch13-C580Y substitutions conferring artemisinin resistance[END_REF][START_REF] Tirrell | Pairwise growth competitions identify relative fitness relationships among artemisinin resistant Plasmodium falciparum field isolates[END_REF][START_REF] Mathieu | Local emergence in amazonia of Plasmodium falciparum K13 C580Y mutants associated with in vitro artemisinin resistance[END_REF]). To our knowledge the only in vivo experiments were carried out with Plasmodium chabaudi (a rodent species) and showed no obvious cost of AS in the absence of treatment [START_REF] Pollitt | Rapid response to selection, competitive release and increased transmission potential of artesunate-selected Plasmodium chabaudi malaria parasites[END_REF].

We did not observe any costs associated to the AS-selected lines in the absence of treatment. Experimental evidence of the costs of resistance has been obtained by setting up competition experiments between drug-resistant and susceptible Plasmodium strains. Using in vitro experiments where the erythrocyte stages of a drug resistant P. falciparum strain bearing mutations in the pfmdr1 gene competed against a drug-sensitive strain with the same genetic background, Hayward et al ( 2005) estimated that the loss of fitness of the resistant strain was ca. 25% per generation. In vivo competition experiments using laboratory selected drug resistant strains of the rodent malaria parasite Plasmodium chabaudi have confirmed that drug resistant strains are competitively suppressed by the sensitive strains in the absence of drug treatment (de Roode et al. 2004b;Wargo et al. 2007b;[START_REF] Huijben | Chemotherapy, within host ecology and the fitness of drug resistant malaria parasites[END_REF]. In our experiments the AS-selected lines were not set to compete with the reference line, which may explain the absence of costs. We instead obtained an opposite trend, whereby the drug-selected lines (AS2 in particular) fared better in terms of parasitaemia than its unselected counterparts. One possible explanation for these results, which would warrant further studies, is that drug selection may be expected to select for traits that increase the parasite's survival probabilities in the presence of drugs (Birget et al 2018). Highly replicating parasite lines may be selected for because they can rapidly recover high parasite densities after a bout of drug-induced mortality (Schneider et al. 2008, Schneider et al. 2012). Another possibility is that when confronted with the stressful environment imposed by the drugs, parasites may parasites may exercise a reproductive restraint (Schneider & Reece 2021), that is, sacrifice gametocyte production and prioritize asexual replication to maximise long term survival in the host. Either way, in as much as virulence is related to parasite fitness through high asexual parasite densities and gametocyte production (Mackinnon and Read 2004), this opens the unpalatable possibility of drugs selecting for more resistant but also more virulent parasites. In our experiments, we obtained some indication of a differential virulence between lines. Birds infected with AS1 and AS2 lines had a significantly higher weight loss than those infected with the reference line, but this was not associated to any clear pattern of RBC loss (anaemia). Current evidence for an association between drug resistance alleles and parasite virulence in the field is weak (Tukbasibwe et al. 2017, Cuu et al. 2020), but more work using experimental animal models under controlled conditions are, in our opinion, urgently needed.

Despite its crucial implications for the emergence and spread of drug resistant mutations in the field, work on the transmissibility of artemisinin-resistant parasites, is practically non-existent. [START_REF] St | Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa[END_REF] showed that artemisinin-resistant strains of P. falciparum from Cambodia were able to infect and produce sporozoites in three vector species (An. dirus, An. minimus, An. coluzzi), and (Pollitt et al. 2014) showed that in the rodent malaria species P. chabaudi, the AS-selected line produced more gametocytes (the blood stages of the parasite that are transmitted to the mosquito) than its reference counterpart. Here, we infected mosquitoes with the drug-selected and reference lines in two different experiments. In Experiment 2, all mosquitoes were dissected on a single day (half of the mosquitoes on day 8 and half on day 9). corresponding to the peak ocystaemia as established in previous experiments with the ancestral, reference line In this experiment we observed no significant difference in prevalence between the lines, but a highly significant difference in the number of oocysts between the reference and the two drugselected lines: the latter produced between 3-5 times fewer oocysts than the reference line (Figure 3). This difference could reflect either a true difference in oocyst burden between the lines, or a shift in the withinmosquito dynamics (peak oocystaemia may have occurred earlier or later in the drug-selected lines). To discriminate between these two options a new experiment (Experiment 3) was launched, where both oocysts and sporozoites were quantified at regular, 2-day, intervals, from day 6 to day 20 post infection. This experiment suffered from the caveat of an unusually low infection prevalence (23%), which significantly reduced the statistical power of the burden analyses, and precluded any meaningful analyses of peak oocystaemia. The cumulative frequency of oocysts, however, suggested that oocysts accumulate at a slower rate in mosquitoes infected with the AS-selected lines (Figure 5). Further work should explore this issue by, eg measuring potential differences in the growth rate (size) of oocysts issued from reference and AS-resistant lines. The mean overall prevalence and burden of oocysts and sporozoites (i.e. prevalence and burden averaged over all time points) was also in lower AS1 and AS2 mosquitoes. Sporozoite burden, in particular, was 4-5 lower in the drug-selected lines than in the reference line. The results therefore concur with those obtained in the previous experiment in that AS-selected lines were less infective to the mosquitoes than their reference counterparts. Another potential caveat of these experiments was that, for logistical reasons, we only used 2 bird hosts per line. It would be interesting to repeat them with a higher number of hosts to account for potential between-host variability in mosquito infection rates.

In conclusion, our data using drug-selected strains of P. relictum provides further proof that susceptibility to artesunate can be rapidly lost under drug pressure. The clearance delay and recrudescence observed during and after the AS treatment, respectively, in two of our three AS-selected lines were similar to the resistance phenotype seen in human malaria infections. The genetic underpinnings of artemisinin resistance are extremely complex and not yet entirely elucidated (Behrens et al. 2021). In P. falciparum the resistant phenotype is associated with several mutations in the propeller domain of kelch13 (pfk13), a protein that is essential for the intra-erythrocytc growth of the parasite. There are currently 33 candidate pfk13 mutations associated to artemisinin resistance (WHO Report 2021). In addition, non-synonymous mutations in several other (kelch13-independent) proteins, have also been associated with resistance, either alone or in combination with pfk13 mutations (Behrens et al. 2021). kelch13 is a highly conserved protein amongst Apicomplexans, and orthologous aminoacid sequences have been found in 21 different Plasmodium species, including P. relictum [START_REF] Coppée | Comparative structural and evolutionary analyses predict functional sites in the artemisinin resistance malaria protein K13[END_REF]. The phylogenetic relationship between the k13 in P. relictum and human malaria has not been clearly established [START_REF] Coppée | Comparative structural and evolutionary analyses predict functional sites in the artemisinin resistance malaria protein K13[END_REF], and its role in the resistance to artesunate in avian malaria remains to be demonstrated.

The recent de novo emergence and clonal expansion of a new pfk13 mutation (R561H) in Subsaharan Africa [START_REF] Uwimana | Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda[END_REF] where >90% of malaria cases occur, highlights the urgent need to understand the costs associated to artemisinin-resistant strains both in the vertebrate host and in the mosquito vector.

In our avian malaria model, we observed no obvious costs of the AS-selected lines in the vertebrate host in single infections. Further work should aim to develop markers of resistance that would allow us to explore whether the drug-selected lines are suppressed when in competition with the reference line [START_REF] Crawley | Competitive release of drug resistance following drug treatment of mixed Plasmodium chabaudi infections[END_REF][START_REF] Wargo | Competitive release and facilitation of drugresistant parasites after therapeutic chemotherapy in a rodent malaria model[END_REF][START_REF] Huijben | Chemotherapy, within host ecology and the fitness of drug resistant malaria parasites[END_REF]. Arguably, however, since our AS-selection lines were not clonal, they may have contained a mixture of resistant and ancestral parasites.

Current knowledge on the transmission potential of artemisin-resistant strains is extremely limited. Our results suggest that, under certain experimental conditions, drug-selected strains may generate lower sporozoite prevalences and burdens in mosquitoes, thus potentially impacting their transmission efficiency. Although the relative costs and benefits of resistance likely differ in magnitude between human and avian malaria parasites, these results provide proof of principle of the need for further studies comparing the infectiousness to mosquitoes of artemisinin-resistant and reference strains of human malaria.

ST2: Description of the statistical models used to analyze the costs of AS-resistance in birds and vectors. N gives the number of mosquitoes or birds included in each analysis. "Maximal model" represents the complete set of explanatory variables (and their interactions) included in the model. "Minimal model" represents the model containing only the significant variables and their interactions. N denotes the total number of observations (nb: which is not always the same as the number of independent replicates). Round brackets indicate that the variable was fitted as a random factor. Square brackets indicate the error structure used (n: normal errors, b: binomial errors), day: sampling day, timing: whether it's before, during or after the treatment, status: alive/dead on sampling day, eggs: number of eggs laid, trt: treatment (AS or shamtreated), line: parasite line, para: proportion of infected red blood cells, rbc: number of red blood cells per ml. ST3: Reduction in parasitaemia as a result of the AS treatment for each of the 3 lines. Treatment was given on days 12-15, parasitaemia was calculated 24h after each treatment (days 13-16). The reduction in parasitaemia is calculated as (px-pb)/pb * 100 where px= parasitaemia at a given day, and pb = baseline parasitaemia (parasitaemia immediately before the treatment, day 12). mn=mean, md=median, Q1=25% quartile, Q3= 75% quartile.

Median

Figure 1 :

 1 Figure 1: Experimental design for experiments 1 -3. In Experiment 1 (n= 6 birds per line), samples were taken every two days between day 2 and day 30 post bird inoculation (pbi), except during the treatment course where samples were taken daily. The parameters quantified were: bird parasitaemia, bird weight and RBC count. In Experiment 2 (n = 2 birds per line plus 2 uninfecteed birds) we quantified fecundity (4-7 pmi, ~30 mosquitoes per bird per line), longevity (~150 mosquitoes per bird per line, survival was monitored on a daily basis), and oocyst count (8-9 pmi, 20 mosquitoes per bird). In Experiment 3 (n = 3 birds per line) : we dissected mosquitoes every two days (2-20 pmi) and quantified oocysts (microscopic examination of the midgut) and sporozoites (qPCR of the thorax). pbi = days post bird infection, pmi = days post mosquito infection. US = unselected reference line, AS1, AS2, AS3 = artesunate-treated (putatively resistant) lines.

Figure 2 :

 2 Figure 2: Experiment 1. Parasitaemia (mean ± s.e) in artesunate-treated (A) and untreated (B) birds. Shaded area corresponds to the 4-day treatment period (measurements taken 24h after each injection). Untreated birds were sham injected with the artesunate solvent. Dashed line indicates the baseline parasitaemia on the day immediately before the first injection (day 12). Bars above/below the dashed line indicate an increase/decrease in parasitaemia with respect to day 12.

Figure 3 :

 3 Figure 3: Experiment 2. Mean (± s.e) oocyst prevalence (A) and burden (B) in mosquitoes infected by each parasite line 8-9 days post infection. Burden is represented as a boxplot showing the median (horizontal lines), and the first and third quartiles (box above and below the medians). Vertical lines delimit 1.5 times the inter-quartile range above which individual counts are considered outliers and marked as circles.

Figure 4 :

 4 Figure 4: Experiment 2. Fecundity of Culex pipiens females uninfected (n=60) or infected with each of the different parasite lines (n=60 per line). The number of eggs per raft is represented as a boxplot with the median (horizontal lines), first and third quartiles (box above and below the medians). Vertical lines delimit 1.5 times the inter-quartile range above which individual counts are considered outliers and marked as circles.

Figure 5 :

 5 Figure 5: Experiment 3. Cumulative proportion of oocysts per day in mosquitoes infected with the reference (control) and AS-selected lines. Data is provided separately for each cage/bird.

Figure 6 .

 6 Figure 6. Experiment 3. Oocyst prevalence (A) and burden (B), sporozoite prevalence (C), and burden (D)of mosquitoes infected by each of the parasite lines for different sampling intervals. Prevalence is represented as the mean ± standard error (calculated as sqrt(pq/n)). Oocyst and sporozoite burden are represented as a boxplot with the median (horizontal lines), first and third quartiles (box above and below the medians). Vertical lines delimit 1.5 times the inter-quartile range above which individual counts are considered outliers and marked as circles.

  Bird weight changes (mean ± s.e) in artesunate-treated (A) and untreated (B) birds. Shaded area corresponds to the 4-day treatment period. Untreated birds were sham injected with the artesunate solvent. Dashed line indicates the baseline bird weight at the start of the experiment (day 0). Bars above/below the dashed line indicate an increase/decrease in parasitaemia with respect to day 0. SF3: Bird red blood cell count (rbc) changes (mean ± s.e) in artesunate-treated (A) and untreated (B) birds. Shaded area corresponds to the 4-day treatment period. Untreated birds were sham injected with the artesunate solvent. Dashed line indicates the baseline rbc at the start of the experiment (day 0). Bars above/below the dashed line indicate an increase/decrease in rbc with respect to day 0.

  longevity (proportion surviving to day 14) Median mosquito longevity and, in parenthesis, proportion of mosquitoes surviving to day 14 (peak sporozoite production) for uninfected mosquitoes, and mosquitoes infected with the reference or ASselected lines. Data are presented separately for each bird.

		uninfected	reference	AS1	AS2
	Bird 1	23 (0.64)	21 (0.56)	21 (0.57)	21 (0.57)
	Bird 2	24 (0.62)	26 (0.65)	19 (0.56)	14 (0.41)
	ST4:

Acknowledgments

Preprint version 3 of this article has been peer-reviewed and recommended by Peer Community In Evolutionary Biology (https://doi.org/10.24072/pci.evolbiol.100156). We would like to thank the PCI Evolutionary Biology recommender (Sylvie Huijben) and the two reviewers (Sarah Reece and Marianna Szucs) for their thorough review of our manuscript and for their pertinent and useful suggestions. We also thank Bethsabé Sheid, the insectarium (a.k.a Vectopole) coordinator.

Funding

The experiments were funded through an ANR-16-CE35-0001-01 ('EVODRUG') to Ana Rivero. The Vectopole is one of the platforms of the Vectopole Sud network and is funded through the ANR "Investissements d'avenir" program (ANR-10-LABX-04-01).

Data, scripts, code, and supplementary information availability

Data available online: https://zenodo.org/record/7113241

Conflict of interest disclosure

The authors declare that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.

Appendix

Appendix 1: Supplementary Tables ST1: Proportion of blood cells infected (parasitaemia) immediately before the AS-treatment.