Lise Ropars

Laurence Affre

Lucie Schurr

Floriane Flacher

David Genoud

Clémentine Mutillod

Benoît Geslin

Land cover composition, local plant community composition and honeybee colony density affect wild bee species assemblages in a Mediterranean biodiversity hot-spot

Identifying environmental drivers which structure wild bee species assemblages appears essential in a context of worldwide pollinator decline. During a two-year survey, we studied wild bee species assemblages in an 85km 2 protected area dominated by Mediterranean scrubland, the Calanques National Park, located in southern France. Our objectives were (i) to assess the composition of the wild bee species assemblages, and (ii) to explore the effect of a) land cover composition (anthropogenic zone, low scrubland, forest and rocky land cover), b) local plant community composition, and c) honeybee colony density on wild bee species assemblages. On 17 circular 100m 2 plots, we collected 541 specimens belonging to 87 wild bee species. We found that large bee species were significantly influenced by land cover composition within a 1000m radius. More specifically, we observed that the presence of a diversified land cover composition within a 1000m radius maximized large wild bee species richness, whereas large bee abundance and richness were negatively affected by honeybee colony density. Small wild bee species were structured by the local plant community composition within 100m 2 plots and by land cover composition within a 1000m radius. Their occurrence were related to the local composition of plants growing on deep soils in scrublands. The Calanques National Park, which is the only European park located at the interface with a large city and consequently suffers from anthropic pressure, encompasses several types of land cover which are beneficial for a wide diversity of bees. However, we would like to raise awareness among park managers regarding beekeeping activities within this territory which includes diverse types of land cover favorable for wild bee species.

Introduction

Over the past decades, several studies have reported major losses of insects mostly due to habitat changes, pollution (fertilizers, pesticides, urban and industrial pollutants), invasive species and climate change [START_REF] Burkle | Plant-pollinator interactions over 120 years: loss of species, co-occurrence, and function[END_REF][START_REF] Hallmann | More than 75 percent decline over 27 years in total flying insect biomass in protected areas[END_REF][START_REF] Potts | Declines of managed honey bees and beekeepers in Europe Pérdidas de abejas manejadas y apicultores en Europa[END_REF][START_REF] Sánchez-Bayo | Worldwide decline of the entomofauna: a review of its drivers[END_REF]. Among all insects, pollinators are essential for the maintenance of the pollination function, to support the productivity of agricultural systems and the reproduction of wild plant species [START_REF] Ollerton | How many flowering plants are pollinated by animals?[END_REF][START_REF] Potts | Safeguarding pollinators and their values to human well-being[END_REF]. Their decline has been linked to the decrease in natural habitats and floral resources as a consequence of land cover changes [START_REF] Potts | Declines of managed honey bees and beekeepers in Europe Pérdidas de abejas manejadas y apicultores en Europa[END_REF]. For example, in Britain, over the last thirty years, [START_REF] Powney | Widespread losses of pollinating insects in Britain[END_REF] observed a decline of 33% of hoverflies and wild bee abundance due to habitat loss, climate change, and pesticide use. The European Red List of bees estimated that at least 9.2% of the 1965 wild bees occurring in Europe are threatened (near threatened status -NT) or endangered (vulnerable, endangered and critically endangered status -VU, EN, CR) [START_REF] Nieto | European Red List of Bees[END_REF]. However, for up to 55.6% of wild bee species, there is still not enough data to determine their protection status. Given this reported loss of insects and pollinators, we still need to survey wild bee species and determine what are the main environmental drivers that structure their species assemblages. In France, 961 wild bee species have been described according to the latest version of the French Taxonomic Referential [START_REF] Gargominy | TAXREF v12, référentiel taxonomique pour la France : méthodologie, mise en oeuvre et diffusion[END_REF]. The European Red List of bees estimates that species richness ranges from 556 to 730 in southern France, most being endemic to the Mediterranean basin [START_REF] Gargominy | TAXREF v12, référentiel taxonomique pour la France : méthodologie, mise en oeuvre et diffusion[END_REF][START_REF] Nieto | European Red List of Bees[END_REF]. This region is one of the world's biodiversity T hotspots where plant-pollinator communities are the most diverse [START_REF] Medail | Biodiversity hotspots in the Mediterranean basin : setting global conservation priorities[END_REF][START_REF] Petanidou | A land for flowers and bees: studying pollination ecology in Mediterranean communities[END_REF]. But to date, few studies have attempted to understand what are the local and landscape drivers that structure wild bee species assemblages in these habitats.

At the local scale, abundance and species richness of wild bees can be influenced by their resource needs such as floral rewards (e.g. pollen and nectar) or nesting sites (clay, sand, gravel, wood, pre-existing cavities, soft-pith stems or snail shells) [START_REF] Cane | Substrates and materials used for nesting by north American Osmia bees (hymenoptera: Apiformes: Megachilidae)[END_REF][START_REF] Steffan-Dewenter | Scaledependant effects of landscape context on three pollinator guilds[END_REF][START_REF] Torné-Noguera | Determinants of spatial distribution in a bee community: nesting resources, flower resources, and body size[END_REF][START_REF] Westrich | Habitat Requirements of Central European Bees and the Problems of Partial Habitats[END_REF][START_REF] Xie | Decline of nest site availability and nest density of underground bees along a distance gradient from human settlements[END_REF]. Wild bee richness and abundance can be also linked to land cover composition. For example, the increase in impervious surfaces at the landscape scale due to urbanization could negatively affect wild bee species assemblages [START_REF] Fortel | Decreasing abundance, increasing diversity and changing structure of the wild bee community (hymenoptera: Anthophila) along an urbanization gradient[END_REF][START_REF] Geslin | Plant pollinator networks along a gradient of urbanisation[END_REF][START_REF] Hall | The city as a refuge for insect pollinators[END_REF]; but see [START_REF] Hamblin | Wild bee abundance declines with urban warming, regardless of floral density[END_REF][START_REF] Theodorou | Urban areas as hotspots for bees and pollination but not a panacea for all insects[END_REF] while the proximity and the amount of natural habitats can increase bee abundance and richness [START_REF] Le Féon | Intensification of agriculture, landscape composition and wild bee communities: a large scale study in four European countries[END_REF]. As a general trend, the response of wild bee species assemblages to environmental conditions strongly depends on their body size [START_REF] Bennett | Landscape and local site variables differentially influence pollinators and pollination services in urban agricultural sites[END_REF][START_REF] Bommarco | Dispersal capacity and diet breadth modify the response of wild bees to habitat loss[END_REF]. Larger bees (i.e., total body length > 11.5 mm) are generally more sensitive to land cover modifications within a radius ranging from 750 to 1250m whereas smaller bees (i.e., total body length ≤ 11.5 mm) are more affected by local environments [START_REF] Hopfenmüller | Trait-specific responses of wild bee communities to landscape composition, configuration and local factors[END_REF][START_REF] Tscheulin | Influence of landscape context on the abundance and diversity of bees in Mediterranean olive groves[END_REF].

Finally, wild bee communities can also be influenced by beekeeping activities. For example, a high density of honeybee colonies can lead to indirect competition for floral resources between wild and domesticated pollinators [START_REF] Cane | Gauging the effect of honey bee pollen collection on native bee communities[END_REF][START_REF] Geldmann | Conserving honey bees does not help wildlife[END_REF][START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF][START_REF] Mallinger | Do managed bees have negative effects on wild bees?: a systematic review of the literature[END_REF][START_REF] Ropars | Wild pollinator activity negatively related to honey bee colony densities in urban context[END_REF][START_REF] Valido | Honeybees disrupt the structure and functionality of plant-pollinator networks[END_REF]. Recently, at least two studies in Mediterranean ecosystems have highlighted that high honeybee colony density can reduce the availability of pollen and nectar for wild bees [START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF][START_REF] Torné-Noguera | Collateral effects of beekeeping: impacts on pollen-nectar resources and wild bee communities[END_REF]. [START_REF] Torné-Noguera | Collateral effects of beekeeping: impacts on pollen-nectar resources and wild bee communities[END_REF] showed that visitation rates of wild bees decreased as the distance to the nearest apiary decreased in the El Garraf Natural Park in Spain, and [START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF] showed that the foraging success of wild bees decreased closer to the apiaries in scrubland land cover in southern France.

Here, we studied the effects of local and landscape drivers including honey bee colony density on wild bee species assemblages in a protected national park (Calanques National Park) in the vicinity of Marseille (France). Our goals were to identify the main drivers of bee species assemblages in order to provide tools to land managers to achieve more efficient managing practices. On the basis of a two-year census, we explored the following questions: (i) What is the wild bee richness in the Calanques National Park, located in a Mediterranean biodiversity hotspot? (ii) Are large and small wild bee species assemblages structured by a) land cover composition, b) local plant community composition and c) honeybee colony density? We expected large wild bee species assemblages to respond to environmental drivers at a larger scale (i.e. land cover) than small bee species assemblages which should rather be structured by local drivers (i.e. local plant community composition). We also expected honeybee colony density to negatively affect the richness and the abundance of large wild bee species due to their similar qualitative and quantitative feeding requirements.

Methods

Study site

The study was conducted in 2017 and 2018 in the Calanques National Park (43°13′27.55″ N, 5°28′2.92″E, near Marseille, France) (Fig. 1). This National Park is part of the Mediterranean basin and covers 85km 2 . The climate is Mediterranean, characterized by hot and dry summers. Precipitation mostly occurs in autumn with an annual mean of 515.4mm (see Supplementary material Table 1). The year 2017 was very dry with a total precipitation of 282.6mm, while 2018 was wetter with 816.6mm. The geological substrate is dominated by dolomitic or Urgonian limestone and puddingstone [START_REF] Pires | La flore remarquable des bouches-du-rhône: Plantes, milieux naturels et paysages[END_REF]. The studied area, dominated by Mediterranean scrubland, presents 1936 plant species, subspecies and varieties, with 83 plant species protected at regional or national level (SILENE Database available on http://flore.silene.eu; com. pers. Calanques National Park). Within the western part of this National Park, we selected 17 circular 100m 2 plots within a homogeneous Mediterranean low scrubland cover dominated by Rosmarinus officinalis, Thymus vulgaris, Cistus albidus and Cistus monspeliensis as main floral resources. Each plot included at least 3 individuals of Rosmarinus officinalis and 3 individuals of Cistus albidus. Only one plot, in the eastern part of the park, did not present any Cistus albidus individuals but individuals of a closely related species, Cistus monspeliensis. The nearest distance between two plots ranged from 460m to 3536m. Plot ranged in altitude from 19m to 274m and their distance to the coast from 309m to 7647m (Fig. 1). The number of honeybee colonies around each plot was taken into account to characterize plots near to and far from apiaries.

Bee sampling

Sampling sessions were conducted in 2017 (N = 10) and 2018 (N = 5), from February to June, which is the main flowering period of the plant community. Very few flowers were observed in July and August due to severe droughts. When weather conditions were favorable (no rainy or windy days), we sampled wild bees on the 17 plots, through sweep netting, according to two sampling protocols. Following the definition of [START_REF] Nielsen | Assessing bee species richness in two Mediterranean communities: importance of habitat type and sampling techniques[END_REF], the first method, 'observation quadrats', consisted in sampling each bee visiting a flower on three well-bloomed one-square meter quadrats for 5 min in 2017 and 10 min in 2018, which equates a total of 50 min per plot for each year. The second method, 'variable transect walks', consisted in sampling all wild bees (foraging on flowers or not) on the entire 100m 2 plot for 20 min in order to actively capture a maximum of bee individuals. Bees were then kept in jars filled with cork fragments soaked with ethyl acetate. To account for temperature variations within a day, we alternatively visited each plot in the morning or in the afternoon.

All captured specimens were pinned and dried prior to identification by professional taxonomists (E. Dufrêne for cuckoo bee species, D. Genoud for Andrenidae, Anthophorinii, Colletes sp. and Halictidae and M. Aubert for Megachilidae, Ceratinii and Hylaeus sp.). For statistical analyses, we classified bee species according to their total body length following the identification keys of Amiet [START_REF] Amiet | Apidae 6: Andrena, Melitturga, Panurginus, Panurgus[END_REF][START_REF] Amiet | Apidae 1: Apis, Bombus, Psithyrus[END_REF][START_REF] Amiet | Apidae 4: Anthidium, Chelostoma, Coelioxys, Dioxys, Heriades, Lithurgus, Megachile, Osmia, Stelis[END_REF][START_REF] Amiet | Apidae 5: Ammobates, Ammobatoides, Anthophora, Biastes, Ceratina, Dasypoda, Epeoloides, Epeolus, Eucera, Macropis, Melecta, Melitta[END_REF][START_REF] Amiet | Apidae 3: Halictus, Lasioglossum[END_REF][START_REF] Amiet | Apidae 2: Colletes, Dufourea, Hylaeus, Nomia, Nomioides, Rhophitoides, Rophites, Sphecodes, Systropha[END_REF], with larger body bees > 12 mm which corresponds to the mean body length of A. mellifera worker size and smaller body bees ≤ 12 mm [START_REF] Albrecht | The Swiss agri-environment scheme enhances pollinator diversity and plant reproductive success in nearby intensively managed farmland[END_REF][START_REF] Bosch | Flowering phenology, floral traits and pollinator composition in a herbaceous mediterranean plant community[END_REF][START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF].

We estimated the total bee species richness with Chao1, Jackknife1 and Jackknife2 indexes excluding honeybee species [START_REF] Gotelli | Estimating species richness[END_REF]. We used the function ChaoSpecies() within the Spade-R package on the R version 3.6 software [START_REF] Chao | SpadeR: Species-Richness Prediction and Diversity Estimation with R[END_REF]R Foundation for Statistical Computing, Vienna, n.d.). The species accumulation curve was obtained with the specaccum() function from the vegan package and is provided in Supplementary Materials [START_REF] Oksanen | Package 'vegan[END_REF].

Environmental drivers

Land cover composition

We computed land cover composition within a 500m and 1000m radius around each of our plots using Quantum-GIS (QGIS Development Team, 2019). Radius sizes were chosen based on the literature regarding wild bee mean flight distances [START_REF] Zurbuchen | Maximum foraging ranges in solitary bees: only few individuals have the capability to cover long foraging distances[END_REF] to encompass foraging or dispersal ranges of small and large wild bees L. Ropars, et al. [START_REF] Greenleaf | Bee foraging ranges and their relationship to body size[END_REF][START_REF] Steffan-Dewenter | Scaledependant effects of landscape context on three pollinator guilds[END_REF]. We used the French CORINE Land Cover 2012 data provided by the Calanques National Park to classify land cover. For each scale (500m and 1000m radius), we computed the area of four different land covers from the polygon layer of CORINE Land Cover 2012: anthropogenic zone (agricultural and artificial land cover), low vegetation (heathland, scrublands, lawns and meadows), forest and rocky land cover (rocks, screes and sand, rocky coasts and sea cliffs). The land cover composition was expressed in our statistical analyses as their proportions within each radius. However, as we did not find any response linking land cover composition within a 500m radius and the assemblages of both small and large bee species, we only present hereafter analyses relative to land cover composition within the 1000m radius; this absence of results is nevertheless discussed in the Discussion section.

Local plant community composition

We performed an exhaustive floristic survey on each circular 100m 2 plot to characterize the plant community. Each plant individual growing in the plot was noted and carefully identified to the species level. For each plant species, we specified a slightly modified Braun-Blanquet index which showed the relative area covered by the species within each 100m 2 circular plot (0: absence of the species; +: scarce individuals; 1: 1-10% of the area covered by species; 2: 10-25% of the area covered by species; 3: 25-50% of the area covered by species; 4: 50-75% of the area covered by species; 5: 75-100% of the area covered by species) [START_REF] Maabel | Transformation of cover-abundance values in phytosociology and its effects on community similarity[END_REF][START_REF] Van Der Maarel | The Braun-Blanquet approach in perspective[END_REF].

Apis mellifera density

Twice a year, we listed apiaries in the park and counted the number of colonies. These surveys were supplemented by information provided by the landowners of the Calanques National Park, the French government's veterinary service, the beekeepers and beekeeping associations. Then, we quantified honeybee density using a spatially explicit density score (SEDs hereafter) inspired by the work of [START_REF] Henry | A common pesticide decreases foraging success and survival in honey bees[END_REF]. Several authors report that the average foraging distance achieved by a honeybee is about 1000m and up to 10000m [START_REF] Couvillon | Honey bee foraging distance depends on month and forage type[END_REF][START_REF] Steffan-Dewenter | Importance of habitat area and landscape context fragmented orchard meadows[END_REF]). To take this information into account, the number of colonies located outside the 1000m radius around each plot was weighted by 1/D 2 where D is the distance expressed in kilometers between the apiary considered and a plot. This spatially explicit density score is a distance weighted sum of colony numbers in all apiaries located in the Calanques National Park and this score was calculated for each plot. The complete formula is:

∑ = = SEDs Colony number D X i N i Xi 1 2
With X, a considered plot; i, a considered apiary and N the number of apiaries.

An increase in the number of colonies near the plot will lead to an increase in the SED score. This score allowed us to include both the distance to apiaries and the number of colonies in each apiary.

Data analysis

Covariation with land cover composition and local vegetation

We conducted multivariate analyses to assess the impact of the land cover composition on small and large bee species assemblages. First, we conducted correspondence analyses (CA) on the bee species assemblages per plot (16 plots for small beesone plot had no small bee individualsand 17 plots for large bees). To accurately build the CA analysis, each bee species present in a single plot only was removed from the analysis. Secondly, we built principal component analyses (PCA) relative to land cover composition at 500m and 1000m. Finally, with these two previous analyses, we performed co-inertia analyses between bee data and environmental data. Co-inertia analysis is used to study species-environment relationships and highlights species community composition [START_REF] Dolédec | Co-inertia analysis: an alternative method for studying species-environment relationships[END_REF][START_REF] Le Féon | Intensification of agriculture, landscape composition and wild bee communities: a large scale study in four European countries[END_REF]. Co-inertia significance was obtained using Monte Carlo random permutation tests with 999 repetitions. All these analyses and tests were performed with the ade4 package of the R Cran software [START_REF] Dray | The ade4 package: implementing the duality diagram for ecologists[END_REF]R Foundation for Statistical Computing, Vienna, n.d.). Following the same method, multivariate analyses with the local plant community composition within the 100m 2 plots were performed. As previously stated, a CA was performed on small and large bee species assemblages. Then, we built PCAs relative to the local plant community composition for each plot. In order to prevent distortion, each plant species present in only one plot was removed from the PCA [START_REF] Sydenham | Does multi-level environmental filtering determine the functional and phylogenetic composition of wild bee species assemblages?[END_REF]. Finally, we performed co-inertia analyses between bee and plant data, and significance was also obtained by Monte Carlo random permutation tests with 999 repetitions.

Effect of land cover composition and local vegetation on wild bee richness and abundance

We analyzed richness and abundance of small and large wild bee species (data were pooled on a yearly basis in each plot). To assess the impact of each land cover on small and large wild bee species, we conducted generalized linear models following a Poisson distribution to deal with count data. No random effect was included in these models because the land cover did not vary between the two years. Then, to explore the effect of local plant community composition, we recorded plant richness per plot and used the same generalized linear models following a Poisson distribution. Finally, to assess the effect of honeybee colony density on wild bee species assemblages, we used generalized linear mixed models. We added the year as a random effect since honeybee colony density varied between 2017 and 2018. We used lme4 package to build these models [START_REF] Green | Package "lme4[END_REF]. For all models built, the best models were selected with the lowest AIC criterion, with a ΔAIC ≥ 2 against the null model.

Results

Bee species assemblages

During the two-year survey, we collected 269 and 272 bee specimens in 2017 and 2018 respectively. Net samples over the two years returned 87 wild bee species belonging to five families (Andrenidae -18 species; Apidae -21 species; Colletidae -10 species; Halictidae -20 species and Megachilidae -18 species). The three most captured species were Apis mellifera (21.4%) followed by Rhodanthidium sticticum (7.4%) and Anthophora dispar (6.9%) (see Supplementary Materials, Table 2). Three sampled species were considered near threatened and one endangered (respectively Andrena ovatula, Colletes albomaculatus, Lasioglossum pygmaeum and Lasioglossum soror) according to the European Red List of bees. Finally, 19 of the captured species (21.8%) were considered as 'data deficient' in the European Red List of bees.

Excluding honeybee species, the total estimated bee species richness reached 113 ± 12 species with Chao1 index, and between 121 ± 8 and 133 ± 14 species with Jackknife1 and 2 indexes respectively. The collected bee species richness thus accounted for 65%-77% of the estimated richness obtained by Jackknife2 and Chao1 indexes respectively (72% for Jackknife1). We plotted a species accumulation curve which is provided in the Supplementary Material Fig. 1.

Effect of environmental drivers on large bee species assemblage

Land cover composition

The co-inertia analysis linking the land cover composition within a 1000m radius and the large bee species assemblages on the 17 plots (Fig. 2, Supplementary Material Fig. 2) revealed that land cover composition (Principal Component Analysisthe first two axes representing 88% of the total inertia) were significantly associated (RV coefficient = 0.38; Monte Carlo permutation test p = 0.032) with the large bee species assemblages (Correspondence Analysisthe first two axes representing 51% of the total inertia). The first axis of the coinertia plane accounted for 52.89% of the total inertia, whereas the second axis accounted for 38.88%.

For the first co-inertia axis, the anthropogenic land cover was opposed to rocky land cover which represents an increasing distance between the plots and the urban land cover. Melecta italica, Anthophora mucida, Xylocopa violacea, Melecta albifrons, Nomada succinta, Halictus scabiosae and Eucera nigrecens contributed the most to the construction of the first axis (Fig. 2A). Among those species, Halictus scabiosae was related to anthropogenic land cover, conversely Nomada succinta and Osmia tricornis were related to scrublands (Fig. 2B). For the second coinertia axis, low vegetation was opposed to Mediterranean forests representing a gradient of vegetation closure. Eucera caspica and Anthophora plumipes were particularly related to Mediterranean forests (Fig. 2B).

We recorded a significant decrease in large bee species richness with increasing low vegetation proportions within a 1000m radius around each plot (slope = -0.010, R 2 = 0.126, p = 0.045, see Fig. 2C). Our results did not show any influence of anthropogenic, forest, rocky land cover on the species richness of large bees.

Local plant community composition

The second co-inertia analysis showed that the local plant community composition had no effect on the large bee species assemblages (RV = 0.60, Monte Carlo permutation test p = 0.245). Moreover, plant richness within the 100m 2 plots did not affect the abundance and richness of large bees.

Honeybee density

We found a significant decrease in the richness and in the abundance of large bees with the increase of honeybee colony density (respectively slope = -0.011, R 2 = 0.158, p = 0.006 and slope = -0.010, R 2 = 0.199, p = 0.002, Fig. 3), suggesting a negative correlation between the presence of honeybees and the foraging activity of large bee species.

Effect of environmental drivers on small bee species assemblage

Land cover composition

The co-inertia analysis performed on the 16 plots (Fig. 4A-B, Supplementary material Fig. 3) revealed that the land cover composition within a 1000m radius (Principal Component Analysisthe first two axes representing 88% of the total inertia) had a significant effect (RV coefficient = 0.39; Monte Carlo permutation test p = 0.046) on the small bee species assemblages (Correspondence Analysis -the first two axes representing 35% of the total inertia). The first axis of the coinertia plane accounted for 62.25% of the total inertia, and the second axis accounted for 27.57%.

The first co-inertia axis represented a gradient of vegetation closure with low vegetation cover being opposed to Mediterranean forests. Nomada flavoguttata, Lasioglossum bluethgeni were more related to scrubland and Hylaeus clyperis and Andrena lagopus were more related to Mediterranean forests than other habitats and contributed the most to the construction of the first axis (Fig. 4A-B). For the second co-inertia axis, rocky land cover was opposed to anthropogenic land cover representing an increased in the distance from the urban land cover. Nomada maculicornis and Andrena similis were related to rocky land cover whereas Seladonia gr. smaragdula was related to anthropogenic land cover (Fig. 4A-B).

Local plant community composition

The co-inertia analysis linking the local plant community composition within the 100m 2 plots and the small bee species assemblages on the 16 plots (Fig. 4C-D, Supplementary Material Fig. 4) revealed that the local plant community composition (Principal Component Analysis -the first two axes representing 39% of the total inertia) was significantly associated (RV coefficient = 0.69; Monte Carlo permutation test p = 0.041) with small bee species assemblage (Correspondence Analysis -the first two axes representing 35% of the total inertia). The first axis of the co-inertia plane accounted for 25.25% of the total inertia, whereas the second axis accounted for 14.27%.

For the first co-inertia axis, plant species such as Teucrium chamaedrys, Ulex parviflorus, Euphorbia serrata and Rubia peregrina were opposed to plots dominated by Ruta angustifolia, Coronilla juncea and Biscutella laevigata. We found that bee species such as Nomada flavoguttata, Lasioglossum albocinctum, Lasioglossum bluethgeni, Osmia rufohirta and Ceratina cucurbitina were related to Teucrium chamaedrys, Ulex parviflorus, Euphorbia serrata and Rubia peregrina. We also found that Seladonia gr. smaragdula, Hylaeus clypearis and Andrena lagopus were related to Ruta angustifolia, Coronilla juncea and Biscutella laevigata. For the second co-inertia axis, Centaurea sp. and Rhus coriaria plant species were opposed to Erica multiflora, Fumana thymifolia and Laserpitium gallicum. Osmia versicolor and Nomada sheppardana were related to Centaurea sp. and Rhus coriaria whereas Andrena similis and Hylaeus cf. imparilis were related to Erica multiflora, Fumana thymifolia and Laserpitium gallicum (Fig. 4C-D).

Regression models highlighted that the richness and the abundance of small bees were not significantly affected by land cover composition, plant richness, or honeybee colony density.

Discussion

Bee species assemblages

In this study, we explored which environmental drivers structured the communities of small and large bee species in the Calanques National Park. During a two-year survey, we inventoried 87 species of wild bees in scrubland land cover dominated by Rosmarinus officinalis, Thymus vulgaris, Cistus albidus and Cistus monspeliensis. In a similar habitat, in the El Garraf National Park in Spain (32km 2 ), Torné-Noguera et al. ( 2014) found a similar bee richness with 98 wild bee species. Two previous studies sampled the bee community within the Calanques National Park [START_REF] Geslin | Compte rendu des captures réalisées lors de la formation Européenne à la détermination des abeilles (COST Super-B Project) dans le Parc National des Calanques[END_REF][START_REF] Schurr | Pollination insights for the conservation of a rare threatened plant species, Astragalus tragacantha (Fabaceae)[END_REF], and the bee richness of this protected habitat now amounts to 131 species. We 2020) (Submitted) and the species richness amounts to 192 bee species. As previously stated in the literature, this shows that a single sampling year is insufficient to detect the species richness of a particular location, which should be sampled for several years to precisely account for its richness. For example, [START_REF] Petanidou | Longterm observation of a pollination network: fluctuation in species and interactions, relative invariance of network structure and implications for estimates of specialization[END_REF] showed inter-annual variations in a four-year survey of a pollinator community within Mediterranean scrublands from 183 to 238 insect species collected per year, totaling 661 species. Here, we found almost 10% of the entire French wild bee fauna focusing only on scrubland, the dominant habitat of the terrestrial French Mediterranean. During this survey, we recorded 4 wild bee species which benefit from a protection status: Andrena ovatula (NT), Colletes albomaculatus (NT), Lasioglossum pygmaeum (NT) and Lasioglossum soror (EN). Located at 70km in the East of the Calanques National Park, the Port-Cros National Park host a similar percentage (4%) of threatened wild bee species [START_REF] Gombault | Inventaires des Abeilles sauvages sur les sites de Port-Cros , Bagaud et du cap Lardier[END_REF].

Our sampling could be completed by other methods as we caught only 65% of the bee species estimated richness and as the species accumulation curve is not stabilized. Net sampling is a well-known method to assess the richness of wild bees visiting a local plant community [START_REF] Popic | Evaluation of common methods for sampling invertebrate pollinator assemblages: net sampling out-perform Pan traps[END_REF][START_REF] Rhoades | Sampling technique affects detection of habitat factors influencing wild bee communities[END_REF], but pan trap methods are a good complement to better reveal small wild bee species richness [START_REF] Roulston | A comparison of pan trap and intensive net sampling techniques for documenting a bee (Hymenoptera: Apiformes) fauna[END_REF]. Moreover, as we inventoried a small fraction of the Calanques National Park, other protected habitats such as sandpits or dry lawns should be investigated to better assess the total wild bee richness.

Local and landscape drivers of wild bee species assemblages

In protected areas, nesting and floral resources have been highlighted as the most widely studied drivers of wild bee assemblages [START_REF] Murray | Local-scale factors structure wild bee communities in protected areas[END_REF][START_REF] Potts | Role of nesting resources in organising diverse bee communities in a Mediterranean landscape[END_REF][START_REF] Torné-Noguera | Determinants of spatial distribution in a bee community: nesting resources, flower resources, and body size[END_REF]. In Mediterranean habitats, several authors have demonstrated that the effects of land cover and local plant community composition on wild bees depended on the body size of bees [START_REF] Torné-Noguera | Determinants of spatial distribution in a bee community: nesting resources, flower resources, and body size[END_REF][START_REF] Tscheulin | Influence of landscape context on the abundance and diversity of bees in Mediterranean olive groves[END_REF].

Effect of local plant community composition

Small bee species were affected by the local plant community composition within 100m 2 plots, which depends on soil depth [START_REF] Bernard-Verdier | Community assembly along a soil depth gradient: contrasting patterns of plant trait convergence and divergence in a Mediterranean rangeland[END_REF]. Small generalist bee species such as Nomada flavoguttata, Lasioglossum albocinctum, Osmia rufohirta were related to open scrubland plants that grows on deep soil such as Teucrium chamaedrys, Ulex parviflorus, Euphorbia serrata plant species. Hylaeus clypearis was linked to plant species which are characteristic of rocky scrublands with shallow soils, such as Ruta angustifolia and Biscutella laevigata. These two plant species provide small and open flowers which correspond to the particular floral needs of Hylaeus bee species [START_REF] Westrich | Habitat Requirements of Central European Bees and the Problems of Partial Habitats[END_REF]. As a general trend however, rather than highlighting a direct match between small bee species and the plant species composing their floral diet, we may had highlight here a co-occurrence between small bees species and plant species growing close to their preferential nesting substrates.

A tight correspondence between specialist bees and their related plant species was not observed. As few examples, Lasioglossum bimaculatum preferentially forages on Cistaceae, Andrena lagopus and Lasioglossum transitorium on Brassicaceae (D. Genoud, pers. comm.). This mismatch between bees' diet and their floral resources in our results could be due to the few captured individuals or to a lack of knowledge regarding the ecology of small wild bees. The known floral diet of bees is more related to plant species used to make bee-breads than plant species used to collect nectar. Likewise, numerous small bee species recorded in this survey were generalists and no clear pattern appeared with a particular plant species as they could forage on a wide range of plants. Unlike small bees, large bees were not affected by the local plant community composition. Considering their ability to forage and nest over larger distances than small bees, large bees could feed on distant floral resources which may explain their insensitivity to local flora or habitats.

Effect of land cover composition

Surprisingly, both small and large bees did not respond to land cover composition within a 500m radius. This result is unexpected, as a 500m radius around sampling sites is commonly used to evaluate the land cover effect on wild bee assemblages [START_REF] Dorchin | Patterns and drivers of wild bee community assembly in a Mediterranean IUCN important plant area[END_REF][START_REF] Geslin | Effect of local spatial plant distribution and conspecific density on bumble bee foraging behaviour[END_REF][START_REF] Kovács-Hostyánszki | Local and landscape effects on bee communities of Hungarian winter cereal fields[END_REF][START_REF] Wilson | The effects of urbanization on bee communities depends on floral resource availability and bee functional traits[END_REF]. Other studies such as [START_REF] Tscheulin | Influence of landscape context on the abundance and diversity of bees in Mediterranean olive groves[END_REF], found that the abundance of small bees reacted to the land cover composition within a smaller radius of 250m and the lack of response of large and small bees we observed here, could be due to an inappropriate choice of radius. We explored if a strong homogeneity or heterogeneity in the land cover composition of our sites at a 500m radius could explain this lack of response compared to the 1000m radius. However, we found no difference in the variance of the land cover composition between the two considered scales. Another possible explanation lies in the important number of sampling sites that did not include the four differents land cover we chose within a 500m radius (11 sites). Within the 1000m radius, only 4 of the 17 sites did not include the four differents land cover.

Small bee species were related to land cover composition within a 1000m radius; however, the pattern between small bee species and the land cover is difficult to explain. Even though several studies documented that small bee species have limited dispersal abilities [START_REF] Gathmann | Foraging ranges of solitary bees[END_REF][START_REF] Greenleaf | Bee foraging ranges and their relationship to body size[END_REF], other works have reported that flight distances of small solitary bees could be underestimated and may reach between 500 and 1000m [START_REF] Zurbuchen | Maximum foraging ranges in solitary bees: only few individuals have the capability to cover long foraging distances[END_REF]. Small bee species were distributed across the gradient of vegetation closure with Nomada flavoguttata, Lasioglossum bluethgeni being related to scrubland and Hylaeus clyperis and Andrena lagopus being related to Mediterranean forests. Nomada, Andrena and Lasioglossum genera are often generalist when seeking for nectar and there is no supporting literature regarding their general soil requirements for belowground nesting to confirm or infirm our results.

As expected, large bees respond to land cover composition with the 1000m radius due to their ability to forage within larger area [START_REF] Greenleaf | Bee foraging ranges and their relationship to body size[END_REF]. For several species, it may be linked with their nesting preferences. For example, Halictus scabiosae was related to anthropogenic land cover, and this species generally nests in anthropogenic sites or in disturbed places [START_REF] Ulrich | Flexible social organization and high incidence of drifting in the sweat bee, Halictus scabiosae[END_REF]. Rhodanthidium sticticum was related to rocky land cover as it often nests in snail shells easily detectable on bare soils [START_REF] Moreno-Rueda | Weather and the use of empty gastropod shells by arthropods[END_REF]. For other species, such as Xylocopa violacea, Melecta species, Anthophora mucida and Eucera nigrecens, it was more difficult to link their presence to a particular nesting habitat, as they were mostly related to the interface between anthropogenic zone and scrublands. Likewise, the diversity of land covers within a 1000m radius around our plots was linked with a higher species richness of large bees. We found that large bee species richness decreased when the scrubland land cover increased. This might underline that land cover diversity within a 1000m radius around plots increases the number of plant species and nesting substrates which can shelter large bee species [START_REF] Dafni | Pollination syndromes in the Mediterranean: generalizatoins and peculiarities[END_REF]. Several studies demonstrated a loss of bee species diversity linked to the homogenization of one land cover in agricultural landscapes [START_REF] Holzschuh | Diversity of flowervisiting bees in cereal fields: effects of farming system, landscape composition and regional context[END_REF][START_REF] Tscharntke | Characteristics of insect populations on habitat fragments: a mini review[END_REF]. The bee species richness we observed here might also be linked to the interface between anthropogenic land cover and scrublands. [START_REF] Fortel | Decreasing abundance, increasing diversity and changing structure of the wild bee community (hymenoptera: Anthophila) along an urbanization gradient[END_REF] found a maximum in the species richness of bees at an intermediate proportion of anthropogenic land cover, illustrating the Intermediate Disturbance Hypothesis.

Effect of honeybee colony density

Semi-natural habitats with dominant flowering species, such as scrublands (with Rosmarinus officinalis, Cistus albidus), are also particularly attractive to beekeepers to settle their honeybee colonies [START_REF] Geldmann | Conserving honey bees does not help wildlife[END_REF]. Through the practice of transhumance during spring in the scrublands of southern France, beekeepers can set up apiaries that include large numbers of colonies at the same time for a short period [START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF]. In the Calanques National Park, the mean honeybee colony density is more than twice the national mean (respectively 5.3 colonies/km 2 and 2.5 colonies/km 2 Chauzat et al., 2013). Our results showed a decrease in abundance and richness of large bees with the increase of honeybee colony density, which could be linked to an emerging issue relative to the competition for resources between honeybees and wild bees [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Mallinger | Do managed bees have negative effects on wild bees?: a systematic review of the literature[END_REF]. The intensive installation of beehives may lead to the monopolization of available nectar and pollen resources by honeybees. As demonstrated by several studies, Apis mellifera is a highly generalist species and can easily use resources that are common to the diet of large wild bee species [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF]. Large bee species have greater food requirements for their progeny than smaller bee species [START_REF] Müller | Quantitative pollen requirements of solitary bees: implications for bee conservation and the evolution of bee-flower relationships[END_REF] and they have the capacity to collect larger quantities of pollen than small bee species [START_REF] Greenleaf | Bee foraging ranges and their relationship to body size[END_REF]. Furthermore, large bees can fly further away to avoid honeybee competition and forage where the competitive pressure is low. This may explain why we found fewer individuals and species of large bees when the local density of honeybee colonies was high. [START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF] demonstrated a similar trend with large bees being particularly affected by honeybee colony density, especially for nectar resources.

Conclusion

In this study, we observed that responses of bee species assemblages to land cover composition in the Calanques National Park depend on their body size. The assemblage of small bee species was particularly sensitive to local plant community composition and land cover composition within a 1000m radius. Large bee species assemblages were also sensitive to land cover composition within a 1000m radius with an observed peak of species richness at the interface of different land covers. In this context, the Calanques National Park, by its localization in the vicinity of Marseille, maintains and protects a diversity of land covers which seems essential to preserve and sustain a wide range of bee species. However, the density of honeybee colonies appeared to be a non-negligible determinant acting negatively upon wild bee species assemblages. Our study suggested a negative effect of honeybee colony density on large bee species richness and abundance. As semi-natural protected areas may cover endangered habitats and consequently protected species, we would like to raise awareness among land managers to encourage the limitation of honeybee colonies in protected areas. To better assess the exploitative competition between wild bees and domesticated honeybees, future studies should take into account the quantity of floral resources available for flower-visitors.

Fig. 1 .

 1 Fig. 1. Boundaries and limits of the western part of Calanques National Park. Red circles correspond to each sampling plot. Geographical data were obtained from the Calanques National Park.

Fig. 2 .

 2 Fig. 2. Land cover composition effect on large bees with A Principal Component Analysis projection of land cover and B Correspondent Analysis projection of large bee species on the first factorial plane of the land cover composition co-inertia. C Relationship between the percentage of low scrubland within the 1000m radius and the large wild bee species richness.

Fig. 3 .

 3 Fig. 3. Linear regressions between the honeybee density score SED and A large bee species richness, and B large bee abundance.

Fig. 4 .

 4 Fig. 4. Land cover composition effects on small bees with A Principal Component Analysis projection of land cover and B Correspondent Analysis projection of small bee species on the first factorial plane of the land cover composition co-inertia. Local plant community effects on small bees with C Principal Component Analysis projection of plant species and D Correspondent Analysis projection of small bee species on the first factorial plane of the local plant community composition coinertia.

Acknowledgments

We are thankful to the Calanques National Park and Observatoire Hommes-Milieux Littoral Méditerranéen (OHM-Littmed) for financial support. We also thank the Calanques National Park and its territory owners for access permission. We are grateful to Matthieu Aubert and Eric Dufrêne for bee species identification. We thank Mallaury Hamon, Maxime Berenger, Alexandra Bideau, Manon Panchot, Christel Scagliola, Astrid Malechaux, Anaïs Syx, Virgile Calvert, Benjamin Oursel who participated in bee sampling in the field. We finally thank the two anonymous referees for their constructive comments on a previous version of this manuscript and M. Paul for English improvement.

Declaration of competing interest

The authors state that they have no conflict of interest.


Yan Lin

Yih Ching Ong

Sarah Keller

Johannes Karges

Rafika Bouchene

Eric Manoury

Olivier Blacque

Joachim Müller

Nicoleta Anghel

Andrew Hemphill

Cécile Häberli

Aya C Taki

Robin B Gasser

Kevin Cariou

Jennifer Keiser

Gilles Gasser

Synthesis, Characterization and Antiparasitic Activity of Organometallic Derivatives of the Anthelmintic Drug Albendazole

Keywords: anthelmintics, bioorganometallic chemistry, ferrocene, medicinal inorganic chemistry, medicinal organometallic chemistry

Helminthiases, a group of neglected tropical diseases, affect more than one billion people mainly in tropical and subtropical regions. Moreover, major intestinal protozoa have a significant impact on global public health. Albendazole (ABZ) is a broad-spectrum anthelmintic recommended by the World Health Organisation (WHO). However, drug resistance is emerging due to its widespread use.

In order to tackle this problem, taking into account the spectacular results obtained with the organometallic derivatization of the antimalarial drug chloroquine, we have prepared, in this study, a series of new ferrocenyl and ruthenocenyl derivatives of the organic drug ABZ and assessed their activity against different helminths and protozoans, namely Trichuris muris, Heligmosomoides polygygrus, Schistosoma mansoni, Giardia lamblia, Haemonchus contortus and Toxoplasma gondii.

The ferrocene-containing ABZ analogue 2d exhibited over 70% activity against T. muris adults in vitro at 200 µM and no toxicity to mammalian cells (IC 50 > 100 µM). H. polygyrus adults were not affected by any of the derivatives tested. Against T. gondii, the ferrocene-containing ABZ analogues 1a and 2d showed better in vitro activity than ABZ and low toxicity to the host cells. The activity of the analogous ruthenocenyl compound 2b against S. mansoni and T. gondii in vitro might be attributed to its toxicity towards the host cells rather than a specific antiparasitic activity. These results demonstrate that the derivatives show a species specific in vitro activity and the choice of the organometallic moieties attached to the organic drug is playing a very important role. Two of our organometallic compounds, namely 1b and 2d, were tested in T. muris infected mice. At a 400 mg/kg dose, the compounds showed moderate worm burden reductions but low worm expulsion rates. Overall, this work, which is one of the first studies reporting the potential of organometallic compounds on a very broad range of parasitic helminths and protozoan, is a clear confirmation of the potential of organometallic complexes against parasites of medical and veterinary importance.

INTRODUCTION

Neglected tropical disease (NTDs) affect the poorest populations without adequate water and sanitation in tropical and subtropical regions. More than one billion people, which amounts to onesixth of the world's population, are affected by NTDs within 149 countries. [START_REF] Who | Why are some tropical diseases called 'neglected[END_REF][START_REF] Who | Neglected tropical diseases[END_REF] Even though the morbidity exceeds the mortality, more than half a million deaths per year are caused by NTDs. [START_REF] Hess | Ferrocenyl, Ruthenocenyl, and Benzyl Oxamniquine Derivatives with Cross-Species Activity against Schistosoma mansoni and Schistosoma haematobium[END_REF] Among NTDs, worm infection, or helminthiasis, has been the target of global elimination programs. [START_REF] Campbell | A Critical Appraisal of Control Strategies for Soil-Transmitted Helminths[END_REF] Because of the large number of individuals affected, a resolution was passed by the World Health Organisation (WHO) in 2001 to control several helminth infections through preventive chemotherapy (PC), employing anthelmintics on populations at risk to prevent morbidity. [START_REF] Lo | A call to strengthen the global strategy against schistosomiasis and soil-transmitted helminthiasis: the time is now[END_REF][START_REF] Bethony | Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm[END_REF] Considering the rapid re-infection following treatment and the possible development of resistance in species such as Trichuris or Schistosoma, there is an urgent need for research on novel and structurally different classes of potential anthelmintic drugs. Despite the urgency to develop new anthelmintics, the budget invested in this field of research is limited. The poorest population affected by NTDs is often living on less than US$ 2 per day. Therefore, most of them cannot afford expensive medicines. [START_REF] Bethony | Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm[END_REF] Moreover, drugs in PC programs are donated in large scale treatment programs, which have mitigated effect. [START_REF] Chami | More medicines alone cannot ensure the treatment of neglected tropical diseases[END_REF] Concurrently, there is little motivation for the pharmaceutical industry to develop new drugs that might not provide return on huge research and development investment, making the discovery of new anthelmintics an even greater challenge.

Albendazole (ABZ, Figure 1) is an anthelmintic that was recommended by the WHO to treat soil- transmitted helminth infections. [START_REF] Who | Soil-transmitted helminth infections[END_REF] ABZ shows a broad-spectrum of activity in domestic animals and was subsequently licensed for human use in 1982. [START_REF] Ehteda | Complexation of Albendazole with Hydroxypropyl-β-Cyclodextrin Significantly Improves its Pharmacokinetic Profile, Cell Cytotoxicity and Antitumor Efficacy in Nude Mice[END_REF] Because of its effectiveness, safety and low price, ABZ is one of the main drugs used in PC programs. [START_REF] Horton | Albendazole: a review of anthelmintic efficacy and safety in humans[END_REF] The excellent safety record of ABZ is related to its mechanism of action, which involves selective binding to -tubulin and disruption of microtubule polymerization. [START_REF] Lubega | Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: Binding affinity and anthelmintic efficacy[END_REF] However, to the best of our knowledge, the exact manner how ABZ binds -tubulin is unclear. [START_REF] Horton | Albendazole: a review of anthelmintic efficacy and safety in humans[END_REF] Nevertheless, there are several drawbacks associated with its use, such as poor drug absorption and the lack of water solubility. [START_REF] Lanusse | Clinical Pharmacokinetics and Metabolism of Benzimidazole Anthelmintics in Ruminants[END_REF] Importantly, its widespread use has resulted in reduced susceptibility of helminths of veterinary importance. [START_REF] Waghorn | Prevalence of anthelmintic resistance on 62 beef cattle farms in the North Island of New Zealand[END_REF] Taking these effects into account is at the heart of the One Health initiative, 14 as the development of drugs for human use can imply consequences for veterinary uses and vice-versa.

With this in mind, our research groups have started a program to derivatize ABZ with various organometallic moieties in order to evaluate their activity on various human and animal parasites, including, but not limited to, helminths. Organometallic compounds, which are defined as metal complexes containing at least one direct, covalent metal-carbon bond, have recently [START_REF] Rivas | New heterobimetallic ferrocenyl derivatives: Evaluation of their potential as prospective agents against trypanosomatid parasites and Mycobacterium tuberculosis[END_REF][START_REF] Hillard | Ferrocene Functionalized Endocrine Modulators as Anticancer Agents[END_REF][START_REF] Golbaghi | Organoruthenium(II) Complexes Bearing an Aromatase Inhibitor: Synthesis, Characterization, in Vitro Biological Activity and in Vivo Toxicity in Zebrafish Embryos[END_REF] found numerous applications in medicinal chemistry, [START_REF] Barry | Exploration of the medical periodic table: towards new targets[END_REF][START_REF] Gasser | The potential of organometallic complexes in medicinal chemistry[END_REF][START_REF] Gasser | Organometallic Anticancer Compounds[END_REF][START_REF] Brown | Medicinal organometallic chemistry -an emerging strategy for the treatment of neglected tropical diseases[END_REF] including against helminths [START_REF] Hess | Assessment of the nematocidal activity of metallocenyl analogues of monepantel[END_REF][START_REF] Hess | Synthesis, Characterization, and Biological Activity of Ferrocenyl Analogues of the Anthelmintic Drug Monepantel[END_REF][START_REF] Hess | Organometallic Derivatization of the Nematocidal Drug Monepantel Leads to Promising Antiparasitic Drug Candidates[END_REF] and NTDs like schistosomiasis. [START_REF] Hess | Ferrocenyl, Ruthenocenyl, and Benzyl Oxamniquine Derivatives with Cross-Species Activity against Schistosoma mansoni and Schistosoma haematobium[END_REF][START_REF] Ong | Metal Compounds against Neglected Tropical Diseases[END_REF][START_REF] Patra | Ferrocenyl Derivatives of the Anthelmintic Praziquantel: Design, Synthesis, and Biological Evaluation[END_REF][START_REF] Hess | Toward organometallic antischistosomal drug candidates[END_REF] For instance, Brocard, Biot and co-workers synthesized ferroquine (FQ, Figure 1), a ferrocenyl derivative of chloroquine (ACQ, Figure 1), which is currently undergoing phase II trials against malaria. [START_REF] Biot | Synthesis and Antimalarial Activity in Vitro and in Vivo of a New Ferrocene-Chloroquine Analogue[END_REF][START_REF] Biot | The antimalarial ferroquine: from bench to clinic[END_REF] It was demonstrated that ferroquine has an activity against both chloroquine-susceptible and chloroquine resistant P. falciparum. Ferrocene is a small, rigid, lipophilic molecule that can easily cross cellular membranes. [START_REF] Dombrowski | Metallocenes in biochemistry, microbiology & medicine[END_REF] These characteristics, along with its reversible electrochemical behavior, make ferrocenyl derivatives interesting candidates for biological applications and further drug design. For ferroquine or ferrocifen (Fc-TAM, Figure 1) -an anticancer drug analogue of tamoxifen (TAM, Figure 1) developed by the Jaouen group [START_REF] Hillard | Ferrocene Functionalized Endocrine Modulators as Anticancer Agents[END_REF] -the addition of a ferrocenyl moiety into a known organic drug has allowed a remarkable increase in activity compared to the parent organic compounds. Inspired by these successes, we herein report our findings concerning the antiparasitic activity of five ferrocenyl and two ruthenocenyl derivatives of ABZ with the aim to bypass drug-resistance pathways and/or to increase the potency and therapeutic scope of ABZ. The ruthenocenyl derivatives were synthesized because ferrocene and ruthenocene are isoelectronic and have a similar geometry and steric demand, yet they exhibit completely different electrochemical behavior and redox properties.

RESULTS AND DISCUSSION

Synthesis and characterization

In this study, two strategies were used to synthesize ABZ analogues incorporating a metallocene moiety (Scheme 1). In the first strategy, (route A) the N-methylcarbamate group was left untouched and the metallocenyl fragment added on the nitrogen. In the second (route B), the carbamate function was replaced by an acyl metallocene group (with various linkers). Compounds 1a, 1b and 1c were prepared by direct treatment of ABZ with (ferrocenylmethyl)-trimethylammonium iodide, (ruthenocenylmethyl)-trimethylammonium iodide and (2-ferrocenylvinyl)methyltrimethylammonium iodide, respectively, with K 2 CO 3 in acetonitrile using a similar synthetic method employed by Spiccia and co-workers. [START_REF] Gasser | Products of hydrolysis of (ferrocenylmethyl)trimethylammonium iodide: Synthesis of hydroxymethylferrocene and bis(ferrocenylmethyl) ether[END_REF] Route B started by the diethylenetriamine-mediated cleavage of the unactivated carbamate in ABZ to give ABZ-NH 2 in good yield. [START_REF] Noshita | Diethylenetriamine-Mediated Direct Cleavage of Unactivated Carbamates and Ureas[END_REF] Compound ABZ-NH 2

was then converted into 2a-d by treatment with the ferrocene-(2a,c,d) or ruthenocene-(2b) containing carboxylic acid derivatives via a TBTU mediated amide coupling reaction. Comparing the activities of ruthenocenyl analogues (1b, 2b) with their ferrocene counterparts (1a, 2a) should allow detecting whether the iron-mediated redox properties are responsible for the anthelmintic activity.

All newly synthesized compounds were unambiguously characterized by 1 H, [START_REF] Waghorn | Prevalence of anthelmintic resistance on 62 beef cattle farms in the North Island of New Zealand[END_REF] CNMR spectroscopy, IR spectroscopy and ESI mass spectrometry. The purity of individual compounds was confirmed by microanalysis. Details on synthesis and characterization of the complexes can be found in the Supporting Information.

S N Additionally, complexes 1a, 1b, 1c were characterized using single-crystal X-ray crystallography (Figure 2). Details on the acquisition of the structures can be found in the Supporting Information. The thermal ellipsoids are drawn at the 30% probability level.

H N NH O O (i) or (ii) (iii) S N H N N O O M S N H N N O O Fe M = Fe (1a), Ru (1b) 1c S N H N NH 2 S N H N N H O M S N H N H N Fe S N H N H N O O Fe M = Fe (2a), Ru (2b) 2c 2d (iv) (v) or (vi) (vii) (viii

Stability in human plasma

In order to assess the stability of our metallocene-containing ABZ derivatives under simulated biological conditions, we first studied their behavior in human plasma. Human plasma stability is a crucial parameter of the stability profile for drug discovery, as compounds unstable in plasma are generally not suitable drug candidates, with the exception of some prodrugs or antedrugs. [START_REF] Keller | A tutorial for the assessment of the stability of organometallic complexes in biological media[END_REF] In order to do so, a rapid, accurate and economic method based on HPLC and LC-MS technique was chosen, as recently reported by our group. [START_REF] Patra | Ferrocenyl Derivatives of the Anthelmintic Praziquantel: Design, Synthesis, and Biological Evaluation[END_REF]36 Caffeine, which is known to be stable in human plasma, was chosen as an internal standard. After incubation in human plasma at 37°C for various times, methanol was added to quench the plasma solution. Afterwards, the recovery of the internal standard, the test compound and the eventual metabolites was performed by liquid extraction with dichloromethane. The obtained residue was analyzed by RP-HPLC (See Supporting Information for details) and the results of these analyses are gathered in Figure 3. ABZ and ABZ-NH 2 proved to be relatively stable after 24 h incubation in human plasma -they are decomposed by only 2% and 17%, respectively. The metallocene-containing ABZ derivatives showed moderate to good stability after 6 h but significant decay was observed after 24 h. The ferrocene-containing compound 2d displayed the best stability of the derivatives in human plasma after 24 h, with only 23% degradation.

Compound 2b showed the second best stability since it only decomposed by 26% after 24 h incubation. Nearly 50% degradation was measured after 24 h for compounds 1a-1c and 2a.

Figure 3. Stability of the metallocene-containing ABZ in human plasma. The ratio of the test compound peak area to the internal standard peak area was set as 100% at t = 0. This ratio was plotted with regards to the time of incubation.

Compound 2c was shown to decompose at a highest rate (ca. 77% release after 24 h incubation).

The following order for stability in human plasma can be made from the most stable to the least stable: 2d > 2b > 1a > 1b > 1c ≈ 2a > 2c. We thought the electron-withdrawing functional group attached to the metallocene, as carbonyl, might help to stabilize the organometallic derivatives. To gain further insight on the nature of the decomposition products, LC-MS analyses were performed.

In the case of 1b, the main degradation compound was identified to be the result from the oxidation of 1b. Hypothesizing that the oxidation site would be the sulfur atom, 1b-Ox was synthetized from ABZ by a two-step oxidation/substitution sequence (Scheme 2). Its identity as the degradation product from 1b was then confirmed by measuring its retention times under the same HPLC conditions (see Supporting Information). As shown in Scheme 2, the sulfoxide analogue 2d-Ox was also synthetized by acylation of ABZ-NH 2 -Ox that was itself obtained from ABZ-Ox. As expected, 2d-Ox was confirmed to be the major metabolite of 2d in human plasma. The metabolites of ABZ and ABZ-NH 2 were also confirmed to be ABZ-Ox and ABZ-NH 2 -Ox, respectively (see Supporting Information). [START_REF] Dayan | Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics[END_REF] The metabolites of 1a, 1c, 2a and 2b were not unambiguously identified, yet, the mass spectra are consistent with oxidized species, presumably also occurring on the sulfur atom.

For compound 2c, despite its rather moderate stability, no degradation peak could be found by HPLC and LC-MS. This study is an excellent indication that these metallocene-containing ABZ derivatives were exclusively metabolized to their oxidized species, as neither peaks corresponding to the free metal complexes nor to released ABZ could be observed.

Antiparasitic activities

A broad spectrum screening against a range of helminths was conducted. In a One Health approach, the chosen panel of parasites included species able to colonize both human and animals:

Heligmosomoides polygygrus (rodents), Trichuris muris (mice, analogous to the human parasite Trichuris trichiura), Schistosoma mansoni (human), Haemonchus contortus (ruminants such as sheep) as well as protozoan parasites that can affect humans: Giardia lamblia and Toxoplasma gondii.

Helminths. Results obtained when testing the derivatives against adult species of the hookworm H. polygygrus, the whipworm T. muris and the schistosome S. mansoni in vitro are summarized in Table 1. Interestingly, compound ABZ-NH 2 was the most active compound showing high activity against all three species tested at concentrations of 100-200 µM. Against S. mansoni, ABZ-NH 2 achieved even death of all worms at 200 µM. On the other hand, ABZ, the drug of choice for hookworm and whipworm infections lacked activity against all 3 species tested confirming previous findings. [START_REF] Buchter | Preparation, Physicochemical Characterization and In Vitro and In Vivo Activity Against Heligmosomoides polygyrus of Novel Oral Formulations of Albendazole and Mebendazole[END_REF] Compounds 1b and 2d showed activity (resulting in reduced motility) against T. muris at the highest concentration tested while 2b affected S. mansoni. None of the compounds showed activity against H. polygyrus adults. In the in vitro cytotoxicity assay using L6 rat cells, compounds 2a and 2d were found to be the least toxic against mammalian cells. Given these promising in vitro findings, compounds ABZ-NH 2 , 1b and 2d underwent testing in T. muris infected mice. At a 400 mg/kg dose compounds showed moderate worm burden reductions but, unfortunately, only low worm expulsion rates (Table 2). All compounds were well tolerated by mice despite the low IC 50 of 1b in the cytotoxicity L6 assay. 4A). Dose-response evaluations estimated the potencies of individual compounds on xL3 motility after 72 h (Figure 4B). An inhibition of xL3 motility was observed for compounds 1b and 2b, but no greater than 27.2% inhibition was observed at 100 µM, and ABZ exhibited xL3 motility by 16.0%. The two reference compounds, monepantel and moxidectin, exhibited the expected reduction in xL3 motility (> 85.0%). All six other compounds were ineffective at the highest concentration tested (100 µM). Giardia lamblia. As expected, cultivation in the presence of 0.1 and 1 µM ABZ resulted in complete inhibition of resazurin reduction, indicating loss of viability in 100% of all parasites, while the presence of 0.01 µM ABZ caused a 70% reduction of viability when compared to the solvent control (Figure 5). Compound ABZ-NH 2 was largely ineffective at all three concentrations, while for compound 1a a reduction in viability to approximately 60% was measured at 0.01 µM, but viability was increased at higher concentrations. For 1c, 2a, and 2c, the presence of these compounds impaired viability to levels of 40-60%, except for 2d, which resulted in a reduction of >95% when 1 µM was applied. Compounds 1b and 2b were ineffective under these conditions.

Toxoplasma gondii.

As can be seen in Figure 6, compounds ABZ, ABZ-NH 2 , 1a, 2d and 2b severely impair T. gondii proliferation when applied at 10 M, and when applied at lower concentrations, a clear dose-dependent effect is evident. However, the inhibition seen with 2b and 2d at 10 M can be partially attributed to the impact these compounds have against HFF host cells (see below). In order to discriminate between the activity on the parasite and on the host cell, the toxicity of the compounds was also evaluated on HFF cells alone (Figure 7). With the exception of compound 2b and 2d at the highest concentration, none of these compounds presented cytotoxicity against HFF monolayers. Thus, with the exception of 2b, and 2d, the effects seen against T. gondii can be attributed to mechanisms that are targeting the parasites.

CONCLUSIONS

In this study, we synthesized and characterized a series of ferrocene-and ruthenocene-containing organometallic analogues of the anthelmintic drug ABZ. In human plasma, the metallocenecontaining ABZ derivatives showed moderate to good stability after 6 h, with ferrocenyl derivative 2d displaying the best stability after 24 h incubation. As for ABZ, the major metabolites for the compounds turned out to be the corresponding sulfoxides, as demonstrated by the syntheses of the oxidized forms of 1b and 2d, namely 1b-Ox and 2d-Ox. These findings are a good indication that the active forms were not due to the release of ABZ or of a free organometallic moiety. In vitro activity of the metallocene-containing ABZ derivatives against H. polygygrus adult, T. muris adult and S. mansoni adult showed that 1b and 2d displayed over 70% activity against T. muris at 200 µM.

Compound 2b also showed over 70% activity against S. mansoni at the highest concentration.

Encouraged by these promising in vitro results, compounds 1b and 2d underwent testing in T. muris infected mice at a single dose of 400 mg/kg and exhibited interesting, albeit moderate, worm burden reductions and worm expulsion rates. However, the anthelmintic activity against H. contortus was negligible for most compounds, compared to the high efficiency of ABZ itself.

Although not better than ABZ, the activity of four analogues (1c, 2a, 2c and 2d) against protozoan G. lamblia was also promising. Finally, against the protozoan T. gondii, compounds 1a and 2d showed better activity than ABZ at 1 µM, while they mostly showed cytotoxic effects on not infected HFF host cells at 10 µM. This suggests that the ferrocene-containing compounds 1a and 2d are promising anti-toxoplasma candidates for in vivo evaluation. Overall, this study demonstrates that organometallic ABZ derivatives can outperform ABZ as interesting leads for further development not only as a new class of anthelmintic agents but also against other types of parasites due to the attached organometallic portions. Further studies would be warranted to be better elucidate the mechanism of action of these organometallic derivatives, investigated the effect on metal-specific properties, like ROS production, redox activation, and lipophilicity, etc. Nonetheless, these compounds present excellent starting points that help advance the field of organometallic medicinal chemistry in the area of neglected tropical diseases.

MATERIALS & METHODS

In vitro assays on H. polygyrus and T. muris adult worms

Female NMRI mice were infected with 88 H. polygyrus L3. Two weeks post-infection, mice were dissected. Seven weeks post-infection of female C57BL/6NRj, T. muris adult worms were collected from the intestines. Three to four adult worms were placed in each well of a 24-well plate and exposed to the test drugs at concentrations ranging from 200-10 µM. The highest percentage of DMSO used in the assays served as a control. The assay was conducted in duplicate. Worms were incubated at 37°C / 5% CO 2 up to 72 h, after which the drug effect was evaluated. The condition of the worms was microscopically evaluated using a viability scale from 3 (normal activity) to 0 (dead) following stimulation with 500 µl hot water (≈80°C).

In vitro assays on adult S. mansoni

Mice were dissected seven weeks post-infection with 100 cercariae. At least three worms (both sexes) were incubated with 200 and 100 μM of the derivatives in 2 ml RPMI 1640 (Gibco, USA) supplemented with 5% (v/v) FCS and 1% (v/v) pen-strep at 37°C / 5% CO 2 for 72 hours h. Details on the viability scale used for scoring are published elsewhere. [START_REF] Lombardo | Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni[END_REF]

Animals

Three-week-old female C57BL/6NRj mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and female mice (NMRI strain; age 3 weeks-old; weight ca. 20-22 g) were purchased from Charles River, Germany. Rodents were kept under environmentally-controlled conditions (temperature: 25°C, humidity: 70%, light/dark cycle 12 h /12 h) with free access to water (municipal tap water supply) and rodent food. Rodents were allowed to acclimatize for 1 week before infection.

The current study was approved by the local veterinary agency, based on Swiss cantonal and national regulations (permission no. 2070).

T. muris in vivo studies

C57BL/6NRj mice were orally infected with 200 embryonated T. muris eggs. Mice were immunosuppressed with dexamethasone (1 mg/ml) in their drinking water until 2 days prior to treatment. At 42 days post-infection, the stool was collected and checked for the presence of eggs.

The compounds were administered orally at a single oral dose of 400 mg/kg. Four untreated mice served as controls. The stool was collected from 0-72 h post-treatment from each mouse to determine the worm expulsion rate. Six to seven days post-treatment, animals were killed, the intestine was dissected and adult worms were collected and counted.

Cytotoxicity testing against L6 cells

Rat skeletal myoblast L6 cells were exposed to the test drugs. Podophyllotoxin (with 0.05 µg/ml as highest concentration) was used as a positive control. After a 70-h incubation at 37°C / 5% CO 2 , resazurin dye (Alamar Blue) was added and the plates were incubated for an additional 2 hours Fluorescence and the cytotoxic concentration (CC50) were measured at 530 nm excitation and 590 nm emission wavelength using a SpectraMax M2 (Molecular Device, Sunnyvale CA, USA; Softmax version 5.4.6). Experiments were conducted in duplicate and repeated once.

In vitro assessment of inhibitory activity on larval development of Haemonchus contortus

All derivatives and ABZ were individually tested for their ability to inhibit the motility of exsheathed third-stage larvae (xL3) of H. contortus and the development of these larvae to fourth-stage larvae (L4) using established assays. [START_REF] Preston | Low cost wholeorganism screening of compounds for anthelmintic activity[END_REF] Compounds were tested in an 18 point, two-fold dilution series, starting at 100 µM. The reduction in xL3 motility was measured at 72 h, and inhibition of L4 development was evaluated after 7 days. For the xL3 motility assay, monepantel (Zolvix, Elanco Animal Health, Australia) and moxidectin (Cydectin, Virbac, France) were included as positive controls. Culture medium, i.e. Luria Bertani broth supplemented with 100 IU/ml of penicillin, 100 µg/ml of streptomycin and 0.25 µg/ml of amphotericin B (Gibco, Thermofisher Scientific, USA), with

20

 20

Figure 1 .

 1 Figure 1. Structures of albendazole, chloroquine and tamoxifen and their metallocene derivatives.

Scheme 1 .

 1 Scheme 1. Synthesis of the metallocene analogues of albendazole. Reagents and conditions: (i) (ferrocenylmethyl)-trimethylammonium iodide, K 2 CO 3 , dry MeCN, 90°C; (ii) trimethylammoniomethylruthenocene iodide, K 2 CO 3 , dry MeCN, 90°C; (iii) (2-ferrocenylvinyl) methy-trimethylammonium iodide, K 2 CO 3 , dry MeCN, 90°C; (iv) diethylenetriamine, 130°C; (v) ferrocenecarboxylic acid, TBTU, DIPEA, dry DMF; (vi) ruthenocenecarboxylic acid, TBTU, DIPEA, dry DMF; (vii) ferroceneacetic acid, TBTU, DIPEA, dry DMF; (viii) 3-Ferrocenoylpropionic acid, TBTU, DIPEA, dry DMF.

Figure 2 .

 2 Figure 2. A: molecular structure of 1a; B: molecular structure of 1b; C: molecular structure of 1c.

Scheme 2 .

 2 Scheme 2. Synthesis of the oxidized analogues of 1b and 2d. Reagents and conditions: (i) 37% H 2 O 2 , glacial acetic acid; (ii) trimethylammoniomethylruthenocene iodide, K 2 CO 3 , dry MeCN, 90°C; (iii) diethylenetriamine, 130°C; (iv) 3-Ferrocenoylpropionic acid, TBTU, DIPEA, dry DMF.

Figure 4 :

 4 Figure 4: In vitro activity of ABZ derivatives against exsheathed third-stage larvae (xL3) H. contortus in vitro. (A) Development of xL3 to fourth-stage larvae (L4) at 100 µM after 7 days. (B)

Figure 5 .

 5 Figure 5. In vitro screening of G. lamblia trophozoites. The viability of G. lamblia trophozoites is measured by Resazurin assay. Compounds were applied at 1, 0.1 or 0.01 µM for a period of 72 h.

Figure 6 .

 6 Figure 6. In vitro screening of T. gondii β-gal tachyzoites cultured in HFF host cells. The proliferation of T. gondii tachyzoites was quantified by measuring β-galactosidase activity. Compounds were applied at 0.1, 1 or 10 M for a period of 72 h. The compound activity is shown as percentage in relation to the DMSO controls which represent 100% of parasite growth.

Figure 7 .

 7 Figure 7. In vitro screening of non-infected HFF host cells. The viability of HFF was quantified by measurement of resazurin reduction. Compounds were applied at 0.1, 1 or 10 M for a period of 72 h. The viability of HFF is shown as percentage in relation to the DMSO controls which represent 100% viability.

Table 1 . in vitro activity of derivatives against Heligmosomoides polygygrus, Trichuris muris and Schistosoma mansoni

 1

	Compound	T. muris adult		H. polygygrus adult	S. mansoni adult	Cytotoxic L6 cells a
		200 µM 100 µM	10 µM	200 µM	100 µM	200 µM	100 µM	IC 50 (µM)
	1a	31.4 (9.3)		38.3 (22.4)		48.9 (11.8)		14.7
	1b	74.5 (9.3) 23 (11.8)		46.7 (11.8)		57.1 (12.5)		4.3
	1c	35.3 (37.5)		41.7 (9.3)		56.8 (18.6)		18
	2a	19.6 (9.3)		40.0 (0.0)		26.5 (0.0)		> 100 µM
	2b	11.8 (12.5)		31.7 (9.3)		73.4 (9.3)	24.8 (12.5)	3.8
	2c	27.5 (9.3)		30.0 (0.0)		20.3 (12.5)		27
	2d	78.4 (9.3) 19.2 (0.0)		46.7 (11.8)		59.2 (11.8)		> 100 µM
	ABZ	19.6 (9.3)		28.3 (9.3)		56.8 (18.6)		2
	ABZ-NH2	90.2 (9.3) 84.6 (11.8) 40.0 (12.5) 70.0 (0.0) 33.3 (14.4)	100 (0.0)	52.8 (14.4)	28.9

a 2 h after adding Resazurin dye. Table reports mean activity values (%) and standard deviation (SD).

Table 2 . Activity of 1b and two derivatives in T. muris infected mice when administered at 400 mg/kg

 2

	Compound	Mice cured/investigated	Mean number of worms in intestine (SD)	Mean number of worms in stool (sd)	Worm burden reduction*	Worm expulsion rate **
	Control	0/4	114.0 (9.3)	0.5 (0.6)	--	--
	ABZ-NH2	0/4	48.8 (5.7)	0.3 (0.5)	57	0,5
	1b	0/4	36.3 (2.2)	2.0 (1.8)	68,1	5,2
	2d	0/4	47.5 (11.2)	0.8 (0.1)	58,1	1,6
	* decrease in mean worm burden of treatment groups compared to the mean burden of the control
	group
	** Mean number of worms expelled within 72 hours after treatment
	All nine derivatives, including ABZ, were tested against H. contortus xL3s (a helminth affecting

ruminants such as sheep) in the whole-organism assay. The parent compound, ABZ, and its derivatives ABZ-NH 2 and 2a all decreased L4s development over 7 days using 100 µM of each compound. ABZ,

[START_REF] Hall | The dose response of several benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubriformis selected with thiabendazole[END_REF]

which has previously been evaluated, fully inhibited L4 development at this concentration. Compounds ABZ-NH 2 inhibited L4 development by 35.8% (p=<0.0001), whereas compound 2a reduced this development by 19.1% (p=0.001; Figure

Funding Information

This work was financially supported by the Swiss National Science Foundation (Grant Sinergia CRSII5_173718, to G.G.), an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679) and has received support under the program Investissements d'Avenir launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). Y.L. thanks to the China Scholarship Council for financial support. Funding from the Australian Research Council, Yourgene Health and The University of Melbourne is gratefully acknowledged.

matched concentrations of DMSO (0.25%), served as negative controls in the assays. Each assay has three technical replicates and was repeated three times on different days. For each compound, dose-response curves were determined by fitting the log 10 -transformed concentration to a variable slope four-parameter model. The data were analysed with reference to the negative controls using non-parametric one-way ANOVA and Dunnett's multiple comparison tests employing the GraphPad Prism (v.8.3.1).

In vitro activity against the protozoan Giardia lamblia

G. lamblia trophozoites were grown under anaerobic conditions in 10 ml culture tubes (Nunc, Roskilde, Denmark) as described. [START_REF] Muller | In Vitro Effects of Thiazolides on Giardia lamblia WB Clone C6 Cultured Axenically and in Coculture with Caco2 Cells[END_REF] Cultures with adherent confluent trophozoite lawns were incubated on ice for 15 min, suspended motile trophozoites were counted in a Neubauer chamber at 200 x magnification), and were distributed into 96 well plates in 0.2 ml Keister-medium containing 1000 trophozoites per well in the presence of the compounds (0.01, 0.1 or 1 µM, all in triplicates) dissolved in DMSO (10 mM stock solutions) or with the corresponding DMSO solvent control. They were then cultured for 72 h at 37°C in an anaerobic chamber (80% N 2 , 10% H 2 , 10% CO 2 .

Subsequently, plates were centrifuged (2 min, 1000 x g), medium was removed, and cells were washed once with PBS containing 1 g/l glucose. Then, PBS-glucose containing 10 mg/l resazurin was added, and plates were incubated for up to 5 h at 37°C. Plates were read (excitation 530 nm, emission 590 nM) using an Enspire multilabel reader (2300 EnSpire™ Multilabel Reader, Perkin-Elmer, Turku, Finland) at various time points, and the linear increase of fluorescence was used as a marker for cell viability. [START_REF] Bénéré | A new quantitative in vitro microculture method for Giardia duodenalis trophozoites[END_REF]

In vitro cytotoxicity assessment in human foreskin fibroblasts (HFF)

The cytotoxicity of ABZ and its derivatives in HFF host cells was assessed as previously described. [START_REF] Schorer | Di-cationic arylimidamides act against Neospora caninum tachyzoites by interference in membrane structure and nucleolar integrity and are active against challenge infection in mice[END_REF] Briefly, HFF were grown to confluence in 96 well-plates. The medium was removed, and monolayers received 200 μl fresh medium containing either 0.1, 1 or 10 µM of each compound, or the medium was supplemented with the corresponding concentrations of DMSO. Following cultivation at 37°C / 5% CO 2 for 72 h, the medium was aspirated, and cells were washed once with PBS. Subsequently, 200 µl PBS containing resazurin (10 mg/ml) were added per well. Plates were read (excitation 530 nm, emission 590 nM) using an Enspire multilabel reader (2300 EnSpire™ Multilabel Reader, Perkin-Elmer, Turku, Finland) at different timepoints between 0 and 5h. The cytotoxicity was calculated and is represented as percentage of the DMSO control.

In vitro activity against the protozoan Toxoplasma gondii

For the assessment of drug efficacy against Toxoplasma tachyzoites, transgenic T. gondii β-gal tachyzoites (RH background, constitutively expressing β-galactosidase) [START_REF] Mcfadden | Use of Toxoplasma gondii Expressing ␤-Galactosidase for Colorimetric Assessment of Drug Activity In Vitro[END_REF] were cultured in human foreskin fibroblasts (HFF) host cells as previously described. [START_REF] Barna | In Vitro Effects of Novel Ruthenium Complexes in Neospora caninum and Toxoplasma gondii Tachyzoites[END_REF] Assays were carried out in 96 well plates. 5 x 10 3 HFF / well were grown to confluency in 200 µl phenol-red free culture medium at 37 °C and 5% CO 2 . Monolayers were infected with freshly isolated T. gondii β-gal tachyzoites (1 x 10 3 / well) and drugs were added concomitantly with infection. Assessment of drug efficacy was done by exposing parasite cultures to 0.1, 1, 10 M (quadruplicates) of each compound (40 mM stock solutions), and respective concentrations of DMSO were added to control wells. After 72 h of culture in the presence of the drugs at 37°C/ 5% CO 2 , the medium was removed by aspiration, wells were washed once with 200 μl PBS and cells were permeabilized with 90 μl PBS containing 0.05% Triton X-100. After addition of 10 µL of 5 mM chlorophenol red-β-D-galactopyranoside (CPRG; Roche Diagnostics, Rotkreuz, Switzerland) dissolved in PBS, the absorption shift was measured at 570 nm wavelength at various time points on a EnSpire® multimode plate reader (Perkin Elmer). The activity, measured as the release of chlorophenol red over time, was calculated as percentage from DMSO control, which represented 100% of T. gondii growth.

ASSOCIATED CONTENT

Supporting Information contains detailed synthetic procedures and characterizations of the compounds and copies of the NMR spectra, details concerning the X-ray analyses and copies of the HPLC chromatograms for the plasma stability assays.

AUTHOR INFORMATION

Corresponding Authors

*e-mail: kevin.cariou@cnrs.fr * e-mail: jennifer.keiser@swisstph.ch * e-mail: gilles.gasser@chimieparistech.psl.eu

ORCID:

Yan Lin: 0000-0001-7808-5444 Yih Ching Ong: 0000-0003-0411-1114 Sarah Keller: 0000-0003-2667-9157

Author Contribution

All authors were involved with the design and interpretation of experiments and with the writing of the manuscript.