Stanislas Talaga
email: stantalaga@free.fr

Olivier Dézerald

Alexis Carteron

Céline Leroy

Jean-François Carrias

Régis Céréghino

Alain Dejean

Urbanization impacts the taxonomic and functional structure of aquatic macroinvertebrate communities in a small Neotropical city

Keywords: Aedes aegypti, Bioindicator, Diversity, Functional traits, Tank bromeliads, Urban ecology

Due to habitat fragmentation, resource disruption and pollution, urbanization is one of the most destructive forms of anthropization affecting ecosystems worldwide. Generally, human-mediated perturbations dramatically alter species diversity in urban sites compared to the surroundings, thus influencing the functioning of the entire ecosystem. We investigated the taxonomic and functional diversity patterns of the aquatic macroinvertebrate communities in tank bromeliads by comparing those found in a small Neotropical city with those from an adjacent rural site. Changes in the quality of detrital inputs in relation to lower tree diversity and the presence of synanthropic species are likely important driving forces behind the observed structural changes in the urban site. Leaf-litter processors (i.e., shredders, scrapers) were positively affected in the urban site, while filter-feeders that process smaller particles produced by the activity of the shredders were negatively affected. Because we cannot ascertain whether the decline in filter-feeders is related to food web-mediated effects or to competitive exclusion (Aedes aegypti mosquitoes were present in urban bromeliads only), further studies are necessary to account for the effects of intra-guild competition or inter-guild facilitation.

Introduction

Urbanization is one of the most destructive forms of anthropogenic disturbance experienced by ecosystems worldwide. Generally, species diversity is altered dramatically in urban sites compared to the surroundings. The loss of natural habitat, habitat fragmentation, resource disruption and pollution are among the main causes; moreover, urban areas are hotter and drier than rural ones ('urban heat island effect') because the vegetation is supplanted by impervious surfaces, reducing evaporation and evapotranspiration [START_REF] Mckinney | Urbanization, biodiversity, and conservation. The impacts of urbanization on native species are poorly studied, but educating a highly urbanized human population about these impacts can greatly improve species conservation in all ecosystems[END_REF][START_REF] Mckinney | Effects of urbanization on species richness: a review of plants and animals[END_REF]. Also, the greater density of artificial, man-made habitats (e.g., ponds, domestic gardens) fosters colonization by either native or synanthropic species [START_REF] Santoul | Gravel pits support waterbird diversity in an urban landscape[END_REF][START_REF] Talaga | A bromeliad species reveals invasive ant presence in urban areas of French Guiana[END_REF].

Even if the presence and abundance of most species are influenced by urbanization, community-level responses cannot only be interpreted as a simplification equivalent to a decrease in α diversity [START_REF] Mcintyre | Ecology of urban arthropods: a review and a call to action[END_REF]. Indeed, the negative or positive effects of urbanization upon community diversity differ markedly between taxonomic groups, and explanations for the contrasting patterns lie in the nature of the processes acting on the focal taxa studied [START_REF] Henle | Predictors of species sensitivity to fragmentation[END_REF]). For example, the habitat fragmentation inherent to urban areas adversely affects vertebrates due to gene flow disruption between populations [START_REF] Delaney | A rapid, strong, and convergent genetic response to urban habitat fragmentation in four divergent and widespread vertebrates[END_REF], whereas invertebrates are less affected given the small habitat size needed to sustain a population [START_REF] Jones | Invertebrates in urban areas: a review[END_REF]. Moreover, a higher β diversity can be detected within urban areas where habitat fragmentation increases habitat heterogeneity, promotes edge species, and/or creates specific niches for invertebrates [START_REF] Jones | Invertebrates in urban areas: a review[END_REF].

In the Neotropics, tank bromeliads form a highly discontinuous aquatic meta-habitat embedded within a terrestrial matrix. These phytotelmata ('plant-held waters') provide a habitat to aquatic organisms ranging from bacteria to small vertebrates [START_REF] Kitching | Food webs and container habitats: the natural history and ecology of phytotelmata[END_REF][START_REF] Brouard | Understory environments influence functional diversity in tank-bromeliad ecosystems[END_REF]. Interestingly, this spatially discrete microecosystem can be sampled exhaustively, providing unbiased records of community-level diversity and a relevant model system to bring out environmental effects on community assembly [START_REF] Srivastava | Are natural microcosms useful model systems for ecology?[END_REF]). The key environmental determinants structuring aquatic macroinvertebrate communities in tank bromeliads are related to the characteristics of the aquatic compartment (i.e., habitat size and complexity, food resources, presence of a top predator) and to the characteristics of the terrestrial matrix most often evaluated as open versus closed environments [START_REF] Brouard | Understory environments influence functional diversity in tank-bromeliad ecosystems[END_REF][START_REF] Dézerald | Food-web structure in relation to environmental gradients and predator-prey ratios in tank bromeliad ecosystems[END_REF][START_REF] Dézerald | Environmental determinants of macroinvertebrate diversity in small water bodies: insights from tank bromeliads[END_REF][START_REF] Dézerald | Environmental drivers of invertebrate population dynamics in Neotropical tank bromeliads[END_REF][START_REF] Hammill | Predation threat alters composition and functioning of bromeliad ecosystems[END_REF]Trzcinski et al. 2016). Tank bromeliads are common epiphytes in Neotropical cities where the structure of their aquatic communities can be directly or indirectly influenced by humanmediated pressures [START_REF] Talaga | A bromeliad species reveals invasive ant presence in urban areas of French Guiana[END_REF]. The fragmentation of the terrestrial matrix caused by urbanization clearly increases the patchy distribution of trees and, so, that of the epiphytes they host. Consequently, their aquatic compartment might be directly affected by less tree canopy cover that leads to a reduction in incoming detritus (e.g., leaf litter) and to an increase in incident radiation, whereas the characteristics of the terrestrial matrix negatively impact their colonization. Indeed, most macroinvertebrates developing in the tanks of bromeliads are larvae with winged, terrestrial adults that are active dispersers whose range of activity can be limited in the hotter and drier conditions of urban areas (passive dispersers like worms and crustaceans colonize new aquatic habitats through phoresy).

Most studies on urban ecosystems have been conducted in temperate regions in Europe and North America. Those conducted in the Tropics were mostly confined to megacities that are not very different from their temperate counterparts in terms of landscape features. Yet, most tropical urban areas are represented by medium to small urban settlements displaying comparatively low levels of urbanization and are often adjacent to natural habitats [START_REF] Savard | Biodiversity concepts and urban ecosystems[END_REF][START_REF] Grau | A peri-urban neotropical forest transition and its consequences for environmental services[END_REF]. Consequently, a reduction in the range of the natural-to-urban gradient is likely to favor exchanges between the two environments and thus limit the influence of urbanization on communities. To test this hypothesis, we investigated the influence of urbanization on the macroinvertebrate communities living in the tanks of an abundant bromeliad species by comparing their biodiversity, and taxonomic and functional structure between an urban and a rural site using a small Neotropical city as a model. We also verified if urbanization favors the presence of synanthropic and/or exotic species.

Materials and methods

Study area

This study was conducted in the coastal region of French Guiana, in and around the city of Sinnamary (05°22′39″N 52°57′35″W) during the 2011 dry season. The area has an equatorial monsoon climate corresponding to an average of 2800 mm of yearly rainfall distributed over 251 days [START_REF] Peel | Updated world map of the Köppen-Geiger climate classification[END_REF]). There is a major drop in rainfall from mid-July to mid-November (dry season) and a shorter and more irregular dry period in March. Minimum and maximum monthly mean temperatures, relatively stable, vary between 23.6 °C and 31.6 °C.

The residential urban area of Sinnamary, which is home to most of the 3165 inhabitants recorded within the municipality (INSEE 2012), displays a moderate level of urbanization representative of a common form of urbanization in the Neotropics. In an attempt to control the proliferation of adult Aedes aegypti (Linnaeus 1762), a well-known mosquito vector of chikungunya, dengue and zika [START_REF] Christophers | Aedes aegypti (L.) the yellow fever mosquito: its life history, bionomics and structure[END_REF], the city of Sinnamary is ULV-sprayed during selected periods with different insecticide. This was the case throughout the sampling period during which each street of the city were sprayed weekly with a solution of 1 g of deltamethrin (Aqua K-Othrine®, Bayer, Leverkusen, Germany) per hectare (Centre de démoustication de Sinnamary, personal communication).

Impervious surface areas were calculated within the convex hulls formed by the A. aquilega sampled at each site using QGIS software (QGIS Development Team 2015). The percentages of impervious surface areas were 14.23% and 47.59% in the rural and urban sites, respectively, falling into the low (< 20% impervious surface area) and moderate (20-50% impervious surface area) categories proposed by [START_REF] Mckinney | Urbanization, biodiversity, and conservation. The impacts of urbanization on native species are poorly studied, but educating a highly urbanized human population about these impacts can greatly improve species conservation in all ecosystems[END_REF].

The focal plant taxa and sampling methods

In this region, the tank bromeliad Aechmea aquilega (Salisb.) Griseb. naturally occurs along a gradient of anthropogenic disturbance from rural to urban areas. This bromeliad abounds on medium to large trees, either native or exotic species, and mainly comprised of mango trees in the city of Sinnamary [START_REF] Talaga | A bromeliad species reveals invasive ant presence in urban areas of French Guiana[END_REF]. We sampled 26 mature A. aquilega on trees at two sites (13 individuals of very similar size per site): the city of Sinnamary and a rural site situated along a dead-end dirt road (Route de l'Anse) ca. 2 km away (hereafter, 'urban site' and 'rural site'). The city of Sinnamary was built according to a grid street plan, most of the blocks being squares or rectangles with 60 to 120 m-long sides [START_REF] Géoportail | Institut national de l'information géographique et forestière[END_REF]. We randomly selected one tree per block, none of them situated along the city limits; the sum of the surface areas formed by the selected A. aquilega was 10.09 ha. The rural site along the Route de l'Anse was characterized by very few dwellings among large fragments of an old secondary forest; the sum of the surface areas formed by the selected A. aquilega was 9.75 ha (a surface area close to that of the urban site).

Before being collected, each A. aquilega was geolocated with a GPS, its elevation above ground (EG) was measured (in meters), and the percentage of incident radiation (IR) was quantified using the hemispherical photography technique (see [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tank-bromeliad[END_REF]. Each plant was then removed from its supporting tree or building and placed into an individual, sealed plastic bag to prevent spillage and contamination. In the laboratory, the number of leaves forming wells (NW) was recorded, plant height (PH) was measured as the distance from the insertion of the outer leaves to the top of the crown, and plant width (PW) as the maximum distance between the tips of the leaves (both in centimeters). Subsequently, aquatic and semi-aquatic macroinvertebrates were extracted using the following method. First, the bromeliads were turned upside down in a bucket, and the water filtered through a 150 μm mesh. The water filtered from the wells was measured as the total volume of water (in milliliters) held by the plant (WV). The macroinvertebrates retained by the 150 μm net were sorted and kept for identification (see below). The organic matter contained by the plant was separated into two classes: fine particulate organic matter (FPOM; 1000-0.45 μm in size) and coarse particulate organic matter (CPOM; small pieces of fragmented material). Both were expressed in dry mass (in grams) after being placed in an oven until a constant weight was reached.

Diversity assessments

The plants were totally dismantled and each leaf was separated from the base and cleaned with a jet of water directed into a bucket. This technique allowed us to exhaustively collect the remaining aquatic macroinvertebrates, especially benthic organisms living deep in the wells (see [START_REF] Jocqué | How effective are non-destructive sampling methods to assess aquatic invertebrate diversity in bromeliads?[END_REF]. All aquatic and semi-aquatic macroinvertebrate organisms were separated from the organic material under a stereomicroscope at 10X constant magnification. The individuals collected were separated into morphospecies or species, enumerated and preserved in 70% alcohol. Mosquitoes were identified to species level using [START_REF] Lane | Neotropical Culicidae, vol I and II[END_REF] keys and individuals belonging to other families were identified using Merritt and Cummins' larval keys (2008). For convenience, both morphospecies and species are regrouped under the term taxa in the rest of the text.

Because functional diversity, or the diversity of species traits in ecosystems, encompases the complementarity and redundancy of co-occurring species, it is accepted as a good predictor of ecosystem productivity and vulnerability. Among the metrics of functional diversity available, we used three indices based mostly on quantitative traits: functional richness (FRic) or the amount of niche space occupied by species in the community; functional evenness (FEve) or the evenness of the abundance distribution in the occupied niche space; and functional divergence (FDiv) or the degree to which the abundance distribution in the niche space maximizes the divergence in functional characters within the community [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF]). We also used functional dispersion (FDis), an index that takes into account the species' relative abundances that is independent of species richness and represents the average distance of species to the abundanceweighted centroid of all species in the community in the trait space [START_REF] Laliberté | A distance-based framework for measuring functional diversity from multiple traits[END_REF]. All diversity indices were calculated from species scores in a trait space defined by a Fuzzy Correspondence Analysis of the abundance-weighted species traits (see [START_REF] Dézerald | Functional trait responses of aquatic macroinvertebrates to simulated drought in a Neotropical bromeliad ecosystem[END_REF]. The biological traits examined were: maximum body size (BS), aquatic developmental stage (AS), reproduction mode (RE), dispersal mode (DM), resistance forms (RF), food (FD), respiration mode (RM), locomotion (LO), and feeding group (FG). The latter comprises predators (Pr) and detritivores composed of: shredders (Sh) that feed on intact leaves which fall into the phytotelmata and produce fine organic matter; scrapers (Sc) that feed on layers of algae, bacteria and organic matter attached to the substrate; filtering-collectors (FC) that feed by filtering small particles of organic matter and microorganisms from the water column; and gathering-collectors (GC) that feed on the organic matter that accumulates at the bottom of the phytotelmata (see details on the biological traits of each taxon in [START_REF] Merritt | An introduction to the aquatic insects of North America[END_REF][START_REF] Céréghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF][START_REF] Dézerald | Food-web structure in relation to environmental gradients and predator-prey ratios in tank bromeliad ecosystems[END_REF]. The categories for each trait were either ordinal or nominal. The information on the biological traits was then structured using a fuzzy-coding technique [START_REF] Chevenet | A fuzzy coding approach for the analysis of long-term ecological data[END_REF]): scores ranged from 0, indicating 'no affinity', to 3, indicating 'high affinity' for a given species traits category (Appendix S1). This species-traits matrix was analyzed with a Fuzzy Correspondence Analysis (FCA) in order to obtain multivariate scores for the full set of species.

Statistical analyses

Differences in bromeliad descriptors between sites (i.e., EG, IR, PH, PW, WV, NW, FPOM, CPOM) were first tested using Wilcoxon Rank Sum tests. A Non-metric MultiDimensional Scaling (NMDS) ordination was then run on raw abundance data using the Bray-Curtis (abundance-based) and the Jaccard (incidence-based) indices to determine the taxonomic structure among aquatic macroinvertebrate communities between the rural and the urban sites. In order to test for statistical differences in the structure of the communities between the two sites, a Permutational Multivariate Analysis of Variance (PERMANOVA) was run on raw abundance data using the same two indices. Diversity (D) was calculated and partitioned within each site into α, β and γ diversity by generalizing the Shannon entropy [START_REF] Marcon | Generalization of the partitioning of Shannon diversity[END_REF]. The α diversity is defined as the average local community diversity, γ diversity Taxa are listed alphabetically according to a classical system and highlighted in bold. The rank number of a taxon's relative abundance is indicated for the rural and the rural meta-communities. Functional Feeding Group (FFG) for each taxa is abbreviated as follows: Scraper (Sc), Shredder (Sh), Gatheringcollector (GC), Filtering-collector (FC) and Predator (Pr) *Taxa ID as in Appendix S1.

corresponds to the diversity of the meta-community composed of all the communities within each site, and β diversity is understood as the diversity between local communities (or the divergence between each community and the meta-community). Diversity profiles were used to compare α, β and γ diversity between the two sites rather than comparing single diversity indices. Entropy is converted into diversity and plotted against different orders of diversity. The importance given to rare species decreases with the order of diversity, and a community is considered more diverse when its profile is higher than the one being compared [START_REF] Marcon | Generalization of the partitioning of Shannon diversity[END_REF]).

Finally, evenness was calculated as the ratio of diversity (D) on richness (S) [START_REF] Tuomisto | An updated consumer's guide to evenness and related indices[END_REF]). Statistical analyses were conducted in R software (R Development Core Team 2013) using entropart, FD and vegan packages.

Results

Although the water volumes were similar, the number of wells containing the total amount of water in a bromeliad (NW) was significantly higher in the urban site compared to the rural site (Wilcoxon Rank Sum test: W = 20.5, P < 0.05). None of the other variables characterizing bromeliads (i.e., EG, IR, PH, PW, WV, FPOM, CPOM) were significantly different between the two sites.

A total of 11,099 aquatic macroinvertebrate individuals belonging to 36 taxa were extracted from the 26 A. aquilega bromeliads sampled (Table 1). Nine taxa out of the 36 were exclusively found in the urban site (Table 1). Most of the taxa belonged to the order Diptera, but some Coleoptera were also found. Species richness was slightly lower in the rural metacommunity (27 taxa) than in the urban meta-community (30 taxa), with one and three singletons, respectively, and a species turnover of 41.67% (15/36) between the two sites. In addition, two taxa, namely Chironominae sp. and Trentepholia sp., were not noted in the urban site, while they were ranked second and third in terms of relative abundance in the rural site (Fig. 1; Table 1). It is worth noting that the culicid species Aedes aegypti was found in seven A. aquilega in the urban site (63 larvae in total), but was absent from the rural site (Table 1).

The dissimilarity in the taxonomic structure of the aquatic macroinvertebrate communities is represented in Fig. 1. A significant difference between the two sites is shown using both the Bray-Curtis (PERMANOVA; N = 26; F = 8.23; P < 0.001; Fig. 1) and the Jaccard indices (PERMANOVA; N = 26; F = 5.49; P < 0.001).

The local α diversities of the aquatic macroinvertebrate communities tended to be higher in the rural site compared to the urban one for each order of diversity, but the difference was not significant (Fig. 2; Table 2). The analyses of β and γ diversity profiles were less straightforward. While the β diversity in the rural meta-community was higher for orders of diversity between 0.5 and 1, mean values were higher in the urban meta-community for orders of diversity of 0 and 2 (Fig. 2; Table 2). Because diversity measures give more Fig. 1 Non-metric MultiDimensional Scaling (NMDS) ordination based on the Bray-Curtis distance showing the degree of dissimilarity of aquatic macroinvertebrate communities between the rural (black dots) and the urban (gray dots) sites. Ellipses are drawn around rural (solid line) and urban (dashed line) communities with a level of confidence of 95%. Taxa are projected in the same ordination space (red crosses) and abbreviated with the first four letters of the species' names or with the first three letters followed by the number of morphospecies' names as in Table 1. Note that a similar ordination was obtained with the Jaccard distance importance to the contribution of abundant relative to rare species with an increasing order of diversity, the higher β diversity in the urban site is due to rare species (for which q = 0) and the heterogeneous distribution of abundant species (for which q = 2). Also, rural β diversity is always included within the range of variation of urban β diversity (Fig. 2). Indeed, higher β diversity reflects larger variations among local communities, a result which is consistent with the hypothesis that habitat fragmentation increases environmental heterogeneity in the urban site. The γ diversity was greater in the urban meta-community than in the rural one for an order of diversity of 0, the contrary being true for orders of diversity of 1 and 2 (Fig. 2; Table 2). A comparison of evenness (E) was unequivocal and showed higher or equal values in the rural site compared to the urban site for all α, β and γ diversities and for each order of diversity (Table 2).

In terms of functional feeding groups, filtering-collectors and gathering-collectors were significantly more abundant in the rural communities than in the urban ones (Wilcoxon Rank Sum test: W = 125, P = 0.04 and W = 124, P = 0.045, respectively; Fig. 3), the contrary being true for shredders and scrapers (Wilcoxon Rank Sum test: W = 22, P < 0.001 and W = 40, P = 0.024, respectively; Fig. 3). Predators tended to be more abundant in the rural site, but the difference was not significant (Wilcoxon Rank Sum test: W = 113.5, P = 0.140; Fig. 3).

Concerning functional diversity, FDis was significantly lower in urban bromeliads (Welch two sample t-test: t = -13.64, df = 12.38, P < 0.001). The three other indices tested did not reveal significant differences between the two sites (Welch two sample t-test: FRic; t = 0.51, df = 20.84, P = 0.619; FEve; t = -1.20, df = 20.89, P = 0.242; FDiv; t = 1.99, df = 20.54, P = 0.060).

Discussion

Bromeliads represent small and discrete habitats that can be abundant in urban and natural sites. They are, therefore, deemed suitable model systems to bring out the environmental effects of human-impacted landscapes on the taxonomic and functional structures of biological communities in the Neotropics. Shifts in this community structure can be explained in at least five ways: (1) the characteristics of the bromeliad habitat are different, (2) the terrestrial characteristics that filter the adult life stage, (3) top-down processes, (4) bottom-up processes, and (5) the presence of synanthropic species. In this context, our study shows that urbanization can have significant impacts on both the taxonomic and functional structure of aquatic macroinvertebrate communities. Yet, despite these important structural shifts, we did not detect significant patterns of α, β and γ diversity between the two sites.

The characteristics of the bromeliads habitat were mostly not affected by the urban environment although we noted significantly more wells per plant in the urban site. This was due to the fact that, although the selected plants were of a similar size, external leaves contained little water in the urban area while their counterparts in the rural area no longer retained water, and so were not counted as 'wells'. Note that the difference noted had no influence on the total water volume of the plants which was similar between the two sites. Also, contrary to our predictions, the bromeliads held comparable amounts of detritus (CPOM and FPOM) and received similar incident radiation in the urban and the rural sites.

Fig. 2 Profiles of the α, β and γ diversity (D) of the order q computed for the aquatic macroinvertebrate meta-communities in the rural (solid lines) and urban areas (dashed lines). The importance given to rare species decreases with order of diversity and a community is considered more diverse when its profile is higher than the one being compared Urban Ecosyst Because bromeliads characteristics are similar between the two sites, we can assume that terrestrial factors associated with the urban environment itself are the primary drivers of community assembly through trait filtering at the adult life stage. This includes insecticide spraying during the rainy season, something that likely affects the survival and behavior of some terrestrial insects whose larvae develop in A. aquilega wells. The impact on untargeted organisms is unknown, but Chironomidae have proven to be sensitive to the insecticide used [START_REF] Morrill | Impact of deltamethrin insecticide on Chironomidae (Diptera) of prairie ponds[END_REF]. This might explain the complete absence of Chironominae sp. and Trentepohlia sp. in the city while they abounded in the untreated rural site, though other factors linked to urbanization can also affect them. In fact, we can assume that microclimatic conditions associated with the urban environment (i.e., it is hotter and drier) might select edge species relative to sylvan ones, resulting in the disappearance and/or density reduction of some species.

Though the α, β and γ diversity were not affected, it is worth noting that shredders (Sh) and scrapers (Sc), both detritivorous leaf-litter processors, were positively affected in the urban site, while filtering-collectors (FC) and gathering-collectors (GC) that process much smaller particles produced by the activity of the shredders were negatively affected. However, nothing indicates that predators (Pr), here mainly represented by the top predator species Toxorhynchites haemorrhoidalis (Fabricius 1787), are influenced by urbanization (Fig. 3; Table 1). Consequently, the major restructuration observed for detritivores, associated with the absence of significant variation among predators, strongly suggests an alteration in urban communities through bottom-up processes. Dry mass only provides an indication of the quantity of the food resource, not its quality Table 2 Values of diversity (D) and evenness (E) for each order of diversity q partitioned into α, β and γ levels for the rural and urban meta-communities q = 0 P q = 1 P q = 2 P Values for γ diversity of the order q = 0, equivalent to richness (S), are highlighted in bold for each metacommunity. Evenness is calculated as the D:S ratio. Welch's two sample t-tests: no differences were significant; '-': the test was not applicable which can be important to aquatic macroinvertebrates [START_REF] Yanoviak | Effects of leaf litter species on macroinvertebrate community properties and mosquito yield in Neotropical tree hole microcosms[END_REF][START_REF] Yee | Consequences of detritus type in an aquatic microsystem: effects on water quality, micro-organisms and performance of the dominant consumer[END_REF]. Because native tree species occurred in the rural site whereas mango trees greatly dominated in the city, it is likely that the quality and diversity of the leaf litterthat is directly related to urbanization in the study areaaccounts for the observed impacts on detritivores. Despite the change in the relative abundance among functional feeding groups, it should be noted that all of them are present in the two sites, showing that aquatic macroinvertebrates are relatively tolerant of urbanization. At the species level, we noted a turnover of 41.67% between the two sites which reflects changes in the composition of the meta-communities. Yet, the remaining 58.33% corresponds to an overlap of taxa between the two habitats, showing that many species can live in both sites. Concerning functional diversity, the fact that functional dispersion (FDis) is significantly lower in the city compared to the rural site is in line with previous studies showing a reduction in FDis with a higher disturbance intensity [START_REF] Gerisch | More species, but all do the same: contrasting effects of flood disturbance on ground beetle functional and species diversity[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF].

Not surprisingly, we detected the presence of synanthropic species in urban communities. The presence of these species is both the direct consequence of urbanization but can also be viewed as a biotic factor responsible for the alteration of native communities. Here we show for the first time that Ae. aegypti uses tank bromeliads as an oviposition site in French Guiana. The use of phytotelmata as breeding sites has long been known [START_REF] Belkin | Collection records of the project "mosquitoes of middle America". 4. Leeward Islands: Anguilla (ANG), Antigua (ANT), Barbuda (BAB), Montserrat (MNT), Nevis (NVS), St. Kitts (KIT)[END_REF] and was specifically reported for urban tank bromeliads in Argentina [START_REF] Stein | Aechmea distichantha (Bromeliaceae) epiphytes, potential new habitat for Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) collected in the province of Tucumán, northwestern Argentina[END_REF], Brazil [START_REF] Varejão | Criadouros de Aedes (Stegomyia) aegypti (Linnaeus, 1762) em bromélias nativas na cidade de Vitória, ES[END_REF] and Trinidad [START_REF] Chadee | Natural habitats of Aedes aegypti in the Caribbean-a review[END_REF]. The percentage of Ae. aegypti larvae noted in this study, superior to those reported from other Neotropical areas, is likely seasonal as a control of their presence in Sinnamary during the 2013 rainy season resulted in only three larvae gathered from two A. aquilega out of the 32 sampled [START_REF] Talaga | Ecologie, diversité et évolution des moustiques (Diptera: Culicidae) de Guyane française : implications dans l'invasion biologique du moustique Aedes aegypti[END_REF]. Furthermore, during the dry season, both the anthropophily of Ae. aegypti and the variation in the density of 'natural enemies' (e.g., competing native mosquito larvae) in the wells of A. aquilega likely influenced the distribution of Ae. aegypti larvae in the urban site (urban environment plus low density of competing larvae) and their absence from the rural site (non-urban environment plus a high density of competing larvae) (Fig. 4). Indeed, during the rainy season, Ae. aegypti larvae were rare in the urban site although adequate, but the density of competing mosquito larvae was very high (Fig. 4). Lastly, the presence of Ae. aegypti larvae in the wells of urban bromeliads is not necessarily a concern as they rather develop in abundance in artificial containers, so that the role of native tank bromeliads in the production of this vector can be considered marginal [START_REF] Maciel-De-Freitas | Variation in Aedes aegypti (Diptera: Culicidae) container productivity in a slum and a suburban district of Rio de Janeiro during dry and wet seasons[END_REF].

We have therefore shown that if urbanization influences the macroinvertebrate communities living in the A. aquilega tanks in terms of taxonomic and functional structure, this was not the case for biodiversity. Also, at least during the period of this study, urbanization favored the presence of synanthropic and exotic species.

Fig. 3

 3 Fig. 3 Composite food web diagrams showing trophic interactions involving the aquatic macroinvertebrate Functional Feeding Groups (FFGs) in the rural (solid line) and urban (dashed line) metacommunities. FFGs are abbreviated as follows: Scraper (Sc), Shredder (Sh), Gathering-collector (GC), Filtering-collector (FC) and Predator (Pr). Circle diameter represents the relative abundance of each FFG standardized by doubling the relative abundance of the least abundant

Fig. 4

 4 Fig.4Comparison of the mean density of native mosquito larvae in the wells of Aechmea aquilega. Data from the 2011 dry season correspond to this study (13 A. aquilega in each case), those from the 2013 rainy season were taken from Talaga (2016) (32 A. aquilega), the sampling method used being strictly the same. Kruskal-Wallis test (χ 2 = 26.05; df = 2; P < 0.001) followed by a Nemenyi's post-hoc test, different letters indicate significant differences at P < 0.05

Table 1

 1 List of the aquatic macroinvertebrate morphospecies or species occurring in Aechmea aquilega in Sinnamary, French Guiana (13 plants in each of the rural and urban areas)

	Class	Order	Family	Sub-family	Morpho-species/species	Rank No. of taxa	FFG Taxa ID*
						Rural	Urban
	Insecta	Coleoptera	Elatheridae		Elatheridae sp.	21	27	Sh	1
			Elmidae		Elmidae sp.	24	22	Sc	2
			Hydrophilidae		Hydrophilidae sp.1	-	2 3	P r	3
					Hydrophilidae sp.2	-	2 4	P r	4
		Diptera			Brachycera sp.1	-	2 0	G C	5
					Brachycera sp.4	14	9	GC	6
					Brachycera sp.5	20	15	GC	7
					Brachycera sp.6	16	25	GC	8
					Brachycera sp.7	23	-	GC	9
					Brachycera sp.8	-	2 8	G C	1 0
					Brachycera sp.9	12	-	GC	11
					Brachycera sp.10	25	14	GC	12
					Brachycera sp.12	-	2 9	G C	1 3
					Brachycera sp.15	26	17	GC	14
			Ceratopogonidae Ceratopogoninae Bezzia sp.1	7	3	S c	1 5
					Bezzia sp.2	9	5	S h	1 6
					Ceratopogoninae sp.2	18	7	Sh	17
					Ceratopogoninae sp.3	22	30	GC	18
					Dasyhelea sp.	-	1 6	S c	1 9
				Forcipomyiinae	Forcipomyiinae sp.2	6	2	S c	2 0
					Forcipomyiinae sp.5	-	1 8	S c	2 1
			Chironomidae	Chironominae	Chironominae sp.	2	-	G C	2 2
				Tanypodinae	Tanypodinae sp.	27	-	Pr	23
			Culicidae	Culicinae	Aedes aegypti	-	1 2	F C	2 4
					Culex pleuristriatus	8	1 0	F C	2 5
					Culex imitator	11	-	FC	26
					Wyeomyia pertinans	5	1 1	F C	2 7
					Toxorhynchites haemorrhoidalis	10	13	Pr	28
			Tipulidae	Limoniinae	Trentepohlia sp.	3	-	S h	2 9
			Psychodidae	Psychodinae	Psychodinae sp.	-	2 1	S h	3 0
					Telmatoscopus sp.	4	1	S h	3 1
			Tabanidae		Tabanidae sp.	19	19	Pr	32
	Oligochaeta				Oligochaeta sp.1	17	26	GC	33
					Oligochaeta sp.2	13	8	GC	34
		Haplotaxida			Aulophorus superterrenus	1	6	G C	3 5
					Pristina sp.	15	4	GC	36

Acknowledgements We are grateful to Andrea Yockey-Dejean for proofreading the manuscript, the Laboratoire Environnement de Petit Saut for furnishing logistical assistance, and the municipality of Sinnamary (through the Department of the Environment) for permitting us to work inside the city limits. This study has benefited from an Investissement d'Avenir grant managed by the Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-0025). ST and OD were funded by a PhD scholarship (Université Antilles-Guyane for ST; French Centre National de la Recherche Scientifique and the Fond Social Européen for OD).


Marie-Lazarine Poulle
email: marie-lazarine.poulle@univ-reims.fr

Matthieu Bastien

Yolan Richard

Laurence Voutquenne-Nazabadioko

Isabelle Villena

Jenny Knapp

Émilie Josse-Dupuis

Dominique Aubert

Detection of Echinococcus multilocularis and other foodborne parasites in fox, cat and dog faeces collected in kitchen gardens in a highly endemic area for alveolar echinococcosis

Keywords: Carnivore faeces, Environmental contamination, Vulpes vulpes, Felis catus, Toxocara spp., Toxoplasma gondii

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

Introduction

In recent years, the impact that foodborne parasites exert on food safety, food security, quality of life and livelihoods has begun to receive well-deserved global attention [START_REF] Robertson | Have foodborne parasites finally become a global concern[END_REF]. Humans can become infected through the ingestion of food, water or soil that has been contaminated with the infectious stage of a parasite, which is often released into the environment in animal faeces [START_REF] Dorny | Emerging food-borne parasites[END_REF][START_REF] Slifko | Emerging parasite zoonoses associated with water and food[END_REF]. Fresh fruit and vegetables have been identified as a vehicle of transmission for half of the 24 foodborne parasites that rank at the top of the multi-criteria ranking for risk management of foodborne parasites [START_REF] Robertson | Have foodborne parasites finally become a global concern[END_REF]. In this ranking, Echinococcus multilocularis, Toxoplasma gondii and Toxocara spp. rank third, fourth and twentieth, making them especially worthy of concern.

Echinococcus multilocularis is a helminth parasite that is responsible for human alveolar echinococcosis (AE), a rare but severe disease that is considered one of the most serious zoonoses in the Northern Hemisphere [START_REF] Torgerson | Alveolar echinococcosis: from a deadly disease to a well-controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years[END_REF]. Echinococcus multilocularis eggs are excreted in the faeces of a definitive host, which in Europe is mainly the red fox (Vulpes vulpes) [START_REF] Eckert | Alveolar echinococcosis (Echinococcus multilocularis) and other neotropical forms of Echinococcus (Echinococcus vogeli and Echinococcus oligarthrus)[END_REF], although free-roaming domestic dogs (Canis lupus familiaris) and domestic cats (Felis silvestris catus) can also carry this parasite [START_REF] Deplazes | Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis[END_REF][START_REF] Dyachenko | Echinococcus multilocularis infections in domestic dogs and cats from Germany and other European countries[END_REF][START_REF] Umhang | Echinococcus multilocularis infections in dogs from urban and peri-urban areas in France[END_REF][START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of freeranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF]. Toxoplasma gondii is responsible for toxoplasmosis, a disease that is usually subclinical, but which can be fatal in immunosuppressed patients. Furthermore, transplacental transmission of T. gondii may lead to severe congenital infections [START_REF] Robert-Gangneux | Toxoplasmosis: a widespread zoonosis diversely affecting humans and animals, in Zoonoses -Infections Affecting Humans and Animals: Focus on Public Health Aspects, Sing A[END_REF]. Felids, and mostly the domestic cat, are the main definitive host of this parasite spreading its oocysts with their faeces [START_REF] Dubey | Toxoplasmosis of Animals and Man[END_REF]. Lastly, Toxocara spp. are responsible for toxocariasis, a zoonosis that is rarely diagnosed because it is generally asymptomatic, but that can occasionally lead to two main clinical syndromes in humans: ocular larva migrans and visceral larva migrans [START_REF] Overgaauw | Veterinary and public health aspects of Toxocara spp[END_REF]. The species contributing most to environmental contamination by Toxocara spp. eggs in urban, suburban and rural areas are stray cat, dog and fox, respectively [START_REF] Nijsse | Environmental contamination with Toxocara eggs: a quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs[END_REF].

Echinococcus multilocularis eggs, embryonated Toxocara spp. eggs or sporulated T. gondii oocysts can be found in fruit and vegetables intended for human consumption; Toxocara spp. eggs have been detected in produce harvested from the soil in organic farms [START_REF] Kłapec | Contamination of vegetables, fruit and soil with geohelmints eggs on organic farms in Poland[END_REF], and T. gondii and E. multilocularis DNA have been detected in fruit and vegetable samples taken from the environment [START_REF] Lass | The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples[END_REF][START_REF] Lass | The first detection of Echinococcus multilocularis DNA in environmental fruit, vegetable, and mushroom samples using nested PCR[END_REF]. Echinococcus multilocularis and Toxocara spp. eggs, as well as T. gondii oocysts, are very resistant to adverse environmental conditions and can remain viable in the environment for years under optimal conditions of low temperatures and high humidity [START_REF] Azam | Temperature and the development and survival of infective Toxocara canis larvae[END_REF][START_REF] Lélu | Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil[END_REF][START_REF] Veit | Influence of environmental factors on the infectivity of Echinococcus multilocularis eggs[END_REF]. The inadvertent ingestion of eggs or oocysts from unwashed raw fruit and vegetables has been identified as a transmission risk for AE [START_REF] Kern | Risk factors for alveolar echinococcosis in humans[END_REF][START_REF] Piarroux | Populations at risk for alveolar echinococcosis, France[END_REF], toxoplasmosis [START_REF] Alvarado-Esquivel | Toxoplasma gondii infection in workers occupationally exposed to unwashed raw fruits and vegetables: a case control seroprevalence study[END_REF][START_REF] Liu | Toxoplasma gondii infection in pregnant women in China[END_REF] and toxocariasis [START_REF] Fallah | Seasonal study of parasitic contamination in fresh salad vegetables marketed in Shahrekord, Iran[END_REF][START_REF] Gyang | Seroprevalence, disease awareness, and risk factors for Toxocara canis infection among primary schoolchildren in Makoko, an urban slum community in Nigeria[END_REF] in humans. Faecal deposition by infected carnivore hosts in cultivated plots devoted to growing produce could thus be a crucial amplifier of the risk of zoonotic diseases. To prevent pre-harvest contamination of raw fruit and vegetables, data about such deposits are needed.

The aim of this study was to provide information on the deposit of fox, dog and cat faeces, potentially contaminated with E. multilocularis, T. gondii or Toxocara spp., in privately owned kitchen gardens used to grow food for household consumption. The occurrence of foodborne parasites in these faeces was investigated by using molecular analysis to screen for parasitic DNA. The study was conducted in northeastern France, which is a high-risk area for human AE [START_REF] Piarroux | Landscape and climatic characteristics associated with human alveolar echinococcosis in France, 1982 to 2007[END_REF].

Materials and methods

Study area

This study was carried out in a rural area of the Ardennes department (49°25 0 N, 4°50 0 E) in northeastern France. This area was chosen because its cumulative incidence rate for human infection with AE during the 1982-2007 period was between 2.74 and 6.10, one of the highest reported in France [START_REF] Piarroux | Landscape and climatic characteristics associated with human alveolar echinococcosis in France, 1982 to 2007[END_REF]. The 1200 km 2 study area was located in the southern part of the Ardennes, where data about the population dynamics of foxes and the transmission dynamics of E. multilocularis in this population were available [START_REF] Guislain | Fox faeces and vole distribution on a local range: ecological data in a parasitological perspective for Echinococcus multilocularis[END_REF][START_REF] Guislain | Ecological and biological factors involved in the transmission of Echinococcus multilocularis in the French Ardennes[END_REF]. The study area was defined to encompass 19 villages in which a contact on site (mayors and/or other contacts) was in a position to introduce us to local gardeners, given that ''having confidence in the field researcher'' was a prerequisite in obtaining the authorisation to sample privately owned kitchen gardens. Depending on the size of the ''trust network'' of each contact, between 1 and 16 kitchen gardens were sampled per village.

The study area was characterised by small villages (most with fewer than 200 inhabitants) scattered in a landscape of cultivated fields, pastureland and woodland (consisting of oak Quercus spp., beech Fagus sylvatica, hornbeam Carpinus betulus and spruce Picea abies). Red fox density was about 3-4 foxes/km 2 during the 2003-2006 period [START_REF] Guislain | Ecological and biological factors involved in the transmission of Echinococcus multilocularis in the French Ardennes[END_REF] and did not significantly vary from 2004 to 2015 [START_REF] Bastien | Contamination des terrains potagers par Echinococcus multilocularis, Toxoplasma gondii et Toxocara spp, parasites responsables de zoonoses transmises par l'alimentation[END_REF]. The cat population was censused in a 460-ha area that encompassed two villages of the study area (Boult-aux-Bois and Briquenay); it reached 142 individuals (~30 cats/km 2 ) during the 2008-2010 period [START_REF] Gotteland | Assessing the homogeneity of individual scat detection probability using the bait-marking method on a monitored freeranging carnivore population[END_REF], around ten times the abundance of the red fox population. The dog population did not exceed 30 individuals in these two villages (Poulle, pers. obs), and was therefore at least four times smaller than the cat population. The prevalence of E. multilocularis in the vulpine population in the study area was 53% during the 2001-2005 period [START_REF] Guislain | Ecological and biological factors involved in the transmission of Echinococcus multilocularis in the French Ardennes[END_REF], and 36% between 2005 and 2010 in the whole Ardennes department [START_REF] Combes | Westward spread of Echinococcus multilocularis in foxes, France, 2005-2010[END_REF].

Sampling

From October 2011 to January 2012, 34 privately owned kitchen gardens in six villages in the study area were surveyed once a month to search for fox, dog and cat faeces. From February to April 2013, the sampling area was enlarged, including 16 out of the previously sampled 34 kitchen gardens (from 4 out of the 6 previous villages), plus 60 others from 13 different villages, making a total of 76 kitchen gardens for this period, and 94 privately owned kitchen gardens from 19 villages over the whole study period. These 94 kitchen gardens were only used for growing food (not for ornamental plants), providing vegetables and fruit such as lettuce, potatoes, carrots, leeks, cabbages, aromatic herbs, strawberries, etc. for one household. The size of the kitchen gardens -calculated by walking their perimeter with a Global Positioning System (GPS) device -was highly variable, ranging from 7 m 2 to 2862 m 2 for a mean of 360.5 ± 424.3 m 2 . The distance from a sampled kitchen garden to the closest dwelling or barn -also estimated by walking with a GPS device -ranged from 3 m to 202 m and averaged 24.7 ± 2.7 m (Fig. 1). Of the 94 sampled kitchen gardens, 32 (34%) were enclosed with a fence and were thus in principle not accessible to foxes, while the 62 others (66%) had open access to foxes, dogs and cats due to the absence of continuous fencing.

Sampling was conducted from October to April, i.e. outside the gardening period to avoid damaging seedlings. In that period, kitchen gardens are deserted, which increases the probability of carnivores visiting them, and plant cover is sparse, which makes faeces detection easier. Furthermore, E. multilocularis infection pressure in foxes is probably highest during the cold months of autumn and winter in northern France, as recently shown in Zurich (Switzerland) [START_REF] Otero-Abad | Mathematical modelling of Echinococcus multilocularis abundance in foxes in Zurich, Switzerland[END_REF], thus increasing the likelihood of contamination at this time of year. It should be noted that parasite eggs or oocysts shed in kitchen gardens between October and April stand a good chance of still being present and viable during the following gardening period (May-September) due to the optimal conditions (low temperatures and high humidity) for these parasite stages during winter in the Ardennes.

We sampled 16 kitchen gardens six times (autumn-winter 2011-2012 and winter 2013), 75 three times (17 during autumn-winter 2011-2012, 58 during winter 2013), one five times, and two just twice. Each survey consisted of a walked transect that allowed for a visual scan of the whole kitchen garden surface area. The size of the sampled area was calculated per kitchen garden by multiplying its surface area with the number of times it was sampled. It ranged from 147 m 2 to 17,172 m 2 , averaging 1411.9 ± 2136.8 m 2 per kitchen garden, for a total sampled area size of 13.27 ha.

Fox, dog, cat or unidentified faeces were first discriminated visually in the field on the basis of shape and size, and then more accurately categorised by identifying the host species through faecal real-time PCR [START_REF] Knapp | Development of a real-time PCR for a sensitive one-step coprodiagnosis allowing both the identification of carnivore feces and the detection of Toxocara spp. and Echinococcus multilocularis[END_REF] (described below). The field researcher's accuracy in discriminating cat, dog and fox faeces was verified by comparing the identification from morphological criteria to the identification of host species from molecular analysis. Faeces were collected with disposable gloves and put into separate labelled plastic bags. All collected faeces were decontaminated for 5 days at À80 °C and then stored at À20 °C before analysis.

Molecular analyses

Copro-DNA extraction DNA from copro-samples was purified using the QIAamp Fast DNA Stool Mini kit (Qiagen, Hilden, Germany). We used 0.5 g of each copro-sample and DNA purification was carried out as described in the manufacturer's protocol.

PCR inhibitor control

The presence of PCR inhibitors was checked using a solution of 10 5 spores/mL of Geotrichum candidum (strain no. 6560, from the Belgian Coordinated Collections of Micro-organisms (BCCM, http://bccm.belspo.be/about/ihE. multilocularis.php) as an internal control added to the copro-sample in the first step of the extraction protocol. Then, a qPCR was performed with an expected cycle threshold (Cq) value under 34 cycles, as previously described by Knapp et al. [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF]. When inhibitors were detected, qPCR was performed a second time on copro-DNA diluted 1:10 and 1:100, respectively.

E. multilocularis and Toxocara spp. qPCR diagnosis

In order to detect the presence of E. multilocularis and Toxocara spp. parasites in the copro-samples, quantitative polymerase chain reactions (qPCRs) were performed on copro-DNA extracts, as previously described by Knapp et al. [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF] for E. multilocularis, and Knapp et al. [START_REF] Knapp | Development of a real-time PCR for a sensitive one-step coprodiagnosis allowing both the identification of carnivore feces and the detection of Toxocara spp. and Echinococcus multilocularis[END_REF] for Toxocara spp. Briefly, a duplex qPCR was performed using TaqMan technology, with hydrolysis probes and TaqMan Gene Expression Master Mix 2X (Life Technologies, Foster City, CA, USA) to simultaneously detect the parasite and the PCR inhibitors (rrn-E. multilocularis primers and probe were associated with Geo primers and probe, and Tox primers and probe were associated with Geo primers and probe, Table 1). The qPCRs were performed with 45 cycles for the two parasites as previously described, in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Results were analysed using 7500 2.0.5 software. A qPCR with Cq 45 cycles allowed us to determine the presence of parasites but qPCR results did not enable estimation of the amount of eggs or oocysts per faecal sample, and it was therefore not possible to estimate the amount of absolute contribution of fox, dog and cat to the contamination of gardens. Molecular analyses were performed at the Chronoenvironment Laboratory, UMR 6249, UBFC, Besançon, France.

Toxoplasma gondii diagnosis

DNA extracts were subjected to a PCR targeting a specific repeated element of 529 bp [START_REF] Reischl | Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes[END_REF] and performed using an iQ5 instrument (Bio-Rad, Hercules, CA, USA) as follows. A Toxoplasma gondii-specific target region (GenBank Accession No. AF487550) was amplified with a labelled TaqMan probe (5 0 6FAM-ACGCTTTCCTCGTGGTGATGGCG-3 0 TAMRA) and DNA oligonucleotide primers (5 0 -AGAGACACCG-GAATGCGATCT-3 0 and 5 0 -CCCTCTTCTCCACTCTT-CAATTCT-3 0 ) [START_REF] Lélu | Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil[END_REF]. The amplification mixture was composed of 12.5 lL of 2• reaction mixture (Platinum Quantitative PCR SuperMix-UDG, Invitrogen, Carlsbad, CA, USA), 3.5 mM MgCl 2 , 0.5 lM of each oligonucleotide primer, 0.2 lM TaqMan probe, 1 lL of 1% bovine serum albumin to increase the PCR performance, and 5 lL of template DNA, for a final volume of 25 lL. The reaction mixture was initially incubated for 3 min at 50 °C to allow for uracil-N-glycosylase (UNG) activity. This first incubation was followed by a second incubation of 3 min 30 s at 95 °C to denature the DNA template, inactivate the UNG enzyme and activate the Platinum Taq DNA Polymerase. Samples were amplified as follows: 45 cycles of denaturation at 95 °C for

0 -CTGTGATCTTGGTGTAGTAGTTGAGATTT3 0 [25] rrn-Rev 5 0 -GGCTTACGCCGGTCTTAACTC-3 0 rrn-probe 5 0 -TGGTCTGTTCGACCTTTTTAGCCTCCAT-3 0 Toxocara spp. Cox1 Toxo-Fwd 5 0 -AAAATAGCCAAATCCACACTACTACCA-3 0 [26] Toxo-Rev 5 0 -GGTGTGGGACTAGTTGAACTGTGTA-3 0 Toxo-probe 5 0 -CCCCATAGTCCTCAAAG-3 0 Toxoplasma gondii 529-rep Tg-Fwd 5 0 -AGAGACACCGGAATGCGATCT-3 0 [32] Tg-Rev 5 0 -CCCTCTTCTCCACTCTTCAATTCT-3 0 Tg-probe 5 0 -ACGCTTTCCTCGTGGTGATGGCG-3 0 Vulpes vulpes CytB Vv-Fwd 5 0 -ACCTTCCCGCACCATCAAA-3 0 [26] Vv-Rev 5 0 -TGTTGCAATCTGTAGAATAAGGCATA-3 0 Vv-probe 5 0 -CTGCCTGATGGAACTTCGGGTCCC-3 0 Canis l. familiaris CytB Cf-Fwd 5 0 -CCACCCACTAGCCAAAATTGTT-3 0 [26] Cc-Rev 5 0 -AAGTTCCATCAAGCAGAGATGTTAGA-3 0 Cf-probe 5 0 -ATAACTCATTCATTGACCTCCCAGCGCC Felis s. catus CytB Fc-Fwd 5 0 -CCCTTCTAGGAGTCTGCCTAATCTT-3 0 [26] Fc-Rev 5 0 -CGGTTATTGTGTCTGATGTGTAGTGT-3 0 Fc-probe 5 0 -AAATCCTCACCGGCCTCTTTTTGGC-3 0 Geotrichum candidum CytB Geo-Fwd 5 0 -CACCGCCCGTCGCTAC-3 0 [25] Geo-Rev 5 0 -AGAAAAGTTGCCCTCTCCAGTT-3 0 Geo-probe 5 0 -TCAATCCGGAAGCCTCACTAAGCCATT-3 0 4 M.-L.

Host faecal test

The field researchers' morphological identification of carnivore faeces was confirmed by a molecular host faecal test developed by Knapp et al. [START_REF] Knapp | Development of a real-time PCR for a sensitive one-step coprodiagnosis allowing both the identification of carnivore feces and the detection of Toxocara spp. and Echinococcus multilocularis[END_REF], using TaqMan probes and based on qPCR technology. Briefly, duplex qPCRs were performed to identify a stool as that of a red fox, dog or cat (Table 1) in the expected ranges of Cq values, as previously described.

Statistical analysis

Correlations between the number of faeces found and the sample sizes, and between the number of faeces testing positive for parasites and the total number of faeces found, were tested by the Spearman correlation test. The frequencies of faeces with positive qPCR results were given with their 95% confidence intervals and were compared using chi-square tests. A Fisher's exact test was carried out when sample size was not sufficient. All statistical analyses were performed with R version 3.1.3.

Results

A total of 85 faeces were collected from October 2011 to January 2012, and 169 faeces from February to March 2013, resulting in 254 faeces collected over the 2011-2013 period. Of the 254 faeces analysed, 194 (76.4%) were visually identified in the field from morphological criteria (size and shape), 191 (75.2%) provided usable DNA and 143 (56.3%) allowed for identification of the emitter species using qPCR analysis. Of the 104 faeces with a specific identification based both on morphological criteria and qPCR analysis, 19 out of 23 faeces (82.6%) and 54 out of 62 faeces (87.1%), respectively, attributed to fox and cat in the field were confirmed by qPCR analysis (Table 2). Thus, morphological criteria alone were considered reliable for the identification of cat and fox faeces. Consequently, the 50 faeces attributed to cat in the field but that did not permit qPCR identification of the emitter were considered as cat faeces, and the 26 faeces attributed to fox based on their morphological identification without molecular confirmation were considered as fox faeces. In contrast, as only 12 out of 19 (63.2%) faeces attributed to dog in the field were confirmed as dog stools by qPCR analysis (Table 2), morphological identification alone was not considered reliable enough to attribute faeces to dog. For that reason, only the 22 faeces identified as dog faeces by molecular analysis (among them some that were unidentified or misidentified in the field) were considered for further analysis. Of the 60 unidentified faeces in the field, 39 were identified by qPCR. In the 62 stools in which the Geotricum qPCR was inhibited, 40 could be identified by morphological and/or molecular analysis. Of these, 37 were attributed to cat, one to dog and two to fox. Cat faeces therefore accounted for almost all the identified faeces that had inhibitors.

To sum up, 219 out of 254 (86.2%) of the collected faeces could be attributed to a species based on qPCR analysis and/or visual identification. Of these, 127 (58%) were cat faeces, 70 (32%) were fox faeces and 22 (10%) were dog faeces.

No stools were found in 39 of the 94 sampled gardens (41.5%), whereas 1-21 stools were collected in the 55 others (Fig. 2A). The kitchen garden where the highest number of faeces were collected (21 faeces: 16 fox stools, 3 cat stools, 1 dog stool and 1 undetermined stool) was the farthest from buildings or barns (by a distance of 234 m). The 23 out of 94 kitchen gardens (24.5%) where at least four stools were found were the source of 191 out of 254 (75.2%) faeces. As might be expected, the number of faeces found per kitchen garden was significantly correlated with the surface sampled (r = 0.54, p < 0.0001). Of the 52 kitchen gardens where at least one identified carnivore stool was found, 35 (67%) had only cat, fox or dog faeces, 10 (19%) had both cat and fox faeces, 1 (2%) had both dog and fox faeces, and 6 (12%) had cat, dog and fox faeces (Fig. 2A). Of the 23 kitchen gardens that had only cat faeces, 8 (33%) were enclosed. There were four kitchen gardens with more than 10 cat faeces: these were characterised by the close proximity of a site where cats were fed (by the kitchen garden owners or their neighbours).

Of the 74 faeces that yielded positive qPCR or PCR results for the detection of parasite DNA, three tested positive for both Toxocara spp. and E. multilocularis (two fox stools and one cat stool) and two others tested positive for both Toxocara spp. and T. gondii (one cat stool and one dog stool), resulting in a total Table 2. Concordance matrix between faeces identification based on morphological assessment and faeces identification based on molecular analysis (by qPCR). In grey cells: occurrence of faeces per species for which the morphological identification was concordant with the molecular analysis (e.g. 19 out of 23 faeces identified as fox faeces from morphological examination were confirmed in molecular analysis). In other cells: the number and percentage of faeces for which the morphological identification did not correspond with the molecular analysis. Percentages are given with their 95% confidence intervals. of 79 parasite DNA detections (Table 3). The occurrence of Toxocara spp. and E. multilocularis DNA did not significantly differ within the total sampling, with 20.7% and 18.3% of stools testing positive, respectively (Table 3, v 2 test = 0.22, p = 0.64). However, the occurrence of Toxocara spp. and E. multilocularis differed significantly (p < 0.001) between cat, fox and dog faeces that yielded qPCR results (Table 3). Toxocara spp. was the most frequently detected parasite in cat faeces (33.3%), while its occurrence was significantly lower in fox faeces (11.6%) and dog faeces (5.5%) (p < 0.001).

Fox by qPCR

In contrast, E. multilocularis was significantly more frequent in fox faeces (34.8%) than in cat faeces (7%) or dog faeces (11.1%) (p < 0.0001). Toxoplasma gondii was detected in 2 out of 125 cat faeces and 2 out of 21 dog faeces and was not detected in fox faeces (Table 3). The two dog faeces that tested positive for T. gondii PCR were collected within a one-month interval in the same enclosed kitchen garden. They originated from the same privately owned dog. Of the total (A) (B) eight faeces collected from this dog, one was contaminated with both T. gondii and Toxocara spp, one was contaminated with T. gondii and one was contaminated with E. multilocularis. In 7 of the 55 kitchen gardens where at least one stool was found, all the stools had inhibitors, resulting in an absence of qPCR results (Fig. 2B). The 79 detections of parasite DNA originated from 36 of the 94 sampled kitchen gardens (38.3%). The number of parasite detections was correlated to the number of faeces collected per kitchen garden (r = 0.55, p < 0.0001). The 34 faeces that tested positive for E. multilocularis were collected in 19 of the 94 sampled kitchen gardens (20.2%), while 28 of the 94 sampled kitchen gardens (29.8%) contained the 40 faeces that tested positive for Toxocara spp. (Fig. 2B).

Discussion

To our knowledge, this study is the first to provide information about the occurrence of E. multilocularis, Toxocara spp. and T. gondii in cat, dog and fox faeces deposited in privately owned kitchen gardens located in a highly endemic area for AE. As an exploratory study, it is based on an opportunistic sampling design leading to uneven sampling regarding villages, sampling periods and sample size per garden. However, despite this and other methodological issues, this study gives an initial insight into the key role kitchen gardens may play in the transmission of foodborne parasites from carnivores to humans.

The use of molecular screening (PCR and qPCR) allowed the identification of the emitter species of faecal samples as well as the detection of the parasite DNA contained in the sample. The proportion of samples usable for molecular analysis (75.2%) was lower than previously described in Knapp et al. [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF], where less than 2% of the samples contained PCR inhibitors. However, in Knapp et al. [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF], only fox stools were investigated, while in this study we also investigated dog and cat faeces, with cat stools accounting for a very large percentage of the faecal samples with inhibitors. As a high level of calcium is known to alter DNA quality [START_REF] Opel | A study of PCR inhibition mechanisms using real time PCR[END_REF], one might assume that cat and dog copro-samples had a higher level of calcium than fox copro-samples due to the high proportion of pet food in these animals' diet. The correct morphological identification of carnivore stools was high for cat stools (87% of faeces were correctly identified in the field) and fox stools (83% were correctly identified), but was lower for dog stools (63% were correctly identified). These results indicate that identifying copro-material remains difficult, even for specialists, and that confirmation by a second technique is often advisable.

In the majority of previous studies dealing with the detection of foodborne parasites in copro-samples from cat, fox or dog populations, only one faecal sample was collected per individual. In this study, faeces were collected in the field, which means that we could not establish whether multiple samples from the same individual were collected. For this reason, our results have a disadvantage in that they cannot be compared to those from studies on the prevalence of E. multilocularis in fox and dog populations (e.g. [START_REF] Dyachenko | Echinococcus multilocularis infections in domestic dogs and cats from Germany and other European countries[END_REF][START_REF] Umhang | Echinococcus multilocularis infections in dogs from urban and peri-urban areas in France[END_REF]) or the prevalence of Toxocara spp. in fox, dog and cat populations (e.g. [START_REF] Brochier | Echinococcus multilocularis and Toxocara canis in urban red foxes (Vulpes vulpes) in Brussels, Belgium[END_REF][START_REF] Villeneuve | Parasite prevalence in fecal samples from shelter dogs and cats across the Canadian provinces[END_REF]). However, they do serve to provide a general view of the contamination risk in kitchen gardens. Indeed, as discussed by Lass et al. [START_REF] Lass | Fresh fruits, vegetables and mushrooms as transmission vehicles for Echinococcus multilocularis in highly endemic areas of Poland: reply to concerns[END_REF], the detection of parasite DNA in faeces is evidence of their presence in the environment and indicates a potential risk for humans, even though this alone does not allow the level of risk to be determined -this would require investigating the viability of the infective stage of the parasites and the parasite burden per faeces. In line with Conraths and Deplazes [START_REF] Conraths | Echinococcus multilocularis: epidemiology, surveillance and state-of-the-art diagnostics from a veterinary public health perspective[END_REF], we consider that data from faeces collected in the environment has the advantage of yielding more relevant information for preventing foodborne parasite transmission than investigating prevalence in definitive host populations, since humans run a much higher risk of contamination from contact with the environment than from contact with definitive hosts. Furthermore, such data also have the advantage of allowing hotspots of intensive transmission to be identified, some having already been identified in the transmission of E. multilocularis to its intermediate hosts [START_REF] Raoul | Trophic ecology, behaviour and host population dynamics in Echinococcus multilocularis transmission[END_REF] and in the transmission of Toxocara spp. to humans [START_REF] Mizgajska-Wiktor | Distribution and dynamics of soil contamination with Toxocara canis and Toxocara cati eggs in Poland and prevention measures proposed after 20 years of study[END_REF].

Almost one-third of the faeces we collected in kitchen gardens were from fox. This relatively high occurrence of faeces from a wild carnivore in close proximity to humans is not surprising since foxes are known to come close to villages and buildings at night in rural areas [START_REF] Janko | Space use and resting site selection of red foxes (Vulpes vulpes) living near villages and small towns in Southern Germany[END_REF][START_REF] Krauze-Gryz | The good, the bad, and the ugly: space use and intraguild interactions among three opportunistic predators -cat (Felis catus), dog (Canis lupus familiaris), and red fox (Vulpes vulpes) -under human pressure[END_REF]. This is likely to explain why E. multilocularis DNA was detected close to rural homes in Poland, where foxes approach human settlements in search of food [START_REF] Szostakowska | First finding of Echinococcus multilocularis DNA in soil: preliminary survey in Varmia-Masuria Province, northeast Poland[END_REF]. In our study, 66% of the sampled kitchen gardens were in open access, i.e. easily accessible to canids, and most were near pastures, meadows or forest edges where voles, the main fox prey in the area, are generally abundant [START_REF] Guislain | Fox faeces and vole distribution on a local range: ecological data in a parasitological perspective for Echinococcus multilocularis[END_REF]. Accessibility and food availability could thus explain the relatively frequent collection of fox faeces in the sampled kitchen gardens. The detection of E. multilocularis DNA in almost 35% of the kitchen gardens is thus not surprising since more than 50% of foxes necropsied during the 2001-2005 Table 3. Occurrence of cat, fox, dog and unidentified faeces collected in kitchen gardens that yielded positive PCR results for the detection of Echinococcus multilocularis, Toxocara spp. or Toxoplasma gondii DNA as compared to the total number of faeces that yielded PCR results for the given emitter and parasite (i.e. faeces without inhibitors). Percentages are given with their 95% confidence intervals.

E. multilocularis

Toxocara spp. period were found to carry E. multilocularis worms in their intestines [START_REF] Guislain | Ecological and biological factors involved in the transmission of Echinococcus multilocularis in the French Ardennes[END_REF] and there is no reason to assume that the prevalence of this parasite in the vulpine population was lower during the sampling period of our study. Echinococcus multilocularis was also detected in two dog faeces (out of 18) collected in kitchen gardens from two different villages.

Given the high reproductive potential of this parasite in fox and dog [START_REF] Kapel | Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats[END_REF], we can assume that the E. multilocularis DNA detected in fox and dog faeces originated from eggs. Nonetheless, the distinction between viable and non-viable eggs would provide valuable information when considering the infection risk. In any case, given the occurrence of fox faeces in kitchen gardens, the occurrence of E. multilocularis DNA in these faeces as well as in dog faeces, and the non-homogeneous distribution of faeces testing positive for parasites we observed, the results indicate that fruits and vegetables cultivated in some kitchen gardens could be at risk of exposing consumers to E. multilocularis. The detection of E. multilocularis DNA in different environmental matrices in a kitchen garden belonging to an AE patient in Poland [START_REF] Lass | The first detection of Echinococcus multilocularis DNA in environmental fruit, vegetable, and mushroom samples using nested PCR[END_REF] tends to confirm this hypothesis. Similarly, the contamination of market gardens by feral dog faeces was identified as being probably responsible for numerous cases of human cystic echinococcosis in Jordan [START_REF] Dowling | Risk factors associated with human cystic echinococcosis in Jordan: results of a case-control study[END_REF]. Cat faeces in kitchen gardens were characterised by their abundance, accounting for 58% of the 219 faeces identified from morphological and/or molecular identification. This high occurrence probably resulted both from the relatively high density of the cat population, as compared to the dog and fox populations, and to the selection of kitchen gardens by cats to defecate because they seek to cover their faeces with loose soil. Due to this covering behaviour, the detection of cat faeces in kitchen gardens may have been less exhaustive than the detection of fox and dog faeces. However, the possible underestimation of cat faeces was probably low in the November to February sessions because the soil was generally frozen or wet, deterring cats from burying their faeces. It may have been higher in March and April, when the soil had thawed and some cat faeces may have been deeply buried as gardeners often turn over the soil during this period. Although E. multilocularis DNA was detected in almost 7% of the cat faeces collected in kitchen gardens, cats probably play a minor role in the transmission of this parasite since the reproductive potential of E. multilocularis is thought to be low in this species [START_REF] Kapel | Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats[END_REF]. This assumption is supported by the fact that no eggs were found in the 10 out of 321 cat faeces collected in two villages of the study area that tested positive for E. multilocularis in a molecular analysis, as well as by the fact that the infected cats originating from these villages carried only immature worms [START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of freeranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF]. However, Knapp et al. [START_REF] Knapp | Could the domestic cat play a significant role in the transmission of Echinococcus multilocularis? A study based on qPCR analysis of cat feces in a rural area in France[END_REF] reported eggs in cat faeces in eastern France, observing entire eggs, with shell and hooks, under the microscope. The role of cat in E. multilocularis transmission in terms of the infectivity of eggs warrants further study.

The 33.3% (27.0-48.6) occurrence of Toxocara spp. DNA identified by qPCR in cat faeces collected in kitchen gardens is very similar to the 35.7% (31.2-40.1) occurrence of eggs from this parasite detected by the flotation technique on faeces collected in the environment from a stray cat population in Argentina [START_REF] Sommerfelt | Prevalence of Toxocara cati and other parasites in cats' faeces collected from the open spaces of public institutions: Buenos Aires, Argentina[END_REF]. This similarity may be explained by the fact that in our study, as in that of Sommerfelt et al. [START_REF] Sommerfelt | Prevalence of Toxocara cati and other parasites in cats' faeces collected from the open spaces of public institutions: Buenos Aires, Argentina[END_REF], only a small percentage of the individuals from the outdoor cat population depositing the faeces collected in kitchen gardens had been dewormed [START_REF] Gotteland | Assessing the homogeneity of individual scat detection probability using the bait-marking method on a monitored freeranging carnivore population[END_REF]. In contrast, the 11.6% (5.1-21.6) occurrence of Toxocara spp. DNA in fox faeces collected in kitchen gardens was lower than the 22.3% (19.9-24.7) occurrence of eggs from this parasite in 1213 fox faeces collected in the field in Belarus and examined with standard flotation techniques [START_REF] Shimalov | Helminth fauna of the red fox (Vulpes vulpes Linnaeus, 1758) in southern Belarus[END_REF]. The direct observation of eggs would allow for more reliable assessment of the zoonotic risk related to the detection of Toxocara spp. DNA in cat, fox and dog faeces collected in kitchen gardens. Nonetheless, the occurrence of faeces testing positive in our sample suggests that this risk may be significant.

Toxoplasma gondii DNA was detected in 1.6% of the cat faeces collected in kitchen gardens; this is in line with the low proportion of individuals found to excrete oocysts in cat populations [START_REF] Dubey | Toxoplasmosis of Animals and Man[END_REF]. Toxoplasma gondii was also detected in two dog faeces, which was an unexpected result as canids do not usually excrete T. gondii oocysts. Toxoplasma gondii DNA detected by qPCR may originate from bradyzoites of infected prey a cat or dog has just ingested, as has been demonstrated experimentally by Poulle et al. [START_REF] Poulle | Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding[END_REF]. Furthermore, as coprophagy is relatively common in dog [START_REF] Frenkel | An apparent role of dogs in the transmission of Toxoplasma gondii: the probable importance of xenosmophilia[END_REF][START_REF] Nijsse | Environmental contamination with Toxocara eggs: a quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs[END_REF], the T. gondii DNA detected in the two dog faeces may also have resulted from the ingestion of cat faeces with T. gondii oocysts, as has been suggested to explain the detection of Toxocara cati and other atypical parasites in dog faeces [START_REF] Fahrion | Toxocara eggs shed by dogs and cats and their molecular and morphometric species-specific identification: is the finding of T. cati eggs shed by dogs of epidemiological relevance[END_REF][START_REF] Nijsse | Environmental contamination with Toxocara eggs: a quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs[END_REF]. More of a concern could be the detection of E. multilocularis and Toxocara spp. in dog faeces collected in kitchen gardens; particularly the discovery of two faeces yielding positive qPCR results for these parasites in the same kitchen garden. The fact that the dog that deposited these faeces was confined in this kitchen garden indicates that eggs from this infected individual were shed where fruit and vegetables were grown and could thus present a zoonotic risk for the dog's owners and any consumers of the garden's produce.

In conclusion, this study revealed that faecal deposition by cats, foxes and dogs does not appear minor in certain kitchen gardens of northeastern France, nor does the occurrence of foodborne zoonotic parasites in their faeces. In particular, the high occurrence of E. multilocularis DNA in fox and dog faeces emphasises the need to prevent access to kitchen gardens by canids to the extent possible in areas where AE is endemic. This could be done through information campaigns to make gardeners aware of the zoonotic threat, by promoting the enclosure of kitchen gardens with fences, by reducing food availability in proximity and by avoiding using kitchen gardens as dog pens. In addition, the deposition of faeces in kitchen gardens by free-roaming cats can lead to a risk of human exposure to Toxocara spp. that should not be underestimated [START_REF] Deutz | Toxocara-infestations in Austria: a study on the risk of infection of farmers, slaughterhouse staff, hunters and veterinarians[END_REF]. Preventing free-roaming cats from accessing kitchen gardens is difficult as they can easily climb fences, but other recommendations to limit the risk of toxocariasis and toxoplasmosis infections could be promoted, such as washing hands after contact with soil or plants, and cooking or thoroughly washing fruit and vegetables in contact with soil. These recommendations would also be valuable in helping to prevent AE infection.

Figure 1 .

 1 Figure 1. Two kitchen gardens in open access to carnivores and located inside (A) and outside (B) the village (at the forefront: a cat stool dropped close to the garden border).

Figure 2 .

 2 Figure 2. The number of carnivore faeces collected in the 55 (out of 94) sampled kitchen gardens in which at least one stool was collected; (A) the number of cat, fox, dog and unidentified faeces; (B) the number of faeces that yielded positive results for the detection of Toxocara spp., Echinococcus multilocularis or Toxoplasma gondii, as well as the number faeces with DNA inhibitors.

Table 1 .

 1 Primers and hydrolysis probes used to detect Echinococcus multilocularis, Toxoplasma gondii and Toxocara spp. parasites, and carnivore host identification of copro-samples.

	Targeted species	Gene	Sequence name	Nucleotide sequence	Reference
	Echinococcus multilocularis	rrnL	rrn-Fwd	5

  Poulle et al.: Parasite 2017, 24, 29 15 s, and annealing/extension at 60 °C for 1 min. Each sample was tested in duplicate. Negative controls were included from DNA extraction to PCR amplification steps, and each PCR run contained both negative and positive controls. No amplification results were systematically obtained for the negative control. A PCR with Cq 45 cycles allowed us to determine the presence of parasites. Molecular analyses were performed at the Laboratory of Parasitology and Mycology, EA3800 PROTAL, Reims University, France.

Acknowledgements. This work was partly supported by a grant from the Conseil Général des Ardennes. We are very grateful to the 94 Ardennes inhabitants who allowed us to survey their kitchen gardens and to our 19 village contacts who helped us in obtaining these authorisations. We would also like to thank Jordan Carvalho for his contribution to the collection and analysis of faeces, Elise Bradbury for her editorial review, as well as Prof. Emmanuelle Gilot-Fromont and the two anonymous reviewers for their helpful comments on the first draft of the manuscript.

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