Julien Raitif

Manuel Plantegenest

Océane Agator

C Piscart

Jean-Marc Roussel

Annual emergence ~ 4 g drymass m -2 ~4 g drymass m ~4 g drymass m

Keywords: Chironomidae, 25% Ephemeroptera, 19% Trichoptera, 56% agroecosystems, winged stream insects, emergence, aquatic subsidies, 25 environmental factors, seasonal variations 26 Water conductivity, O² Sat. = Oxygen saturation, Temp. = Water temperature, Turb. = Water turbidity. Lower panel: Site-scale variables

Highlights

• Aquatic insects emergence reaches 4 g drymass (DM).m -2 .y -1 in agricultural streams • Trichoptera contributed the most, followed by Chironomidae then Ephemeroptera • Emergence happened throughout the year with taxon-specific patterns • Several parameters linked to agriculture influenced emerging DM of aquatic insects • We estimated potential deposit of aquatic subsidies on land at 4.5 kg DM.

Introduction

Riverine ecosystems host a rich but endangered biodiversity [START_REF] Naiman | THE ECOLOGY OF INTERFACES: Riparian Zones[END_REF][START_REF] Dudgeon | Freshwater biodiversity: importance, threats, status and conservation challenges[END_REF]) that could play a significant role in providing ecosystem services. In a recent review, [START_REF] Hanna | A review of riverine ecosystem service quantification: research gaps and recommendations[END_REF] listed more than 30 types of services that riverine ecosystems provide, and observed a potential distinction between the location and the spatial extent at which these services are produced and delivered. Growing evidence indicates that ecosystems that were previously considered unrelated can interact. Several studies have demonstrated the existence of reciprocal exchanges of matter and energy between aquatic and terrestrial habitats (e.g. [START_REF] Polis | Toward an integration of landscape and food web ecology: the dynamics of spatially subsidized food webs[END_REF][START_REF] Richardson | Resource subsidies across the land-freshwater interface and responses in recipient communities[END_REF][START_REF] Bartels | Reciprocal subsidies between freshwater and terrestrial ecosystems structure consumer resource dynamics[END_REF]). These "aquatic subsidies" can significantly influence the functioning of terrestrial ecosystems [START_REF] Jackson | Secondary Production, Emergence, and Export of Aquatic Insects of a Sonoran Desert Stream[END_REF][START_REF] Havik | Seabird Nutrient Subsidies Benefit Non-Nitrogen Fixing Trees and Alter Species Composition in South American Coastal Dry Forests[END_REF][START_REF] Dreyer | Quantifying aquatic insect deposition from lake to land[END_REF], but this is poorly documented for agricultural landscapes. Aquatic subsidies can also influence several ecosystem services supplied to agriculture (Raitif et al., in revision), and future studies are required to estimate the magnitude of their influence.

Adult aquatic winged insects are effective vectors of aquatic subsidies [START_REF] Bartels | Reciprocal subsidies between freshwater and terrestrial ecosystems structure consumer resource dynamics[END_REF] since they are abundant in almost all freshwater ecosystems, and their ability to disperse is substantial [START_REF] Muehlbauer | How wide is a stream? Spatial extent of the potential "stream signature" in terrestrial food webs using meta-analysis[END_REF]. Several studies have demonstrated their role as prey for terrestrial predators (e.g. birds, [START_REF] Gray | Response of Insectivorous Birds to Emerging Aquatic Insects in Riparian Habitats of a Tallgrass Prairie Stream[END_REF]carabids, Hering and Plachter 1997;spiders, Paetzold et al. 2005), providing a valuable source of nutrients for entire terrestrial ecosystems [START_REF] Dreyer | Quantifying aquatic insect deposition from lake to land[END_REF]. Agricultural practices can regulate the production of benthic macroinvertebrates in streams by modifying water chemistry, eutrophication processes and pollution [START_REF] Sallenave | Secondary production of benthic stream invertebrates in agricultural watersheds with different land management practices[END_REF][START_REF] Liess | Analysing effects of pesticides on invertebrate communities in streams[END_REF][START_REF] Cross | Whole-system nutrient enrichment increases secondary production in a detritus-based ecosystem[END_REF][START_REF] Davis | Nutrient enrichment differentially affects body sizes of primary consumers and predators in a detritus-based stream[END_REF]Johnson et al. 2013a;[START_REF] Beketov | Pesticides reduce regional biodiversity of stream invertebrates[END_REF]; changing the flow regime and aquatic habitats [START_REF] Rabení | Stream invertebrate community functional responses to deposited sediment[END_REF][START_REF] Kennedy | River channelization reduces nutrient flow and macroinvertebrate diversity at the aquatic terrestrial transition zone[END_REF][START_REF] Wagenhoff | Subsidy-stress and multiplestressor effects along gradients of deposited fine sediment and dissolved nutrients in a regional set of streams and rivers: Sediment and nutrients in streams[END_REF][START_REF] Magbanua | Individual and combined effects of fine sediment and glyphosate herbicide on invertebrate drift and insect emergence: a stream mesocosm experiment[END_REF]; and modifying riparian vegetation [START_REF] Deegan | The loss of aquatic and riparian plant communities: Implications for their consumers in a riverine food web[END_REF] and water temperature [START_REF] Nagasaka | The influences of land-use changes on hydrology and riparian environment in a northern Japanese landscape[END_REF][START_REF] Sponseller | Relationships between land use, spatial scale and stream macroinvertebrate communities[END_REF]. Consequently, the influence of agriculture in watersheds on the emergence of stream winged insects could increase or decrease the strength of aquatic subsidies provided to terrestrial ecosystems. For instance, stream insect communities are usually dominated by small taxa with greater ability to disperse by flight in agricultural landscapes [START_REF] Stenroth | Land-use effects on terrestrial consumers through changed size structure of aquatic insects[END_REF][START_REF] Carlson | Strong land-use effects on the dispersal patterns of adult stream insects: implications for transfers of aquatic subsidies to terrestrial consumers[END_REF][START_REF] Greenwood | Influence of hydrological regime and land cover on traits and potential export capacity of adult aquatic insects from river channels[END_REF]. While insect emergence depends on season and weather conditions [START_REF] Corbet | Temporal Patterns of Emergence in Aquatic Insects[END_REF], few studies quantify these influences or analyze their variability in agricultural landscapes. For instance, [START_REF] Shieh | Longitudinal changes in macroinvertebrate production in a stream affected by urban and agricultural activities[END_REF] and [START_REF] Gücker | Urban stressors alter the trophic basis of secondary production in an agricultural stream[END_REF] published seasonal variations of aquatic insect production in two agricultural streams, but not emergence data.

However, information on spatial and temporal variation in the emerging biomass of aquatic insect taxa is essential to accurately estimate the amount of aquatic subsidies annually produced by agricultural streams.

In this study, we quantified the spatial and seasonal emergence of aquatic insects in 12 sites located in intensive agricultural landscapes. We aimed to (i) assess temporal variation in dry mass (DM) for the main emerging aquatic insect taxa and (ii) highlight the influence of environmental parameters at different spatial scales on emergence patterns. We hypothesized that agriculture intensification would drive emergence of aquatic insects at both local and watershed scales, and notably enhance Chironomidae (Diptera) that could emerge throughout the year.

Methods

Study sites

The study was performed in the Ille-et-Vilaine county (Brittany), western France. This area sits on sedimentary rocks (schist and sandstone) with occasional layers of aeolian loam deposits. The climate is oceanic, with a mean annual temperature of 10.5-12.5°C and cumulative annual rainfall of approximately 700 mm (peaking in fall and winter) (Météo France 2017). Mean annual discharge of third-order streams in this part of Brittany is 0.65 m 3 s -1 , ranging from 0.06 m 3 s -1 in September (end of summer) to 2.23 m 3 s -1 in March (end of winter).

Intensive agricultural practices that rely on large amounts of inputs (fertilizers and pesticides) and extreme modifications of the landscape have altered terrestrial and aquatic ecosystems for decades (Piscart et al. 2009). Stream banks, deeply incised into thick arable ground, are destabilized by clearcutting of riparian vegetation, which results in sloughing, bank erosion, siltation and homogenization of stream substratum. Twelve sites were selected from 8 watersheds: Champagne, Roncelinais, Jardière, Rocher, Tertre, Ourmais, Bray, Ormal, Moulin, Fèvre, Vallée and Sauvagère (Table S1). Selection criteria was based according to (1) third-order permanent streams (Strahler classification), and ( 2) approximately 6 m wide and flowing along small-grain cereal fields (winter wheat or barley). We chose third-order streams to avoid summer drying of lower-order streams and fall and winter floods in higher-order streams, which would have impeded aquatic insect sampling. A drought occurred in 2016, resulting in very low discharge in winter 2016/2017 (1 m 3 s -1 ). Mandatory grass strips (~8 m wide) separated streams from fields. This highly enriched buffer zone was dominated by nitrophilous plants belonging to a variety of families (Poaceae, Urticaceae, Apiaceae, etc.).

Riparian vegetation near the stream bank consisted of shrubs, small trees (Salix sp.), and occasional larger trees (Quercus sp., Alnus sp.).

Aquatic insect sampling

We estimated aquatic insect emergence by deploying two emergence traps at each site. The traps consisted of a floating pyramidal tent (1 m² at its base) made of nylon mesh and anchored to shorelines with ropes. Because substratum and water velocity strongly affect aquatic insect communities [START_REF] Tachet | Invertébrés d'eau douce systématique[END_REF], one trap was set in a deep and silty habitat and the other in a shallow gravel-pebble habitat to reflect the natural habitat heterogeneity of each site. Adult insects were collected in a plastic bottle placed at the top of the trap and filled with a mix of water, concentrated detergent and propylene glycol (approx. 20%) to preserve insects. Upon collection, aquatic insects were stored in alcohol (70%).

Six campaigns of sampling were performed in 2016 and 2017: 17 -26 May; 6 -15 June; 27 June -6 July; 12 -21 September; 28 November -5 December; 27 February -9 March. At each site, emergence trap was collected after 7 consecutive days and nights. Ten days per campaign were necessary to proceed the 12 sites because of travel time between sites. The timeline was designed to match aquatic insect emergence in such streams, which occurs mainly in spring (March to June), to a lesser extent in summer and early fall (July to October), and almost stops during cold and high-discharge winter months [START_REF] Corbet | Temporal Patterns of Emergence in Aquatic Insects[END_REF]. Sampling was carried out in December 2017 to confirm that insects do not emerge in coldest times at the end of fall or during winter, but only at one site, to avoid damaging the equipment in windy and flooded conditions. A total of 111 samples, totaling 777 days of aquatic insect emergence, were collected; 11 samples were lost due to strong winds, flash floods or vandalism.

Emergence abundance, dry mass and secondary production

A stereomicroscope was used (1) to identify adult aquatic insects to the order level for Ephemeroptera,Plecoptera,and Trichoptera,[START_REF] Cummins | An Evaluation of Some Techniques for the Collection and Analysis of Benthic Samples with Special Emphasis on Lotic Waters[END_REF] to separate Chironomidae from other Diptera, the most abundant taxon in our sampling. Other insects, mainly Diptera, were not considered because their contribution to total DM was low and did not justify the timeconsuming work required for identification. For Chironomidae, sub-samples (1/2, 1/4 or 1/8) were obtained with a Motoda splitter [START_REF] Motoda | Devices of simple plankton apparatus[END_REF] when abundance was too high. Insects were then dried at 60 °C for at least 24 h. After cooling in a desiccator for another 24 h, each sample of each taxon was weighed to the nearest 0.2 mg (on a Mettler Toledo AG). Insect abundance (ind m -2 d -1 ) and DM (mg DM m -2 d -1 ) were recorded for each site separately and then averaged (all sites combined) on a daily basis. Mean daily values obtained for each campaign were prolonged to adjacent days (both forward and backward) to fill temporal gaps between campaigns. Daily values were then summed to calculate cumulative seasonal emerging DM (mg DM m -2 season -1 ) using solstice and equinox dates as reference. We calculated cumulative annual emerging DM as the sum of all seasonal values (mg DM m -2 y - 1 ). Biomass of emerging insects was then transformed to estimate secondary production at each site using the mean Emergence:Production (E:P) ratio of 0.183 calculated by [START_REF] Poepperl | Benthic secondary production and biomass of insects emerging from a northern German temperate stream[END_REF] for a similar agricultural stream. This ratio is relatively constant across taxa and stream ecosystems [START_REF] Statzner | Multiple-site and-year analyses of stream insect emergence: a test of ecological theory[END_REF][START_REF] Gratton | Flux of aquatic insect productivity to land: comparison of lentic and lotic ecosystems[END_REF].

Water quality, land use and habitat assessment

Water quality data were provided for each study stream by the Agence de l'Eau Loire-Bretagne (2017). Information on water temperature (°C), turbidity (Formazin Nephelometric Units, FNU), conductivity (µS cm -1 ), nitrate (mg NO 3 -L -1 ), ammonium (mg NH 4 + L -1 ), phosphorus (P) (mg total P L -1 ), suspended matter (mg DM L -1 ) and oxygen saturation (%)

were collected to reflect water quality at the watershed scale. Monthly values observed from February 2016 to March 2017 were averaged to account for seasonal variations (Table S1).

Similarly, mean annual discharge was estimated for each stream using QGIS (QGIS Development Team 2009), based on information available from the Réseau Hydrographique Théorique [START_REF] Pella | Le réseau hydrographique théorique (RHT) français et ses attributs environnementaux, Géomorphologie : relief, processus, environnement Piscart C[END_REF]. Watershed-scale variables also included watershed area and land use (% of agricultural, artificial and natural area), which were estimated using QGIS and Corine Land Cover (CLC 2012). For each study site, local habitat features were measured or visually assessed at mid-channel every 5 m along a 20-m reach on both sides of each trap:

surface water velocity (cm s -1 ), water depth (cm), depth of soft sediments on the bottom (cm), stream width (cm), bank height (cm), channel openness (%) and substrate size (Table S1).

Substrate size was visually estimated following a modified Wentworth classification [START_REF] Cummins | An Evaluation of Some Techniques for the Collection and Analysis of Benthic Samples with Special Emphasis on Lotic Waters[END_REF]. A substrate score was calculated by multiplying mean particulate size of each class by its observed proportion. Substrate heterogeneity was assessed by calculating the Shannon index of the proportions of each substrate size class. The embankment score (stream width divided by bank height) was used to estimate stream incision and erosion risk.

Statistical analyses

To study the temporal pattern of emergence, we fitted a linear mixed model (LMM) with a Gaussian distribution, considering the sampling campaign as a fixed effect. The campaign from 28 Nov to 5 Dec 2016 was ignored since data were available for only one site. To study effects of environmental variables on emerging insect DM, another LMM with a Gaussian distribution was applied. The small sample size precluded including both site-and watershedscale variables in a single model. Consequently, two separate models were fitted to the dataset to assess effects of each set of variables.

The corvif() function of R software (R Core Team 2013) was used to verify correlations between variables before model computation [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology with R[END_REF]. Variables were removed when r coefficient of correlation exceeded 0.8 and the variance inflation factor exceeded 4.5.

Accordingly, the following variables were selected and considered as fixed effects at the watershed scale: water temperature, turbidity, conductivity, oxygen saturation, ammonium nitrate concentrations and the percentage of watershed area covered by agriculture (cropland, pastures). At the site scale, the variables selected were water depth, stream width, substratum score, embankment score and channel openness. Prior to modeling, DM was log-transformed (log x + 1) to better meet assumptions of normality. Since quantitative explanatory variables were at different scales, we standardized them (mean=0, σ = 0.5) using the standardize() function of the R package arm [START_REF] Gelman | Data Analysis Using Regression and Multilevel/Hierarchical Models[END_REF]). Samplings were nested in site/watershed and campaign; these effects were thus included in the random part of temporal and spatial models respectively. When a random effect was non-significant (i.e. when deviance explained was null), the model was re-run excluding it.

To identify variables that significantly influenced insect emergence, model averaging was preferred to a best model strategy. Indeed, best model strategy select only one model with one or several parameters and thus potentially exclude other model with different parameters having a similarly good fit [START_REF] Whittingham | Why do we still use stepwise modelling in ecology and behaviour[END_REF]. In an explanatory approach, model averaging is more appropriate as it produces estimates of parameters derived from multiple weighted models. The model-averaging method [START_REF] Burnham | Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach[END_REF] was applied at each spatial scale separately. Full models without interactions were used as starting models for the dredge function of the R package MuMIn [START_REF] Barton | MuMIn: multi-model inference[END_REF]. We retained all models whose Akaike Information Criterion (AIC) corrected for small sample size (AICc; [START_REF] Grueber | Multimodel inference in ecology and evolution: challenges and solutions: Multimodel inference[END_REF] differed by less than 4 from the best model. We also checked for correlation between fixed effects during the dredge process, but found no high correlation (all r < 0.7).

We performed model averaging on the selected subset using the model.avg function of the R package MuMIn. We considered explanatory variables significant when their associated 90% parameter confidence interval (CI) excluded zero [START_REF] Arnold | Uninformative Parameters and Model Selection Using Akaike's Information Criterion[END_REF]. We assessed the significance of their effect on variations in DM of each taxon by considering their effect intensity through parameter estimates (i.e. effect size). Finally, the goodness-of-fit of all best averaged models (R²) was assessed using the procedure developed by [START_REF] Nakagawa | A general and simple method for obtaining R² from generalized linear mixed-effects models[END_REF] for mixed models using only significant explanatory variables. R² marginal approximates the variance explained by the fixed part of the model and R² conditional the variance explained by the complete model (fixed and random parts). All models were fitted using the R package lme4 [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. The same datasets were used for temporal and environmental modeling.

This allowed us to compare the AICc and R² of models for each taxon.

3 Results

Variations in environmental characteristics

Stream discharge in 2016 peaked after heavy rainfall in March, two months before the first sampling campaign at all sites. Discharge rapidly decreased in April and May (< 0.5 m 3 s -1 for all streams), followed by a slight increase in June. No further rainfall increased stream discharge until February 2017, resulting in a low regime (< 0.1 m 3 s -1 ) for all streams from late summer to early winter. After some rainfall events in fall and winter, turbidity and suspended matter concentrations increased sharply to a mean of 16 FNU and 15 mg DM L -1 , respectively. Mean water temperature (± 1 standard deviation) ranged from 17.4 °C (± 2.0 °C) in early summer to 4.1 °C (± 1.5 °C) in winter. Mean water conductivity was highest in fall

(1,103 µS cm -1 ± 1,043) and peaked at 3,370 µS cm -1 in Garun. Oxygen saturation ranged from 75% in summer to 100% in spring, with distinct annual variations among sites (Table S1). Mean nitrate concentration was high throughout the year (20.4 mg NO 3 -L -1 ± 6.6), with minimum values recorded at Illet and Veuvre and maximum values at Yaigne. Similarly, ammonium and total P concentrations varied greatly among streams. Agriculture dominates the landscape, covering a mean of 85% of the total watershed area, but variations in local habitat characteristics were observed among sites. For instance, the channel was narrow with dense riparian vegetation at Ormal but wider and almost completely open (no tree or shrub) at Roncelinais. At Tertre, the stream flowed very slowly (< 0.1 m s -1 ), with an accumulation of fine sediment on the bottom, whereas at Jardière and Bray, water velocity, depth and substratum were more heterogeneous. Mean annual values of site-and watershed-scale variables are listed in Table S1.

Characteristics of aquatic insect emergence

We caught 64,027 insects in emergence traps during the study, equaling 13.1 g of total DM (all taxa combined). Chironomidae dominated total abundance (91%) and contributed 25% of total caught DM. Trichoptera contributed the most to total DM (56%) for only 6% of total abundance. Ephemeroptera represented 19% of total DM and 3% of total abundance.

Plecoptera were rarely captured (only 23 individuals, 33.4 mg DM) and thus were not considered for further analysis. Total emerging DM caught in all sampling campaigns differed among sites and ranged from 435 (at Jardière) to 2,091 mg DM (at Vallée) (Table S2).

Temporal dynamics of insect emergence

Daily emergence varied by taxon among sampling campaigns (Fig. 1a). That of Trichoptera increased in spring and early summer (up to 19.35 mg DM m -2 d -1 ), then decreased sharply and remained low until the end of the study (R² marginal = 34%). Daily emergence of Trichoptera varied greatly among sites in July, ranging from 2.62 to 79.53 mg DM m -2 d -1 at Jardière and Fèvre, respectively (Table S3). Mean daily emergence of Chironomidae varied less among sampling campaigns (R² marginal = 11%), ranging from 2.89-6.44 mg DM m -2 d -1 , with two distinct emergence peaks in May and July. Ephemeroptera emergence decreased continuously (R² marginal = 34%) from spring (max. 8.2 mg DM m -2 d -1 ) to winter. No emergence was observed for Ephemeroptera and Trichoptera in December, and it was very low for Chironomidae (~ 0.17 mg DM m -2 d -1 ). Cumulative DM varied by taxon and season (Fig. 1b). Over the entire study, that of Trichoptera was highest (2,050 mg DM m -2 ), followed by those of Chironomidae (1,120 mg DM m -2 ) and Ephemeroptera (670 mg DM m -2 ). For all taxa combined, cumulative DM reached 3,840 mg DM m -2 y -1 , ranging from 1,445 to 7,374 mg DM m -2 y -1 at Jardière and Fèvre, respectively. Using the Poepperl E:P ratio (2000), mean secondary production was estimated at 21 g DM m -2 y -1 , ranging from 7.9 to 40 g DM m -2 y -1 at Jardière and Roncelinais, respectively.

Influence of environmental characteristics on insect emergence

The environmental variables in the spatial models explained a significant percentage of the variation in emerging insects DM, and noticeable differences were observed among taxa (Table 1). For Chironomidae, the spatial models performed better (R² marginal = 15.9% and 21.4% at the site and watershed scale, respectively) than the temporal model (R² marginal = 10.9%). Among spatial models, the model with watershed-scale variables fit the data better but was less parsimonious than that with site-scale variables. Conversely, the temporal model explained emerging Trichoptera and Ephemeroptera DM best (R² marginal ~ 34%), the spatial models R² marginal ranging from 11.6 to 23.9%, with watershed-scale variables explaining Ephemeroptera better and site-scale variables explaining Trichoptera better.

Among the watershed-scale variables (Fig. 2a), the percentage of watershed area covered by agriculture positively and significantly influenced emerging Chironomidae DM. Emerging

Chironomidae DM also increased significantly with increasing ammonium concentration and decreasing dissolved oxygen concentration. Conversely, emerging Ephemeroptera DM was significantly and negatively influenced by the percentage of watershed area covered by agriculture and, although not significantly, negatively influenced by ammonium concentration and positively influenced by dissolved oxygen concentration. Emerging Trichoptera and Ephemeroptera DM increased significantly with increasing water conductivity and temperature, but that of Trichoptera decreased with increasing turbidity. Among the site-scale variables (Fig. 2b), channel openness positively and significantly influenced emerging DM of all taxa. Emerging Trichoptera and Ephemeroptera DM increased significantly with decreasing stream incision (higher embankment scores), while that of Chironomidae increased significantly with decreasing stream width. Finally, emerging DM of Trichoptera increased significantly with increasing proportion of coarse substrate (i.e. higher substratum scores).

Discussion

We measured the biomass of aquatic insects emerging from third-order streams that drain agricultural landscapes in western France. Mean total emerging DM of the three dominant taxonomic groups living in temperate streams -Chironomidae, Ephemeroptera, and Trichopteraequaled approximately 4.0 g DM m -2 y -1 , ranging from 1.4-7.4 g DM m -2 y -1 depending on the stream. These results fall within the range of those reported in previous studies for emerging aquatic insects in streams (listed in Table 2), although exceptionally larger masses (> 20 g DM m -2 y -1 ) have been observed in desert streams in Arizona, United States (US) [START_REF] Jackson | Secondary Production, Emergence, and Export of Aquatic Insects of a Sonoran Desert Stream[END_REF]. In similar agricultural streams in Germany, [START_REF] Poepperl | Benthic secondary production and biomass of insects emerging from a northern German temperate stream[END_REF] reported lower annual biomass of emerging aquatic insects, especially for Trichoptera and Ephemeroptera. The benthic biomass estimated by [START_REF] Gücker | Urban stressors alter the trophic basis of secondary production in an agricultural stream[END_REF] was approximately 20% that observed in this study. The high biomass estimates in our streams are similar to those in other agriculture-dominated streams in several US states [START_REF] Berg | Secondary production of Chironomidae (Diptera) in a north temperate stream[END_REF][START_REF] Shieh | Longitudinal changes in macroinvertebrate production in a stream affected by urban and agricultural activities[END_REF]Johnson et al. 2013b). Compared to other forestdominated streams, the biomass we estimated were four times greater for all taxa (combined) than in a third order stream in China [START_REF] Yuen | The magnitude and seasonality of aquatic insect subsidies to tropical stream riparia in Hong Kong[END_REF], but five times smaller for Chironomidae production than in a second order stream in Alabama (US) [START_REF] Reynolds | Chironomid production along a hyporheic gradient in contrasting stream types[END_REF].

The influence of water temperature on benthic invertebrate production and emergence [START_REF] Huryn | Life History and Production of Stream Insects[END_REF] was confirmed in our study by its positive correlation with Ephemeroptera and Trichoptera emergence, but not that of Chironomidae. Since Chironomidae species tolerate variations in water temperature better [START_REF] Sponseller | Relationships between land use, spatial scale and stream macroinvertebrate communities[END_REF], the temperature range observed in the study may not have been wide enough to influence their biomass. This study confirmed that several agriculture-related variables can regulate the biomass of emerging stream insects [START_REF] Allan | The influence of catchment land use on stream integrity across multiple spatial scales[END_REF]. Indeed, proportion of agricultural land in catchment is often associated with a decrease of emerging biomass of more sensitive taxa such as Ephemeroptera and Trichoptera (Piscart et al. 2009[START_REF] Piscart | Leaf litter recycling in benthic and hyporheic layers in agricultural streams with different types of land use[END_REF] and the enhancement of tolerant taxa such as Chironomidae [START_REF] Lemly | Modification of benthic insect communities in polluted streams: combined effects of sedimentation and nutrient enrichment[END_REF]. [START_REF] Sponseller | Relationships between land use, spatial scale and stream macroinvertebrate communities[END_REF] also reported that such pattern is correlated with a higher algal biomass in agricultural catchments of second to third order streams. At site scale in our study, the alteration of channel morphology by intensive agriculture practices, approximated by stream width and embankment score, was detrimental for Ephemeroptera and Trichoptera, while favoring Chironomidae biomass as reported in previous studies [START_REF] Quinn | Characterisation and classification of benthic invertebrate communities in 88 New Zealand rivers in relation to environmental factors[END_REF]Hickey 1990, Kennedy and[START_REF] Kennedy | River channelization reduces nutrient flow and macroinvertebrate diversity at the aquatic terrestrial transition zone[END_REF]. Accumulation of silt is commonly reported in such altered streams [START_REF] Wagenhoff | Subsidy-stress and multiplestressor effects along gradients of deposited fine sediment and dissolved nutrients in a regional set of streams and rivers: Sediment and nutrients in streams[END_REF]. Filter-feeding taxa (i.e.

Chironomidae and some Trichoptera) usually take advantage of fine sediment on the river bed, while shredders and scrapers (i.e. most Ephemeroptera and some Trichoptera) are hindered by these less favorable trophic conditions [START_REF] Quinn | Characterisation and classification of benthic invertebrate communities in 88 New Zealand rivers in relation to environmental factors[END_REF]Hickey 1990, Compin and[START_REF] Compin | Spatial patterns of macroinvertebrate functional feeding groups in streams in relation to physical variables and land-cover in Southwestern France[END_REF]. Conversely, we observed that biomass of emerging Ephemeroptera and Trichoptera increased with increasing water conductivity. High conductivity is detrimental to benthic macroinvertebrate biomass and diversity (Johnson et al. 2013b), but high nutrient loads promote biofilm growth on stream substrate. Since biofilm is a major food source for Ephemeroptera and some Trichoptera, their biomass may have increased at sites with both high nutrient load and conductivity (Piscart et al. 2009). The positive correlation between channel openness and emergence further corroborates this hypothesis. Incident light enhances primary production in streams, favoring taxa that feed on algae and diatoms [START_REF] Dance | Some effects of agricultural land use on stream insect communities[END_REF] in each taxonomic groups (e.g. Baetis or Serratella mayflies, Glossomatid caddisflies and many chironomids).

While Trichoptera emerged from early spring to summer, Ephemeroptera emerged only in spring. When sorting samples in the lab, we observed at least 15 morphospecies of Trichoptera (not taxonomically identified), suggesting that it had higher diversity than Ephemeroptera. Since each species has its own timing of emergence, the longer emergence period for Trichoptera could have resulted simply from higher specific diversity.

Chironomidae were caught almost continuously except in the coldest period in December, indicating that they emerged throughout the year, and were dominant taxa of emerging insects in fall, winter and early spring. These temporal patterns suggest that stream insects can subsidize surrounding agroecosystems for almost the entire year.

For accurate estimation of aquatic insect deposition on land, it is necessary to take into account the flux of winged aquatic insects returning to streams. Notably, females of several taxa lay their eggs at the surface or in water and died by drowning afterward [START_REF] Baxter | Macroinvertebrate Drift, Adult Insect Emergence and Oviposition[END_REF]. Nonetheless, several studies estimated that terrestrial mortality of female aquatic insects before oviposition is very high and exceeding 95% on average (reviewed by [START_REF] Huryn | Life History and Production of Stream Insects[END_REF]. This was reported for caddisfly, mortality of adult females reaching up to 80% for Limnephilidae (Tricoptera, Enders and Wagner 1996) and 98.8% for Baetidae (Ephemeroptera, Werneke and Zick 1992), two families commonly found in agricultural streams [START_REF] Friberg | Stream macroinvertebrate occurrence along gradients in organic pollution and eutrophication: Macroinvertebrate response to organic pollution and eutrophication[END_REF]). These observations confirm other investigations on the low percentage of aquatic insects that could ultimately return to the stream (1-3%, [START_REF] Jackson | Secondary Production, Emergence, and Export of Aquatic Insects of a Sonoran Desert Stream[END_REF]Fisher 1986, Gray 1989).

Based on the range of invertebrate production in our study, a stream section 100 m long × 6 m wide could produce 0.9-4.5 kg DM y -1 of emerging insects. Assuming that most winged aquatic insects disperse up to 50 m from the stream on each bank, aquatic insect deposition on land could reach 0.9-4.5 kg DM ha -1 y -1 . Using conversion factors of Evans-White et al.

( 2005), this could result in accumulation of 0.5-2.3, 0.1-0.5 and 0.005-0.03 kg ha -1 y -1 of carbon (C), nitrogen (N) and P on land, respectively, mainly in spring and summer. These subsidies are similar to reciprocal fluxes reported for other ecosystems (see [START_REF] Landry | Could the lateral transfer of nutrients by outbreaking insects lead to consequential landscape-scale effects?[END_REF], notably the subsidies that carnivorous birds provide to freshwater or that deer provide from fields to forests. In addition, terrestrial net secondary production can reach 1.9-4.0 kg C ha -1 y -1 in temperate grasslands [START_REF] Gratton | Flux of aquatic insect productivity to land: comparison of lentic and lotic ecosystems[END_REF], i.e. approximately twice that expected along a stream 6 m wide. No estimate of secondary production exists for temperate cropland, but some studies highlighted that terrestrial insect communities are less diverse and abundant in agricultural areas [START_REF] Benton | Linking agricultural practice to insect and bird populations: a historical study over three decades: Farming, insect and bird populations[END_REF][START_REF] Batáry | Responses of plant, insect and spider biodiversity to local and landscape scale management intensity in cereal crops and grasslands[END_REF].

Taken together, these aspects suggest that aquatic subsidies provided by winged aquatic insects could play a significant role in agroecosystem functioning near streams. Chironomidae emerge throughout the year and can easily disperse over land [START_REF] Muehlbauer | How wide is a stream? Spatial extent of the potential "stream signature" in terrestrial food webs using meta-analysis[END_REF], potentially providing a valuable source of nutrients (C, N, P) to receiving agroecosystems at critical times of the year. Recent studies highlighted the importance of aquatic insects in providing essential fatty acids to terrestrial consumers [START_REF] Martin-Creuzburg | Cross-ecosystem fluxes: Export of polyunsaturated fatty acids from aquatic to terrestrial ecosystems via emerging insects[END_REF][START_REF] Popova | Export of aquatic productivity, including highly unsaturated fatty acids, to terrestrial ecosystems via Odonata[END_REF]. Many terrestrial predators (e.g. birds, [START_REF] Nakano | Reciprocal subsidies: Dynamic interdependence between terrestrial and aquatic food webs[END_REF]spiders and carabids, Stenroth et al., 2015) likely feed on aquatic prey and could later shift to terrestrial prey, including crop pests, as previously observed between fields and uncultivated areas [START_REF] Rand | Spillover edge effects: the dispersal of agriculturally subsidized insect natural enemies into adjacent natural habitats[END_REF].

Conclusion

Multiple influences on aquatic insect emergence were identified at the local and watershed scales, and some were directly related to agricultural practices. Our results suggest that emergence and dispersal of aquatic insects could substantially influence terrestrial ecosystem functioning in landscapes with a dense waterway network. More studies are required on the dispersal of aquatic winged insects and their deposition in terrestrial ecosystems to better understand and quantify the ecological processes involved. This will help to assess effects of the aquatic subsidies that streams provide on ecosystem services supplied to agriculture, such as fertilization, biological control and pollination.

Figure 1 .

 1 Figure 1. Emergence of aquatic insects dry mass (DM) all sites combined. Left panel shows mean (± 1 standard deviation SD) daily values for

Figure 2 .

 2 Figure 2. Standardized effect of environmental variables (mean effect size ± 90% confidence interval) at watershed and site scales on emerging

Table 1 .

 1 Comparison of temporal and spatial fitted mix-models for each taxon. Summarizing statistics are: Akaike Information Criterion score corrected for small sample size (AIC c ), the variance explained by fixed parameters (marginal R²) and both fixed and random parameters (conditional R²) of the model. Null models are intercept-only models, which have no fixed parameter and the same random structure as fitted models. They are used as bases of comparison against fitted models for AICc and R². For a given taxon and scale (i.e. temporal or spatial), lower AICc indicates a better fit of the model to the data, higher marginal/conditional R² indicate greater goodness-of-fit (the amount of variance explained).

			Temporal model	Spatial models
	Taxon	Statistic	Null	Sampling campaign	Null	Watershed	Site
	Chironomidae AIC c	305.1	298.6	301.8	294.9	293.5
		Marginal/conditional R 2 0.0/22.0	10.9/34.9	0.0/32.7	21.4/32.5	15.9/41.9
	Trichoptera	AIC c	323.6	285.4	294.3	292.2	272.0
		Marginal/conditional R 2 0.0/13.3	34.1/52.4	0.0/52.5	11.6/50.5	23.9/57.2
	Ephemeroptera AIC c	225.3	196.1	204.2	195.7	201.7
		Marginal/conditional R 2 0.0/11.4	33.9/52.1	0.0/51.8	18.8/50.9	11.9/43.5

Table 2 .

 2 Mean annual dry mass (with range of values in brackets when available) of emerging aquatic insect or secondary production. Values in italics correspond to estimates based on the[START_REF] Poepperl | Benthic secondary production and biomass of insects emerging from a northern German temperate stream[END_REF] Emergence:Production ratio. EPT = Ephemeroptera, Plecoptera and Trichoptera.

	References	Location	Stream order	Landscape	Annual	Annual secondary	Taxa
			or width		emergence g m -2	production g m -2
	Jackson & Fisher 1986	Arizona, USA	3-4 m	Desert	13.8	58.3	Chironomidae
					4.4	55.5	Ephemeroptera
					2.9	3.6	Trichoptera
	Berg & Hellenthal 1991 Indiana, USA	third order	Agriculture/Forest 5.4	29.7	Chironomidae
	Sallenave et Day, 1991 Ontario, Canada	2-4 m	Agriculture/Forest 2.9 [1.1-4.7]	15.6 [5.8-25.4]	Trichoptera
	Poepperl 2000	Schleswig-Holstein, Germany 6 m	Agriculture	1.1 [0.6-1.3]	5.3 [3-6.1]	Diptera
					0.1 [0.01-0.1] 0.6 [<0.1-0.9]	Ephemeroptera
					0.5 [0.3-0.6]	1.9 [1-2.1]	Trichoptera
	Shieh et al. 2003	Colorado, USA	fifth order	Agriculture	4.2 [3.3-5]	23.2 [17.9-27.2]	Chironomidae
					0.4 [0-0.7]	2.4 [0.02-3.7]	Ephemeroptera
					1.7 [0.4-2.2]	9.5 [2.3-12]	Trichoptera
	Gücker et al. 2011	Berlin, Germany	3-4 m	Agriculture	-	3.9 [0.4-70.9]	EPT + Chironomidae
							+ other taxa
	Reynolds & Benke 2012 Alabama, USA	second order	Forest	5.3 [1.4-12.5] 29.2 [7.8 -68.3]	Chironomidae
	Johnson et al. 2013	Kentucky, USA	6 m	Agriculture/Forest -	21.8	EPT + Chironomidae
							+ other taxa
			6 m	Agriculture/Forest 1.4	7.5	EPT
	Yuen & Dudgeon 2016	Hong Kong, China	third or fourth order Forest	0.9 [0.7-1.2]	4.9 [3.8-6.6]	EPT + Chironomidae
							+ other taxa
	This study	Brittany, France	third order	Agriculture	3.8 [1.4-7.4]	21 [7.9-40.2]	EPT + Chironomidae
					1.1 [0.2-2.2]	6.1 [1.3-11.8]	Chironomidae
					0.7 [0.1-1.6]	3.7 [0.4-7.6]	Ephemeroptera
					2.1 [0.7-4.9]	11.2 [2.9-24.8]	Trichoptera

Table 1 .

 1 Comparison of temporal and spatial fitted mix-models for each taxon. Summarizing statistics are: Akaike Information Criterion score corrected for small sample size (AIC c ), the variance explained by fixed parameters (marginal R²) and both fixed and random parameters (conditional R²) of the model. Null models are intercept-only models, which have no fixed parameter and the same random structure as fitted models. They are used as bases of comparison against fitted models for AICc and R². For a given taxon and scale (i.e. temporal or spatial), lower AICc indicates a better fit of the model to the data, higher marginal/conditional R² indicate greater goodness-of-fit (the amount of variance explained).

			Temporal model	Spatial models
	Taxon	Statistic	Null	Sampling campaign	Null	Watershed	Site
	Chironomidae AIC c	305.1	298.6	301.8	294.9	293.5
		Marginal/conditional R 2 0.0/22.0	10.9/34.9	0.0/32.7	21.4/32.5	15.9/41.9
	Trichoptera	AIC c	323.6	285.4	294.3	292.2	272.0
		Marginal/conditional R 2 0.0/13.3	34.1/52.4	0.0/52.5	11.6/50.5	23.9/57.2
	Ephemeroptera AIC c	225.3	196.1	204.2	195.7	201.7
		Marginal/conditional R 2 0.0/11.4	33.9/52.1	0.0/51.8	18.8/50.9	11.9/43.5

Table Click here to download Table: Raitif-et-al-v2.0_Tables.docx

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References

Acknowledgement

This work received financial support from the Metaprogramm EcoServ of the French National Institute for Agronomical Research (INRA) to JM Roussel, and from the French Ministry for Reseach (PhD grant 2015-2018) to J Raitif. We thank two anonymous reviewers for their constructive comments on the first version of the manuscript.

Location Stream order or width

Landscape

Annual emergence g m -2

Annual secondary production g m -2


PhD Guido Pennings
email: guido.pennings@ugent.

MD Célia Ravel
email: celia.ravel@chu-rennes.fr

MD Jean-Maxime Girard
email: jeanmaxime.girard@chu-rennes.fr

MD Mathilde Domin-Bernhard
email: mathilde.domin.bernhard@chu-rennes.fr

PhD Veerle Provoost
email: veerle.provoost@ugent.be

Attitude towards reciprocity as a motive for oocyte donation

Keywords: altruism, anonymity, oocyte donation, reciprocity, sperm donation

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Many European countries are struggling to recruit a sufficient number of oocyte donors. In some countries, media campaigns are started in order to increase awareness. However, the shortage of gamete donors, and oocyte donors in particular, is not going down. On the contrary, due to the increase of recipients (mainly older women) the gap between demand and supply becomes larger. A frequently suggested solution is payment. However, within Europe, payment for body material is forbidden. Moreover, also ethical objections can be raised against commercial transactions of body material. We believe that within accepted ethical limits, new recruitment systems can be tried. One such system is built on the basic moral rule of reciprocity: people who voluntarily accept to benefit from a system should make a contribution to that system according to their capacities. Transferred to the context of gamete donation, this rule implies that persons who received gametes should, if possible, also donate gametes. This can be done by setting up a system in which the partner of the infertile person donates gametes. This system, also called mirror gamete donation, can be organized in different ways and has been applied in clinics in Bologna (Italy) and Eindhoven (The Netherlands) with great success [START_REF] Ferraretti | Semen donor recruitment in an oocyte donation programme[END_REF][START_REF] Pennings | Mirror gametes donation[END_REF]. The Centre Hospitalier Universitaire of Rennes (France) applies a similar system. In this centre, the possibility to donate eggs is discussed systematically with couples who come back for a subsequent child with donor sperm. If they decide to donate, their waiting period is reduced with about 6 months, which is approximately half of the normal waiting time. No other incentives are offered.

At the time of the research project, the French law only allowed people who already had a child to become donors. The law has changed in January 2016 also allowing persons without

A C C E P T E D M A N U S C R I P T
children to donate. Moreover, oocyte donors can now also preserve a number of eggs for themselves if they do not have a child already [START_REF] France | 3 août 2010 modifiant l'arrêté du 11 avril 2008 relatif aux règles de bonnes pratiques cliniques et biologiques d'assistance médicale à la procréation[END_REF].

In France, the operating principle of gamete donation is solidarity. The initial concept was based on the idea that a couple that already has a child goes to the French national gamete biobank (CECOS: Centre d'études et de conservation des oeufs et du sperme humains) to help another couple. The method of recruitment is mostly relational as sterile couples sensitize their friends or family. By recruiting a donor, they allow faster treatment of couples registered on the waiting list before them [START_REF] Ravel | Genetics Commission of the French Federation of CECOS. Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: large-scale survey of over 10,000 sperm donor karyotypes[END_REF].

The reciprocity system met with fierce opposition from different groups, amongst which professionals who considered this a violation of the altruism principle that should underlie the donation [5]. Although we believe that the proposal is perfectly acceptable on ethical grounds, we know nothing about how patients think about this proposal. The present study wanted to find out to what extend reciprocity and fairness play a role in the decision making of patients who themselves benefitted from sperm donation in the past.

Material and methods

This is a prospective monocentric study in the CECOS of the Centre Hospitalier Universitaire (CHU) of Rennes (France). The questionnaire was given during the consultation to couples who came back for a subsequent child with donor sperm. Couples in which the woman was known to be unsuitable as a donor (because of age or medical or genetic contra-indications) were excluded. From November 2014 to December 2015, 30 couples were invited to participate in the study. Questionnaires were received from 26 women and 27 male partners resulting in a response rate of 87%. The study was stopped before the change of the law because the altered conditions would make it difficult to interpret and compare the data.

For the opinion statements, responses were combined into three categories (totally agree/agree, neutral, disagree/totally disagree). Analysis was conducted using Fisher's Exact (Cytel Studio StatXact-8) to compare the distribution of categorical variables and the independent t-test (Statistical Package of the Social Sciences, SPSS version 23) to compare the mean age between two categories of a particular attitude (totally agree and agree versus neutral, disagree and totally disagree) or reason to participate in the reciprocity system (very important and important versus neutral, unimportant and very unimportant).

The study was approved by the local Ethics Committee of the CHU of Rennes (advice 14-67).

A C C E P T E D

M A N U S C R I P T

Results

Demographic characteristics

The respondents had a mean age of 33 years and were highly educated (close to 60% had a university degree). Most had a full or part-time time job and had one child. The majority knew people whose children were donor conceived but only a few people knew other women who had donated eggs (Table 1).

Reciprocity

About half of the respondents (49%) felt that they should give something back (Table 2). About 40% believed that everyone should contribute to the system of gamete donation. No significant differences between men and women were found regarding the items related to reciprocity. Respondents who felt that they should give something back more often thought that everyone should contribute to the system compared to those who did not hold this feeling (14/25, 56.0% versus 7/27, 25.9; p=0.047). Furthermore, when compared to others, a considerably lower proportion of respondents who had this feeling of a duty to give something back thought that they would often think of the woman who would receive their eggs (2/26, 7.7% versus 10/27, 37.0%; p=0.019) or of the child who would result from their eggs (0/26, 0.0% versus 9/27, 33.3%; p=0.002). They also more often stated that their belief in the reciprocity system was a motivation to come to this centre (21/25, 84.0% versus 10/27, 37.0%; p<0.001).

If they would step into the reciprocity system, the overwhelming majority would do so to help others (94%), to give something back (60%), to obtain the sperm they needed (28%) and/or to get faster treatment (24%). (Table 3) An overwhelming majority (83%) disagreed with the statement that they would not donate if they would not get sperm in return and three out of four (75%) disagreed with the statement that they would not donate if they would not receive faster treatment. Only two men would rather wait longer than have their partner donate. No women shared this view. The majority (two thirds) of the respondents did not perceive their potential donation as an act that was mainly meant to benefit the male partner. There were no statistically significant differences in reasons to participate in the reciprocity system according to level of education or professional activity of the respondents.

Relationship with recipient and offspring

Less than one in four would think often about the recipient of their (partners') eggs. About 17% would think often about the child born from their (partners') donation. A significant

A C C E P T E D M A N U S C R I P T
difference exists between men and women in this regard: significantly more women would think about the recipient (42 vs. 37%) and about the offspring (30.9 vs. 3.7%). About one in four would like to know whether a child was born from their donation and one woman would like to know the child. Many more women than men were interested to know whether a child was born due to the donation (42.3 vs. 7.7%, P=0.009).

Anonymity

The majority of the patients (69%) saw anonymity as a necessary condition for them to donate. Two persons (4%) believed that the child born from their eggs should be able to know them. The respondents with a college or university degree more often considered anonymity as a necessary condition to donate compared to the group with a lower educational level (24/30, 80.0% versus 10/50, 50.0%; p=0.034). Respondents who felt that they should give something back less often considered anonymity a necessary condition to donate (14/26,53.8% versus 22/26, 84.6%; p=0.034).

Beliefs about the donation

About 36% considered the donation as an extra burden on top of their own treatment and more than 40% saw it as a big effort. Patients who held a college or university degree significantly more often thought that donating eggs was an extra burden compared to respondents who held a lower degree (19/21, 90.5% versus 15/30, 50.0%; p=0.003). About one in five (19%) thought that the donation would reduce their chances of getting pregnant and two women (4%) thought that donating eggs would have negative effects on their health.

Disclosure and approval of others

Patients would generally be open to their social environment about their donation. Five respondents (10%) would not tell anyone that they had used donor sperm and about three (6%) would not tell anyone that they/their partner had donated eggs. The majority (69%) believed that their friends and family would approve of their donation if they would find out.

Patients who were unemployed significantly less often thought that, if told, their friends and family would approve of them donating eggs (2/7, 28.6% versus 34/45, 75.6; p= 0.023).

Respondents who would not tell anyone that they or their partner donated eggs were significantly older (mean of 38.33 years) than the others (mean of 33.09 years; p=0.032).

Discussion

A C C E P T E D M A N U S C R I P T
Within the field of gamete donation, and by extension donation of body material, one focusses almost solely on altruism as the primary value. However, other moral considerations, such as fairness and reciprocity, may also constitute valuable principles to found the transaction [START_REF] Pennings | The central role of altruism in the recruitment of gamete donors[END_REF].

Our data show that about half of the patients held a personal moral conviction of reciprocity and 40% believed that there is a general obligation for patients to contribute to the system they benefitted from. The statements about the reciprocity system indicate that although people get a reduction in waiting time in return for their (possible) donation, the majority would not donate in order to receive this reduction. They seemed to consider their part mainly in relation to the sperm they already received or would receive. This may explain why the incentive was not decisive. However, one should be cautious since it is possible that another or stronger incentive would have a larger impact on the decision. One such adaptation could be to reserve a number of eggs for immediate fertilization and replacement so that the donor would benefit from a higher success rate (IVF compared to IUI) and a complete abolition of the waiting time. Moreover, although the majority did not donate with the reduction in mind, about one in four (28%) indicated that their reason to participate in the reciprocity scheme was to obtain the sperm they needed and 24% to obtain treatment sooner. The low importance attributed to the incentive can be explained by the fact that only five persons (10%) were concerned about getting sperm in time. With a mean age of 33 years, they may have thought that they still had some time left before their chances would really go down and also because they already had one child.

The patients showed a high degree of openness both about using donor sperm and donating donor eggs. There are a number of studies about telling the child about the donor conception but there are no systematic studies about telling others. The studies are also very difficult to compare since in many countries the recipient population mainly consists of single women and lesbian couples. Since these persons are excluded from donor insemination by law in France, we looked for studies with data on heterosexual couples exclusively or separately. In general, French couples seem to be open about the donor conception towards their child [START_REF] Jouannet | La majorité des couples procréant par don de sperme envisagent d'informer l'enfant de son mode de conception, mais la plupart souhaitent le maintien de l'anonymat du donneur[END_REF]. It is important to know that the counselors in the CECOS centres council directively for parents to be open. A recent study on disclosure to the offspring in French couples who used donor sperm also contained information on telling others [8]. 85% of those who responded had told other people (mainly family and friends) about the use of donor sperm. That leaves 15% who had not told others but this percentage is likely higher since there was a response rate of 65% Donor anonymity is imposed by law since 1994 in France. In a 2006 study among a large sample of parents with a child from donor sperm (a sample very similar to ours), more than 90% was in agreement with donor anonymity and less than 10% wanted to change the law on this point [START_REF] Jouannet | La majorité des couples procréant par don de sperme envisagent d'informer l'enfant de son mode de conception, mais la plupart souhaitent le maintien de l'anonymat du donneur[END_REF]. Also in another study on French parents, 96% of the respondents agreed with the 'anonymous and free' rule of the current system [9]. The findings of our study confirm the wish of parents to maintain donor anonymity.

It is interesting to note that the only three questions on which the answers were significantly different between men and women concerned the results of the donation. Women would think more often about the potential recipient and the child and they more often wanted to know whether children were born from their donation. These findings are consistent with data from oocyte donors elsewhere [10]. However, it is very difficult to compare data from different studies since both the type of donor (known, commercial, identifiable etc.) and the legal setting vary. For instance, the majority of volunteer donors (86%) in a study with identifiable donors wanted to know if their donation had succeeded and 49% would think about the child resulting from their donation [11]. The fact that these donors were identifiable, contrary to our sample, may have affected their attitudes. Men seem far less implicated and seem to distance themselves from the potential gift of their partner. This attitude may express their ideas about the importance of the genetic link: the contribution of their partner is closed (finished business) after the donation. Not wanting to know about the outcome and not thinking about child and recipient means that no relationship is established. The men probably expects the sperm donor to adopt the same attitude towards their gift. Lowering the importance of the genetic link indirectly strengthens their position as social parents.

Limitations of the study. This is a small sample of patients in a specific setting. It is not possible to extrapolate these findings to other oocyte donors since the elements of the context (previous child from sperm donation, waiting time …) are crucial. Moreover, we should be aware of the possibility of social desirability in answering the questions. Regrettably the guarantee of anonymity did prevent us from finding out which patients eventually also decided to donate. However, we do know that in the total group of egg donors in this centre about 45% are recruited within the reciprocity system [12]. As such, it is an important source of extra donors.

A C C E P T E D

M A N U S C R I P T Conclusion
The data indicate that 'giving something back' is a moral norm for about half of the patients who benefitted from donor sperm in the past. This norm may be an important element in the decision to donate. The data also indicate that a reduction in waiting time is not considered as important by the majority of the patients. Two possibilities exist: either the incentive was too weak (not attractive enough) or patients wanted to give something in return for what they had received. In the latter case, they do not give in order to receive an advantage and increasing the advantage would not make much difference.

  and people who intend to keep the treatment to themselves are more likely to refuse participation in a study, as the authors themselves remarked.

  Figure Caption

a

  Percentage of respondents agreeing or totally agreeing. b Data missing for one case except for the following two statements: 'I believe that donating eggs will have negative effects on my/her health' (data missing in two cases); 'I am very concerned that we will not get sperm in time' (data missing in 3 cases) *p ≤ 0.05; **p ≤ 0.01; ***p ≤0.001.

Table 1 : Demographic characteristics (N=53) a

 1

	n	%

Table 2 : Attitudes towards gamete donation and the reciprocity system (N=53)

 2

		All respondents				Gender
		(totally)	neutral	(totally)	Woman	Man
		agree			disagree
		n	%	n	%	n	%	% a	% a
	Reciprocity
	I prefer to wait longer than (let my	2	3.8	17 32.1	34	64.2	0.0	7.4
	partner) donate.
	I would mainly donate to help my	8	15.4	9	17.3	35	67.3	12.0	18.5
	partner/I believe that my partner would
	mainly donate to help me. b
	I would not (let my partner) donate if we	3	5.8	6	11.5	43	82.7	7.7	3.8
	would not receive other gametes in
	return. b
	I/my partner would not donate if we	5	9.6	8	15.4	39	75.0	11.5	7.7
	would not receive treatment sooner. b
	I feel that we should give something	26 49.1	6	11.3	21	39.6	42.3	55.6
	back.
	I believe that everyone should contribute	21 40.4	17 32.7	14	26.9	44.0	37.0
	to the system. b
	Effort
	Donating eggs is an extra burden on top	19 35.8	7	13.2	27	50.9	46.2	25.9
	of our own treatment.
	Donating eggs is a big effort for me/her.	23 43.4	9	17.0	21	39.6	34.6	51.9
	Feelings and beliefs
	I am very concerned that we will not get	5	10.0	13 26.0	32	64.0	12.5	7.7
	sperm in time. b
	I fear that donating eggs reduces our	10 19.2	7	13.5	35	67.3	26.9	11.5
	own changes of getting pregnant. b
	I believe that donating eggs will have	2	3.9	5	9.8	44	86.3	8.0	0.0
	negative effects on my/her health. b
	Beliefs about result of the donation
	I would often think of the woman who	12 22.6	17 32.1	24	45.3	42.3***	3.7
	would receive my/her eggs.
	I would often think of the child who may	9	17.0	12 22.6	32	60.4	30.8*	3.7
	result from my/her eggs.
	I would like to know whether a child was	13 25.0	8	15.4	31	59.6	42.3**	7.7
	born from my/her donation. b
	Disclosure and approval of others
	I will not tell anyone that we used donor	5	9.6	8	15.4	39	75.0	7.7	11.5
	sperm. b
	I will not tell anyone that I/my partner	3	5.7	8	15.1	42	79.2	3.8	7.4
	donated eggs.
	If told, my friends and family would	36 69.2	10 19.2	6	11.5	65.4	73.1
	approve of me/my partner donating
	eggs. b
	Anonymity
	Anonymity is a necessary condition for us	36 69.2	9	17.3	7	13.5	65.4	73.1
	to donate. b
	I think that the child who results from	2	3.8	11 21.2	39	75.0	3.8	3.8
	my/her donation should be able to know
	me/her. b
	I would like to know the child who results	1	1.9	7	13.5	44	84.6	3.8	0.0
	from my/her donation. b

Table 3 . Reasons to participate in the reciprocity system (N=53)

 3 percentage of respondents finding this reason important or very important. b Data missing for one case except for the reason: 'To obtain the sperm I need' (data missing in 3 cases)

		All respondents					Gender
		(Very)		neutral		(Very)		Woman	Man
		unimportant			important
		n	%	n	%	n	%	% a	% a
	To obtain faster treatment	21	39.6	19	35.8	13	24.5	34.6	14.8
	To help others b	2	3.8	1	1.9	49	94.2	92.3	96.2
	To give something back b	15	28.8	6	11.5	31	59.6	61.5	57.7
	To obtain the sperm I need b	30	60.0	6	12.0	14	28.0	36.0	20.0

a