Emma Jeavons

Ondine Chevrie

Cécile Le Lann

David Renault

Maeva Floch

Thomas Bourgeois

Remi Bodiguel

Thierry Fontaine-Breton

Joan Van Baaren

Maëva Floch

Rémi Bodiguel

Exploitative competition for floral resources reduces sugar intake but differently impacts the foraging behaviour of two non-bee flower visitors

Keywords: Aphid parasitoid, Buckwheat, Bumblebee, Hoverfly, Natural enemy, Resource discrimination

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

Introduction

In natural environments, the spatio-temporal availability of trophic resources varies in terms of quality, quantity and distribution [START_REF] Possingham | The distribution and abundance of resources encountered by a forager[END_REF]. To maintain optimal fitness, organisms have developed a range of responses, including adjustments of their foraging strategy to provide the highest benefit (in terms of reproduction or energy intake) at the smallest cost (energy loss or acquisition risk) (the Optimal Foraging Theory [START_REF] Charnov | Optimal foraging, the marginal value theorem[END_REF][START_REF] Pyke | Optimal foraging: A selective review of theory and tests[END_REF]). The plastic nature of this nutritional strategy has been demonstrated in a range of organisms and is adjusted in response to abiotic (e.g. temperature) or biotic (e.g. the presence of predators or competitors) constraints (e.g. [START_REF] Reader | The effects of predation risk from crab spiders on bee foraging behavior[END_REF]).

Ecological interactions among organisms sharing common trophic resources may alter their subsequent fitness [START_REF] Tilman | Resource Competition and Community Structure[END_REF]. Often, the outcome of this interaction is deleterious to one or both competitors, as each can decrease the availability, distribution and quality of the resource to other foragers [START_REF] Tilman | Resource Competition and Community Structure[END_REF]. As such, this competition, known as exploitative competition, represents an important selection driver of the foraging strategies of insects, that triggers adaptations that reduce the level of inter-specific competition. For instance, reduced foraging time on depleted resources, spill-over on more rewarding resources, and diet breadth expansion, have been observed in honeybees (Apis mellifera) and bumblebees (Bombus spp.) [START_REF] Balfour | Exploitative competition alters bee foraging and flower choice[END_REF][START_REF] Fontaine | Generalist foraging of pollinators: diet expansion at high density[END_REF][START_REF] Walther-Hellwig | Increased density of honeybee colonies affects foraging bumblebees[END_REF].

Importantly, these responses require the perception and integration of environmental cues allowing the detection, and thus avoidance, of patches of resources that are supposedly less rewarding [START_REF] Almohamad | Discrimination of parasitized aphids by a hoverfly predator: effects on larval performance, foraging, and oviposition behavior[END_REF][START_REF] Stout | Repellent scent-marking of flowers by a guild of foraging Bumblebees (Bombus spp.)[END_REF].

Competition for floral resources, in particular pollen and nectar, is likely to occur as they are exploited by a wide variety of organisms, mostly dominated by four insect orders: Hymenoptera, Lepidoptera, Diptera, and Coleoptera [START_REF] Brown | Competition between Hummingbirds and Insects for the Nectar of Two Species of Shrubs[END_REF]. This is especially true in agroecosystems, where floral resources are less abundant and less diverse than in natural ecosystems [START_REF] Fried | Arable weed decline in Northern France: Crop edges as refugia for weed conservation?[END_REF][START_REF] Robinson | Post-war changes in arable farming and biodiversity in Great Britain[END_REF]. When already exploited, flowers can be, at least temporarily, less rewarding to other foragers due to diminished nectar and/or pollen resources. For instance, when visited by the insect community, buckwheat (Fagopyrum esculentum: Polygonaceae) flowers are empty after midday [START_REF] Lee | Nectar availability and parasitoid sugar feeding[END_REF]. These authors warn that this exploitative competition for buckwheat nectar resources may limit the ability of buckwheat strips to support flowervisiting natural enemies. In accordance with the Optimal Foraging Theory, some flower visitors have the ability to discriminate and avoid flowers that have been previously exploited by another organism [START_REF] Goulson | Foraging strategies of insects for gathering nectar and pollen, and implications for plant ecology and evolution[END_REF][START_REF] Goulson | Foraging bumblebees avoid flowers already visited by conspecifics or by other bumblebee species[END_REF], thus increasing their foraging efficiency [START_REF] Stout | Repellent scent-marking of flowers by a guild of foraging Bumblebees (Bombus spp.)[END_REF]. Such avoidance of the supposedly less rewarding resources seems to rely on the perception of cues indicating that flowers were recently depleted. These signals can be perceived visually (such as a modification of the flower colour [START_REF] Willmer | Bidirectional flower color and shape changes allow a second opportunity for pollination[END_REF]) or by olfaction, as for instance the perception of scent marks by bumblebees deposited by previous visitors [START_REF] Stout | Repellent scent-marking of flowers by a guild of foraging Bumblebees (Bombus spp.)[END_REF][START_REF] Wilms | Foraging scent marks of bumblebees: footprint cues rather than pheromone signals[END_REF]).

Surprisingly, there has been little investigation on the existence of behavioural avoidance of formerly exploited flowers in flower visitor insects other than bees (Apoidea: Hymenoptera).

Indeed, up to now, the honeybee Apis mellifera [START_REF] Reader | Inter-order interactions between flower-visiting insects: foraging bees avoid flowers previously visited by hoverflies[END_REF][START_REF] Stout | The use of conspecific and interspecific scent marks by foraging bumblebees and honeybees[END_REF], several bumblebee species [START_REF] Goulson | Foraging bumblebees avoid flowers already visited by conspecifics or by other bumblebee species[END_REF][START_REF] Stout | The use of conspecific and interspecific scent marks by foraging bumblebees and honeybees[END_REF], and a few solitary bee species [START_REF] Yokoi | Recognition of scent marks in solitary bees to avoid previously visited flowers[END_REF] are the only ones for which behavioural avoidance capacity has been explicitly tested. Most of those insects have the capacity to recognise cues emitted by both conspecific and heterospecifics [START_REF] Stout | The use of conspecific and interspecific scent marks by foraging bumblebees and honeybees[END_REF]. Honeybees also possess the ability to detect and avoid flowers formerly visited by insects from other orders, e.g. Syrphidae (Diptera) [START_REF] Reader | Inter-order interactions between flower-visiting insects: foraging bees avoid flowers previously visited by hoverflies[END_REF], thus suggesting that they can rely on general cues for detecting the most rewarding resources. Nevertheless, it is still unknown if this capacity is widespread, and thus exists in other flower-visiting insects [START_REF] Reader | Inter-order interactions between flower-visiting insects: foraging bees avoid flowers previously visited by hoverflies[END_REF], even though this information is crucial for understanding their population dynamics. Indeed, the amount and quality of pollen and nectar strongly drives reproduction and survival performance of many insect species [START_REF] Wäckers | Pick and mix: selecting flowering plants to meet the requirements of target biological control insects[END_REF]. This is particularly important for synovigenic insects for which the development of reproductive tissues mainly depends on the acquisition of resources by adults (Branquart and Hemptinne, 2000a;[START_REF] Godfray | Parasitoids: Behavioral and Evolutionary Ecology[END_REF]. For instance, adult hoverflies (Diptera: Syrphidae), which are common flower-visitors, allocate nitrogenous elements provided by pollen and nectar for the maturation of their reproductive system (Branquart and Hemptinne, 2000a) and the production of eggs [START_REF] Gilbert | Foraging ecology of hoverflies: morphology of the mouthparts in relation to feeding on nectar and pollen in some common urban species[END_REF]. Most flower-visiting insects are also highly dependent on carbohydrates such as sucrose, glucose and fructose, which constitute the main metabolites of nectar [START_REF] Nicolson | Nectar chemistry[END_REF], for survival and flight [START_REF] Azzouz | Effects of feeding frequency and sugar concentration on behavior and longevity of the adult aphid parasitoid: Aphidius ervi (Haliday) (Hymenoptera: Braconidae)[END_REF]Branquart and Hemptinne, 2000b;[START_REF] Gilbert | Foraging ecology of hoverflies: morphology of the mouthparts in relation to feeding on nectar and pollen in some common urban species[END_REF][START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF][START_REF] Wäckers | Pick and mix: selecting flowering plants to meet the requirements of target biological control insects[END_REF]. For some insects, such as some parasitoids and hoverflies, carbohydrates are also involved in egg production and maturation [START_REF] Araj | Comparing existing weeds and commonly used insectary plants as floral resources for a parasitoid[END_REF][START_REF] Leroy | An introduction device for the aphidophagous hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae)[END_REF][START_REF] Olson | Larval crowding and adult nutrition effects on longevity and fecundity of female Trichogramma nubilale Ertle & Davis (Hymenoptera: Trichogrammatidae)[END_REF]. In this context, GC-MS nutrimetabolomics, i.e. the description of the metabolic responses of organisms to dietary resources, represents a valuable tool for linking energetic allocations to nutritional foraging patterns of insects.

In this study, we aimed at understanding the physiological and behavioural consequences of exploitative competition for floral resources in two natural enemy species: a frequent flower visitor, the aphidophagous hoverfly Episyrphus balteatus (Diptera: Syrphidae) and a more occasional flower visitor, the aphid parasitoid Aphidius colemani (Hymenoptera: Braconidae) [START_REF] Charles | Fitness effects of food resources on the polyphagous aphid parasitoid, Aphidius colemani Viereck (Hymenoptera: Braconidae: Aphidiinae)[END_REF][START_REF] Gilbert | Foraging ecology of hoverflies: morphology of the mouthparts in relation to feeding on nectar and pollen in some common urban species[END_REF]. To meet their energetic requirements, adults of these two species feed on flower resources, which has been shown to increase females' fecundity, longevity and dispersal capacities [START_REF] Charles | Fitness effects of food resources on the polyphagous aphid parasitoid, Aphidius colemani Viereck (Hymenoptera: Braconidae: Aphidiinae)[END_REF][START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF][START_REF] Pinheiro | Effects of pollen, sugars and honeydew on lifespan and nutrient levels of Episyrphus balteatus[END_REF][START_REF] Van Rijn | Nectar accessibility determines fitness, flower choice and abundance of hoverflies that provide natural pest control[END_REF]. For both species, longevity without nectar intake is only one or two days [START_REF] Charles | Fitness effects of food resources on the polyphagous aphid parasitoid, Aphidius colemani Viereck (Hymenoptera: Braconidae: Aphidiinae)[END_REF][START_REF] Pinheiro | Effects of pollen, sugars and honeydew on lifespan and nutrient levels of Episyrphus balteatus[END_REF]. Two competitors were used: the same hoverfly species E. balteatus and the bumblebee Bombus terrestris (Hymenoptera: Apidae). To address if the recognition capacity varies among species and with the previous visiting species, we compared the effects of competition between conspecifics (E. balteatus -E. balteatus) and between heterospecifics from the same taxonomic order (A. colemani -B. terrestris) and from a different order (E. balteatus -B. terrestris and A. colemani -E. balteatus). Our specific expectations were that 1) individual insects fed on recently exploited flowers should be characterised by lower metabolite concentrations as compared with those fed on unexploited flowers; for instance, lower amounts of fructose and glucose sugars are expected due to the lower quantity of available nectar in exploited flowers. 2.a) If trophic resources are limiting when the flowers have been previously exploited by competitors, we expect secondary foragers to discriminate patch quality, and favour unexploited patches over exploited ones. 2.b) If the exploited patches are not completely avoided, we expect that foragers will spend less time foraging and feeding on patches that are less rewarding (i.e. the exploited ones). 3) The physiological consequences for the metabolic profiles and foraging patterns of the tested species might vary among the different species due to their different physiological needs and ecology, ability to recognise cues, and the previous competitor species.

Material and methods

Biological models

The foraging behaviour of two flower-visitor species, Episyrphus balteatus and Aphidius colemani (hereafter referred to as tested insects), on buckwheat flowers (Fagopyrum esculentum) was studied in response to previous exploitation of the flowers by two competitors, E. balteatus and Bombus terrestris (hereafter referred to as competitor insects).

Insect rearing:

Tested insects. Pupae of hoverflies and mummies of parasitoids were supplied by Katz Biotech® and Biobest France®, respectively. The insects were kept in a climatic chamber at 20±2 °C, 70±4% RH and a 16:8 h (Light: Dark) photoperiod until adult emergence. Pupae of hoverflies were individually stored on a layer of vermiculite in microcages (H=10.5cm, Ø=4.5cm) sealed with a net to allow a sufficient airflow. Mummies of parasitoids were individually stored in 1.5 mL safe-lock microtubes with caps pierced with two small holes. Only unmated female that were less than 24 hours-old were used in the experiments for both species. At this age, female hoverflies are sexually immature [START_REF] Geusen-Pfister | Studies on the biology and reproductive capacity of Episyrphus balteatus Deg. (Dipt., Syrphidae) under greenhouse conditions[END_REF], and we thus assumed that they would not be attracted by male pheromones, as reported for many Diptera species [START_REF] Katsoyannos | Male sex pheromone of Rhagoletis cerasi L. (Diptera, Tephritidae): Factors affecting release and response and its role in the mating behavior[END_REF][START_REF] Lima | Volatile substances from male Anastrepha fraterculus wied. (Diptera: Tephritidae): identification and behavioural activity[END_REF][START_REF] Robacker | Attraction of a laboratory strain of Anastrepha ludens (Diptera: Tephritidae) to the odor of fermented Chapote fruit and to pheromones in laboratory experiments[END_REF]. As most of the individuals emerged overnight, a cotton ball soaked with water was added at 8 AM in all microcages and tubes containing newly emerged individuals, providing them with a water supply prior to sucrose feeding.

Competitor insects. Adult hoverflies used as previous competitors were raised in a Plexiglas cage (L=50cm, l=50cm, H=100cm) kept at room temperature with natural light. They were provided with water, honey and plants of buckwheat (Fagopyrum esculentum: Polygonaceae, "Billy" cultivar), white mustard (Sinapis alba: Brassicaceae, "Rota" cultivar) and phacelia (Phacelia tanacetifolia: Hydrophyllaceae, "Stella" cultivar). Both males and females were used as competitors.

Adult bumblebees (B. terrestris) were provided by Biobest France® and kept in the greenhouse at 21±3 °C, 53±8% RH and a 16:8 h (Light: Dark) photoperiod. Bumblebees had access to the same three plant species as described above for hoverflies. The insects were supplied with water and supplementary pollen obtained from ground organic honeybee pollen loads.

Plants:

Following [START_REF] Leadbeater | Do inexperienced bumblebee foragers use scent marks as social information?[END_REF], natural flowers were used to maximise foraging behaviours as it has been previously reported that inexperienced insects require larger exploration periods before foraging when maintained on artificial flowers. All plants used for the experiments were 7-8 week old inflorescences of buckwheat, corresponding to the age at which flower and nectar production reaches its optimum [START_REF] Cawoy | Morphology of Nectaries and Biology of Nectar Production in the Distylous Species Fagopyrum esculentum[END_REF]. Buckwheat nectar is characterized by high sugar concentrations, which can represent up to 55% of the composition of nectar [START_REF] Cawoy | Morphology of Nectaries and Biology of Nectar Production in the Distylous Species Fagopyrum esculentum[END_REF]. This plant is commonly used in biological conservation programs [START_REF] Lavandero | Enhancing the effectiveness of the parasitoid Diadegma semiclausum (Helen): Movement after use of nectar in the field[END_REF][START_REF] Lee | Nectar availability and parasitoid sugar feeding[END_REF][START_REF] Quinn | Floral strips attract beneficial insects but do not enhance yield in Cucumber fields[END_REF] and is visited by a large number of insects for nectar and pollen feeding in agroecosystems [START_REF] Ambrosino | Relative frequencies of visits to selected insectary plants by predatory hoverflies (Diptera: Syrphidae), other beneficial insects, and herbivores[END_REF][START_REF] Hogg | Attractiveness of common insectary and harvestable floral resources to beneficial insects[END_REF][START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF][START_REF] Lee | Nectar availability and parasitoid sugar feeding[END_REF].

Buckwheat has also been found to be one of the plant species that best increases A. colemani and E. balteatus females' longevity, parasitism rate of A. colemani females, and duration of oviposition of E. balteatus individuals [START_REF] Jado | Floral resources to enhance the potential of the parasitoid Aphidius colemani for biological control of the aphid Myzus persicae[END_REF][START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF]. In a preliminary experiment aimed at describing buckwheat nectar production dynamics (Supplementary Information I.A and I.B), we found that nectar volume per flower is low (maximum of 0.16 µL), but that sucrose concentration is high (between 60 and 70% of sucrose in nectar mass). For our experiments, buckwheat plants were raised in a greenhouse at 21±3 °C, 53±8% RH and a 16h:8 Light: Dark photoperiod. They were placed under a net (L=200cm, l=100cm, H=180cm) to prevent any contact between plants and insects.

Preparation of exploited and unexploited flowers:

Inflorescences were freshly cut (less than 1 hour) before the experiments, and were checked to ensure that no aphids (which are prey for hoverfly larvae and hosts for parasitoids) were present on the plant. Parasitoid longevity was shown to be similar when individuals were fed with excised flowers as when fed with intact flowers [START_REF] Wade | Excised or intact inflorescences? Methodological effects on parasitoid wasp longevity[END_REF]. Exploited buckwheat inflorescences were obtained by placing them into Plexiglas cages (L=35cm, l=35cm, H=50cm) with either 2 bumblebee workers (patch visited by bumblebees) or 10 hoverflies (patch visited by hoverflies). Once all the flowers had been visited at least once, the individuals were left to forage on the flowers for an additional 15 minutes. To make sure hoverfly competitors fed on the inflorescences, they were previously left fasting for 17-23h.

Bumblebees did not need any fasting to quickly forage on the inflorescences. This design allowed us to obtain relevant experimental conditions, with flowers visited several times and possibly by different organisms, as observed in natura (e.g. [START_REF] Lee | Nectar availability and parasitoid sugar feeding[END_REF]. Exploited inflorescences were presented to tested insects in the choice and no-choice experiments in the 15 minutes following their exploitation by competitors. Unexploited flowers were kept for the same duration and in the same conditions without competitors. A preliminary experiment showed that intact buckwheat flowers only moderately replenish after nectar removal, with a maximum of 34% of the initial nectar volume being restored two hours after nectar removal (Supplementary Information I.C).

No-choice experiment: metabolite contents of secondary foragers:

To assess if flower exploitation by competitors subsequently limited resources available to a secondary forager, metabolic profiles of individuals fed on exploited or unexploited flowers were compared. To that aim, newly emerged females (hoverflies and parasitoids) were exposed to one of the four following experimental treatments: (i) water (Water), (ii) water and unexploited flowers (Unexploited), (iii) water and flowers exploited by bumblebees (Exploited B), or (iv) water and flowers exploited by hoverflies (Exploited H). Water was provided by a soaked cotton ball in all microcages and tubes. The floral resources consisted of an inflorescence of five flowers for each hoverfly, and a single flower for each parasitoid. All tested individuals were left feeding for 15 minutes after the beginning of the feeding behaviour was first observed. For each treatment, 16 to 22 hoverflies and 30 to 45 parasitoids were tested. Individuals were then immediately frozen in liquid nitrogen, and stored at -80 °C until processing. After having freeze-dried for over 48h, dry mass of the insects was measured with a Mettler Toledo XP2U precision balance with 0.1 μg resolution.

For GC-MS analyses, insect dry masses must be above 0.2 mg to ensure reliable detection and quantification of several primary metabolites. Hoverflies' dry mass ranged from 2.373 mg to 5.787 mg, and parasitoids' dry mass ranged from 0.0213 mg to 0.1258 mg. Therefore, hoverfly samples contained one individual, while parasitoids were pooled in groups of 2-5 individuals, resulting in 16-22 replicates for hoverflies and 10-13 replicates for pooled parasitoids per treatment. The samples were homogenized in 900μL [300μL] (respectively for hoverflies and

[parasitoids]) of methanol-chloroform solution (2:1), and a volume of 600μL [220μL] of ultrapure water was added to each sample. After centrifugation, a 120μL [220μL] aliquot of the upper aqueous phase, which contains polar metabolites, was transferred to new glass vials. The GC-MS procedure used was first described by [START_REF] Khodayari | Deciphering the metabolic changes associated with diapause syndrome and cold acclimation in the two-spotted spider mite Tetranychus urticae[END_REF], and modified by [START_REF] Thiébaut | Variation of thermal plasticity for functional traits between populations of an invasive aquatic plant from two climatic regions[END_REF]. Concentrations of each metabolite were divided by the dry mass of the individual tested (or the sum of the masses of pooled individuals), and are presented in nmol per mg of dry mass.

Choice experiment: Does previous flower exploitation impact patch choice, foraging and feeding behaviours of secondary foragers?

To determine the innate feeding preference of the secondary foragers for exploited and unexploited flower patches, a choice test was carried out with newly emerged insects. A preliminary sucrose feeding was carried out, as E. balteatus and A. colemani individuals are likely to die after 24 hours when they are not offered sugar intake [START_REF] Charles | Fitness effects of food resources on the polyphagous aphid parasitoid, Aphidius colemani Viereck (Hymenoptera: Braconidae: Aphidiinae)[END_REF][START_REF] Pinheiro | Effects of pollen, sugars and honeydew on lifespan and nutrient levels of Episyrphus balteatus[END_REF]. A cotton ball soaked with 0.5 mL of a 70% solution of sucrose was placed in each microcage or tube. Ten minutes after the individual had started feeding on the sucrose solution, the cotton ball was replaced by another one containing water only. Individuals that did not feed on the cotton ball with sugar were not used for the choice test. For both tested species (hoverfly and parasitoid), the feeding occurred at 5 PM on 1-23 h old individuals.

The choice test experiment was carried out in an air-conditioned room, 16-20 h after the preliminary sucrose feeding to stimulate the foraging behaviour of the tested insects (Fig. 1).

The two resource patches used during the choice test were an unexploited buckwheat inflorescence and an exploited one by either hoverflies or bumblebees. Each inflorescence contained five flowers (the other flowers were cut), and were selected to have a similar architecture. Both patches were placed under a Plexiglas cage (L=27cm, W=15cm, H=17cm) at a 10 cm distance from one another (Fig. 1). A 2 cm diameter hole was pierced at the centre of the cage, so that the tested individual (hoverfly or parasitoid) entered at an equal distance from both patches. The position of the two inflorescences was alternated from one test to the other to avoid an effect of the position of the patches on the insect's choice. The soil surface was covered with white paper changed between each test to prevent the presence of olfactory marks deposited by the individual previously tested. For the same reason, the Plexiglas cages used were washed with distilled water between each test and with ethanol at the end of each day. Individuals that did not forage within three minutes of their entrance into the Plexiglas cage were removed and tested again later. Each individual was given a maximum of three trials, after which they were discarded from the experiment.

The behaviour of each tested individual was observed for 10 minutes after it entered the cage (head out of tube) and was recorded with the sequenceR package [START_REF] Hervé | SequenceR : une interfce d'encodage de séquences comportementales[END_REF] of the R software. All observed behaviours are described in Table 2. At the end of the assay, individuals were frozen in liquid nitrogen and stored at -80 °C before being processed. After the samples were freeze-dried for 48 h, dry mass was measured with a Mettler-Toledo XP2U balance with 0.1μg resolution.

In total, 55 hoverfly females (N=29 in the hoverfly competitor trial and N=26 in the bumblebee competitor trial) and 40 parasitoid females (N=23 in the hoverfly competitor trial and N=17 in the bumblebee competitor trial) were tested.

Statistical analyses

All statistical analyses were conducted with the RStudio software (v.1.2.1335).

Metabolite contents of secondary foragers:

To identify the metabolites that contributed to the separation of the four experimental treatments, Linear Discriminant Analyses (LDA) were performed on the log-transformed concentrations of all quantified metabolites for each tested species (hoverfly and parasitoid) (lda function from the "MASS" package) [START_REF] Colinet | Metabolic effects of CO 2 anaesthesia in Drosophila melanogaster[END_REF]. The effect of the treatment on metabolite concentrations was tested for each metabolite family (Table 1), as well as fructose, glucose and trehalose. When the concentrations of the metabolites/metabolite families were close to a Normal distribution (amino acids for both species, organic acids for hoverflies and polyols for parasitoids), a linear model was fitted to the data (lm function). For all other metabolites/metabolite families, the response variable was log-transformed. For hoverflies, the testing day was added as a random factor (lmer function from the "lme4" package), but not for parasitoids due to the pooling of individuals. When the effect of the experimental treatment was significant, a Tukey post-hoc test was carried out to distinguish which of the four treatments differed from the others with the "multcomp" package (glht and cld functions) [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF].

Patch choice of the secondary forager:

The probability of first landing or feeding on an unexploited patch compared to an exploited patch was tested with an independent binomial test (function binom.test) for each tested species (hoverfly or parasitoid) and each previous competitor species (hoverflies or bumblebees). All observations were assumed to be independent and the null hypothesis was that individuals chose a patch at random (probability of 0.5 to choose the unexploited patch). As very few parasitoid individuals fed on the inflorescences (N=3 during the bumblebee competitor trial and N=7 during the hoverfly competitor trial), it was not possible to analyse feeding behaviours for parasitoids (neither first feeding patch choice nor feeding duration, see below). For hoverflies only, the number of visits per patch was analysed with GLMMs with a lognormal distribution (glmer function from the "lme4" package [START_REF] Bates | Fitting Linear Mixed-Effects Models using lme4[END_REF]). The patch quality (i.e. exploited by a competitor species or unexploited) and the side of the visited patch (right or left) were considered as fixed factors, while the tested individual dry mass was added as a covariate. Each patch visited (the unexploited and the exploited) was considered an observation, so the same tested individual could have been observed twice if both patches were visited during the choice test. Such a behaviour was recurrent for hoverflies, so individual identity was added as a random factor in the models. The number of visits per patch was not analysed for parasitoid individuals as among the 39 individuals observed, only 15, all treatments included, visited the same patch several times.

Foraging and feeding behaviours of the secondary forager:

The durations of foraging and feeding behaviours per patch were analysed with Cox proportional hazard models, using the coxph function from the "survival" package. Explanatory variables were the same as for the models testing the number of visits per patch. Similar to above, hoverfly identity was added as a random factor as one individual often visited both patches. For parasitoids, the only individual who visited both patches during the choice test was removed from the data for this analysis, so each observation corresponded to one individual, and therefore the models did not contain any random factors. Feeding duration was not analysed for parasitoids as too few individuals actually fed on the inflorescences (see above). The mean number of flower changes per patch visit was analysed for hoverflies and parasitoids with mixed generalized linear models (GLMMs, glmmPQL function from the "MASS" package) and GLMs following quasipoisson distributions (log link function), respectively.

All models were simplified by removing variables (except "patch quality" as it was the main tested variable) for which significance was above the significance threshold (0.05). A Fisher test (for LMs) and a Wald Chi-square test (for LMMs and GLMMs) were used assuming a type II sum of squares (Anova function from the "car" package). Final model validity was checked to meet model assumptions according to [START_REF] Crawley | Statistics: An introduction using R[END_REF].

Results

Metabolite content of secondary foragers

Nineteen metabolites were quantified from Episyrphus balteatus and Aphidius colemani (Table 1). Four additional metabolites were found from hoverflies only and fifteen from parasitoids only (Table 1). When quantified in a given species, the metabolite was detected in all individuals from the four treatments. Both LDAs performed on the metabolite contents of hoverflies and parasitoids revealed a separation according to the feeding treatment experienced by individuals on the two first axes (representing 90.7% and 93.3% of the variance for hoverflies and parasitoids respectively) (Fig. 2). In both species, sugars (in particular glucose, fructose and trehalose), many amino acids, and sorbitol highly contributed to separate tested individuals between treatments on LD1 (Fig. 2B,2D). In particular, waterreared individuals were characterised by lower concentrations of metabolites in general, compared with flower-fed individuals (Fig. 2A,2C). For parasitoids, there was a clear differentiation of the metabolic fingerprint of individuals fed with flowers exploited by bumblebees and those fed with flowers exploited by hoverflies (Fig. 2C).

Figure 2. Sample projections of hoverflies (A) and parasitoids (C) onto the first discriminant plane of the multivariate analysis LDA (representing 90.7% and 93.3% of the variance for hoverflies and parasitoids, respectively) depending on their metabolic composition and treatment (Water (crosses), patch exploited by bumblebees (Exploited B, squares); patch exploited by hoverflies (Exploited H, round dots), or an unexploited patch (Unexploited, triangles)). Normal confidence ellipses are represented. The correlation circles (B, D) depict the normalized relation between each metabolite and LDA axes for hoverflies (B, 23 log-transformed variables) and parasitoids (D, 34 log-transformed variables).

For readability, only metabolites contributing by more than 30% to both dimensions are presented.

Total sugar concentration was higher in insects fed on flowers compared with those reared on water, and in those fed with unexploited flowers compared with exploited ones (χ 2 =83.1; df=3; p<0.001 for hoverflies and F=19.3; df1/df2=3/43; p<0.001 for parasitoids) (Fig. 3A,3B). For both tested species, there was no difference in total sugar concentration between individuals fed on flowers previously exploited by bumblebees or by hoverflies. Hoverflies ingesting water or flower resources previously exploited by bumblebees consistently exhibited higher amino acid (χ 2 =16.0; df=3; p=0.001) and organic acid (χ 2 =10.1; df=3; p=0.017) concentrations than their relatives fed on unexploited flowers (Fig. 3C,3E).

Parasitoids reared on water had higher amino acid concentrations than individuals fed with unexploited flowers (F=3.38; df1/df2=3/43; p=0.027); no difference was found among individuals from the two other treatments (Fig. 3D). Parasitoids fed on flowers exploited by bumblebees showed lower concentrations of organic acids than those fed with flowers previously exploited by hoverflies, but no concentration difference was detected with waterreared parasitoids or parasitoids fed with unexploited flowers (F=3.65; df1/df2=3/43; p=0.020) (Fig. 3F). Water-reared hoverflies exhibited lower concentrations of polyols than hoverflies fed with flowers exploited by bumblebees (χ²=9.57; df=3; p=0.023) (Fig. 3G). In parasitoids, polyol concentration was not affected by the feeding treatment (F=1.22; df1/df2=3/43; p=0.314) (Fig. 3H).

Fructose and glucose concentrations were higher for hoverflies fed on unexploited flowers than for those fed on exploited ones, themselves higher than for water-reared individuals (Fructose: χ²= 139.0; df=3, p<0.001; Glucose: χ 2 =57.0; df=3; p<0.001) (Fig. 4A,4C). For parasitoids, fructose and glucose concentrations were higher for individuals fed on unexploited flowers than those fed on flowers exploited by bumblebees, but not on flowers exploited by hoverflies (Fructose: F=37.5; df1/df2=3/43; p<0.001; Glucose F=12.4; df1/df2=3/43; p<0.001) (Fig. 4B,4D). No difference between treatments was detected for trehalose concentrations in hoverflies (χ²=8.0; df=3; p=0.047) (Fig. 4E). The concentration of trehalose was higher in parasitoids fed on unexploited flowers than on the three other treatments (F=11.0; df1/df2=3/43; p<0.001) (Fig. 4F).

Secondary forager patch choice

Hoverflies preferentially landed on the flower patch that had already been exploited by conspecifics (probability to first land on the patch exploited by hoverflies=0.83; p<0.001, Fig. 5A). This preference was not found when previous competitors were bumblebees (probability to first land on the patch exploited by bumblebees=0.38; p=0.327) (Fig. 5A). No preference was recorded for the first feeding patch exploited by hoverflies whatever the nature of the first competitor (probability to first feed on the patch exploited by hoverflies=0.61; p=0.405 and probability to first feed on the patch exploited by bumblebees=0.29; p=0.144).

Furthermore, hoverflies visited the patch exploited by conspecifics significantly more times than the unexploited one (χ 2 =20.69; df=1; p<0.001); this difference was not observed when the patches were previously exploited by bumblebees (χ²=2.19; df=1; p=0.139).

Parasitoid individuals had no innate landing preference in our experimental design (probability to first land on a patch exploited by hoverflies=0.44; p=0.678; probability to first land on a patch exploited by bumblebees=0.35; p=0.332) (Fig. 5B).

Foraging and feeding behaviours of secondary foragers

Foraging duration was similar in hoverflies visiting unexploited patches and patches previously exploited by other hoverflies (χ²=2.27; df=1; p=0.132). However, hoverflies spent more time feeding on unexploited patches than on those previously exploited by their conspecifics (χ²=16.8; df=1; p<0.001) (Fig. 6A,6C). Additionally, hoverflies spent more time foraging and feeding on unexploited patches compared with patches previously exploited by bumblebees (foraging duration: χ²=16.6; df=1; p<0.001; feeding duration: χ²=4.10; df=1; p=0.043) (Fig. 6B,6D).

For both competitor species, the foraging duration of parasitoids was not significantly different between exploited and unexploited patches (hoverfly: χ²=3.52 df=1; p=0.060; bumblebee: χ²=3.55; df=1; p=0.059) (Fig. 6E,6F).

Hoverflies moved more from one flower to another within unexploited inflorescences than within inflorescences exploited by conspecifics (χ²=7.77; df=1; p=0.005) or by bumblebees (χ²=9.00; df=1; p=0.003). The number of flowers visited by parasitoids was not significantly different on unexploited and exploited patches (exploited by hoverflies: χ²=0.25; df=1; p=0.618; exploited by bumblebees: χ²=0.79; df=1; p=0.373).

Figure 6. Probability of a hoverfly to still be foraging (A, B) or feeding (C, D) and of a parasitoid to still be foraging (E, F) after a given amount of time (in seconds) on unexploited patches (blue lines) or patches exploited by either hoverflies (A, C, E) or bumblebees (B, D, F) (red lines). P-values from Cox

proportional hazard models are given.

Discussion

This study aimed to determine the effect of exploitative competition on the foraging behaviours of two common natural enemy species, the hoverfly Episyrphus balteatus and the aphid parasitoid Aphidius colemani, both flower visitors. As we considered two species with different physiological needs and ecology, yet both relying on the same floral resources for their reproduction and survival, we were expecting that exploitative competition would trigger behavioural responses in favour of the most rewarding foraging strategy. The assumption that exploited flowers would limit food availability, and thus nutrient ingestion, was verified for sugars for both species. In particular, glucose and fructose levels were drastically reduced in insects fed on flowers previously exploited by conspecifics or heterospecifics. Nevertheless, and surprisingly, we did not find evidence of an innate avoidance of previously exploited inflorescences for either species. Our results even showed that hoverflies were more attracted to inflorescences previously exploited by conspecifics for their first landing, although they spent more time feeding on unexploited patches. Parasitoids did not seem to show any innate preference, nor adjustment in their foraging and feeding behaviours, according to patch quality. Differences in energy requirements of the two species may explain these different responses, as discussed below.

To assess if already exploited flowers limit the uptake of nutrients by secondary foragers, we performed nutrimetabolomics on hoverflies and parasitoids fed on previously exploited or Nevertheless, exploited flowers were not fully depleted of nectar and sugars, as individuals fed on exploited flowers consistently exhibited significantly higher sugar concentrations than water control individuals. This can be explained by the high sugar concentrations of buckwheat nectar (Cawoy et al., 2008, Fig. SI.B). Buckwheat nectar is considered to be hexoserich, according to the Percival's classification (1961), thus mainly encompassing fructose and glucose, at the detriment of sucrose. Accordingly, the decrease of sugar contents in hoverflies and parasitoids fed on exploited flowers resulted from an impoverishment of glucose and fructose compared to insects fed on unexploited flowers. On the other hand, trehalose, which is often found in high amounts in insects' haemolymph, is not found in buckwheat nectar [START_REF] Wäckers | Hymenopteran parasitoids synthesize 'honeydew-specific' oligosaccharides[END_REF], yet this compound can be directly synthesized from glucose molecules in many insect species [START_REF] Thompson | Trehalose -The Insect 'Blood' Sugar[END_REF]. Thus, the higher trehalose concentrations in parasitoids fed with unexploited flowers suggest that A. colemani can also synthesize this molecule. Trehalose concentrations were similar in hoverflies in all treatments, suggesting that E. balteatus individuals did not synthesized additional trehalose directly after feeding on floral resources in our experiment.

As buckwheat nectar and pollen are also sources of amino and organic acids for foragers [START_REF] Pinheiro | Effects of pollen, sugars and honeydew on lifespan and nutrient levels of Episyrphus balteatus[END_REF], we expected hoverflies and parasitoids to be more concentrated in these two families of metabolites when fed on unexploited flowers. In particular, during the choice experiments, hoverflies were often observed feeding on pollen (but this was not the case for parasitoids). However, the opposite pattern was reported as individuals fed on unexploited flowers had identical or lower concentrations of organic acids and amino acids than the ones reared on water and exploited flowers. Such result could be explained by a direct metabolite use of those compounds for female egg maturation (Branquart and Hemptinne, 2000a).

Sugar content of the hoverflies and parasitoids was not influenced by either species of previous competitor. Nevertheless, the nutrimetabolomics approach and the subsequent discriminant analysis revealed a high differentiation of metabolic fingerprints between parasitoids fed on flowers exploited by bumblebees and flowers exploited by hoverflies. This suggests that the effect of competition on parasitoid physiology may vary depending on the previous competitor species.

Exploitative competition due to sugar resource limitation could lead to fitness costs for insects. [START_REF] Pinheiro | Effects of pollen, sugars and honeydew on lifespan and nutrient levels of Episyrphus balteatus[END_REF] found that a higher glucose concentration of E. balteatus individuals increases their longevity, and the same result was reported for Aphidius ervi, a parasitoid species closely related to A. colemani [START_REF] Azzouz | Effects of feeding frequency and sugar concentration on behavior and longevity of the adult aphid parasitoid: Aphidius ervi (Haliday) (Hymenoptera: Braconidae)[END_REF]. Buckwheat nectar has also been shown to increase hoverfly and parasitoid females' longevity and fecundity [START_REF] Jado | Floral resources to enhance the potential of the parasitoid Aphidius colemani for biological control of the aphid Myzus persicae[END_REF][START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF][START_REF] Vattala | The influence of flower morphology and nectar quality on the longevity of a parasitoid biological control agent[END_REF]. Therefore, in an environment where floral resource availability is limited, exploitative competition among flower visitors could potentially impact the fitness of E. balteatus and A. colemani. For instance, exploitative competition is likely to happen in summer, as already evidenced in agroecosystems for other flower visitors [START_REF] Couvillon | Summertime blues: August foraging leaves honey bees empty-handed[END_REF]. This is particularly true in agroecosystems with intensive beekeeping activities, as the honeybee Apis mellifera has been demonstrated to be a high floral resources competitor [START_REF] Henry | Controlling the impact of the managed honeybee on wild bees in protected areas[END_REF]. Under these conditions, we could expect hoverfly and parasitoid foragers to show a preference for flowers which are more rewarding in terms of glucose content, i.e. that were not previously exploited, as shown for other flower visiting insects [START_REF] Reader | Inter-order interactions between flower-visiting insects: foraging bees avoid flowers previously visited by hoverflies[END_REF][START_REF] Stout | The use of conspecific and interspecific scent marks by foraging bumblebees and honeybees[END_REF][START_REF] Yokoi | Recognition of scent marks in solitary bees to avoid previously visited flowers[END_REF].

However, we did not observe such pattern. Parasitoid females did not show any preference between exploited or unexploited flowers, or did not have the capacity to discriminate between these two types of flowers. Conversely, hoverfly females showed a preference for flowers previously exploited by their conspecifics, in terms of first visited patch and also of numbers of visits per patch. The fitness cost incurred by hoverflies feeding on previously exploited resources might not be as strong as expected. Indeed, Charles & Paines (2016) found that the longevity and fecundity of A. colemani were not different when individuals were fed with honeydew or nectar, even if nectar was more concentrated in fructose and glucose.

Therefore, the exploitation of flowers may lower the amount of nectar sugars available for A.

colemani parasitoids, but not to a sufficient extent to incur fitness costs in the short term (as glucose and fructose contents were still higher than in the water treatment). Also, A. colemani are small insects (usually less than 0.1 mg dry mass), and their energetic needs, in particular for sugars, are lower than those of larger insects, as supported by the low amount of feeding behaviours in the trials. As sugar content in hoverflies showed exactly the same pattern, the same hypothesis could be proposed for this species. Nevertheless, it is less probable that hoverflies reached their energetic needs with exploited inflorescences, as it is known that hoverflies visit many flowers in sequence when foraging in nature [START_REF] Gilbert | Diurnal activity patterns in hoverflies (Diptera, Syrphidae)[END_REF]. This observation is strengthened by our behavioural assays. Indeed, hoverflies spent more time foraging (in the trials where bumblebees were the previous competitor) and feeding (in both the trials where hoverflies and bumblebees were the previous competitors) on unexploited patches, showing that there were more resources available on those patches and that the amount of resources on exploited patches was not sufficient. Therefore, hoverflies might be more attracted to patches exploited by conspecifics at distance, but once on the patch, we suggest that they possess the ability to discriminate those flowers that are less rewarding.

Floral resources may not be sufficiently limiting in nature to impose selective pressure on hoverflies and parasitoids strong enough for the species to have adopted innate strategies to increase resource foraging efficiency. Among insects, the use of social information can either be genetically determined or acquired by learning processes [START_REF] Dukas | Evolutionary biology of insect learning[END_REF]. [START_REF] Leadbeater | Do inexperienced bumblebee foragers use scent marks as social information?[END_REF] suggested that innate rejection of previously visited flowers could be weakly selected, as the abundance and frequency of scent marks encountered in natura can allow quick and context-specific learning to occur. Accordingly, inexperienced bumblebees do not show any preference, nor rejection, of flowers previously visited by conspecifics, but such behaviour has been shown to develop over time by learning [START_REF] Leadbeater | Do inexperienced bumblebee foragers use scent marks as social information?[END_REF].

Moreover, learning also depends on the competitive context: for instance, bumblebees learn to follow conspecific cues when competitive pressure is low, while they learn how to avoid conspecifics when competition pressure is high [START_REF] Baude | Conspecifics as informers and competitors: an experimental study in foraging bumble-bees[END_REF][START_REF] Leadbeater | Bumble-bees learn the value of social cues through experience[END_REF]. Our study species are non-social, contrary to bumblebees, which makes them difficult to compare. Nevertheless, concerning foraging for host/oviposition sites, some parasitoids and hoverflies can learn to detect and avoid competitors [START_REF] Almohamad | Discrimination of parasitized aphids by a hoverfly predator: effects on larval performance, foraging, and oviposition behavior[END_REF][START_REF] Le Lann | Do past experience and competitive ability influence foraging strategies of parasitoids under interspecific competition?[END_REF], 2011) by relying on olfactory cues for instance (Benelli et al 2004). Future work will have to be conducted to determine if the foraging strategies adopted by flower-visitors facing competition for floral resources are modified with experience resulting from learning processes.

Hoverfly preference for patches exploited by conspecifics was consistent between the first patch chosen for landing and the number of visits to the patch. Such attraction by inexperienced individuals suggests the use of cues, possibly scent marks, deposited (actively or not) by conspecifics. No such attraction was found for bumblebee-visited patches, which suggests that specialist cues are involved. Nevertheless, the experimental design does not allow us to identify which cues were used by female hoverflies for patch choice. We identify three different resource types that female hoverflies could be attracted to: males for mating, aphids for oviposition, and pollen/nectar foraging for feeding. Females used in this experiment were not sexually mature, as they were less than 24h old and unmated [START_REF] Geusen-Pfister | Studies on the biology and reproductive capacity of Episyrphus balteatus Deg. (Dipt., Syrphidae) under greenhouse conditions[END_REF].

In many Diptera species, immature females are not attracted to male pheromones [START_REF] Katsoyannos | Male sex pheromone of Rhagoletis cerasi L. (Diptera, Tephritidae): Factors affecting release and response and its role in the mating behavior[END_REF][START_REF] Lima | Volatile substances from male Anastrepha fraterculus wied. (Diptera: Tephritidae): identification and behavioural activity[END_REF][START_REF] Robacker | Attraction of a laboratory strain of Anastrepha ludens (Diptera: Tephritidae) to the odor of fermented Chapote fruit and to pheromones in laboratory experiments[END_REF]. Moreover, plants were protected by a net and checked before use to ensure they were not infested with aphids [START_REF] Almohamad | Assessment of oviposition site quality by aphidophagous hoverflies: reaction to conspecific larvae[END_REF]. Therefore, it is very unlikely that one-day-old females were attracted by conspecific cues for mating or oviposition, but rather for feeding. It is known that E. balteatus individuals (and syrphids in general) preferentially use olfactory cues over visual cues to localize floral resources [START_REF] Majetic | The sweet smell of success: floral scent affects pollinator attraction and seed fitness in Hesperis matronalis[END_REF][START_REF] Primante | A syrphid fly uses olfactory cues to find a non-yellow flower[END_REF], and that they are able to detect conspecific olfactory cues (eggs or larval) when foraging for oviposition sites [START_REF] Almohamad | Assessment of oviposition site quality by aphidophagous hoverflies: reaction to conspecific larvae[END_REF][START_REF] Almohamad | Assessment of oviposition site quality by aphidophagous hoverflies: reaction to conspecific larvae[END_REF][START_REF] Scholz | Oviposition site selection of Episyrphus balteatus[END_REF]. Nevertheless, to our knowledge, this is the first evidence of conspecific use of information for floral resource foraging in E.

balteatus. Such behaviour may be beneficial for inexperienced individuals to find rewarding patches of food (i.e. the presence of conspecifics on a patch may be a good indication that resources are present on the patch), and in that sense could be part of a learning process to forage more efficiently on flowers. It is interesting that this behaviour is innate in a non-social species, whereas social species such as bumblebees do not show this innate attraction to conspecifics [START_REF] Leadbeater | Do inexperienced bumblebee foragers use scent marks as social information?[END_REF].

Conclusion

By linking physiological and behavioural approaches, this study provides the first evidence that exploitative competition for floral resources can generate cascading effects on the metabolic intake and the foraging behaviour of two flower-visiting natural enemies. It demonstrates that even insects with very small nectar requirements, such as the parasitoid studied, can be physiologically affected by heavy exploitative competition for floral resources. Nevertheless, these flower-visiting insects do not innately discriminate the most rewarding resource, which could make natural enemies even more exposed to exploitative competition in nature. With high levels of competition for floral resources in agroecosystems linked to honeybee management and low flowering plant abundance and diversity, such experimental results suggest that we can expect flower-visiting natural enemies to suffer from exploitative competition in nature. Such understanding could help to improve the success of floral resource enhancement strategies to conserve the insects that provide essential ecosystem functions such as pollination and herbivore control.

Figure 1 .

 1 Figure 1. Experimental design of the choice test. Female Episyrphus balteatus and Aphidius colemani previously fed on sucrose were tested in a choice arena. The arena contained two resource patches: an unexploited inflorescence of buckwheat and an inflorescence previously exploited by bumblebees; or an unexploited inflorescence of buckwheat and an inflorescence previously exploited by hoverflies.

Figure 3 .

 3 Figure 3. Effect of feeding treatment (Water (white), patch exploited by bumblebees (Exploited B, light grey); patch exploited by hoverflies (Exploited H, dark grey), or an unexploited patch (Unexploited, black)) on the concentrations (nmol.mg -1 ) of total sugars (A, B), amino acids (C, D), organic acids (E, F) and polyols (G, H), of the hoverfly E. balteatus (A, C, E, G) and parasitoid A. colemani (B, D, F, H). On

Figure 4 .

 4 Figure 4. Effect of feeding treatment (Water (white), patch exploited by bumblebees (Exploited B, light grey); patch exploited by hoverflies (Exploited H, dark grey), or an unexploited patch (Unexploited, black)) on the concentrations (nmol.mg -1 ) of fructose (A, B), glucose (C, D), and trehalose (E, F) of the hoverfly E. balteatus (A, C, E) and parasitoid A. colemani (B, D, F). On each boxplot, horizontal lines represent the median, first and third quartiles, the black points are extreme values, the red square is the mean and N the sample size. Different letters between treatments indicate significant differences (Tukey tests, p<0.05).

Figure 5 .

 5 Figure 5. Proportion of hoverflies (A) and parasitoids (B) choosing first the unexploited patch or the patch exploited by either bumblebees (left) or hoverflies (right). N is the sample size. Significant differences between exploited and unexploited patches are represented above the plots: NS: not significant; *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

  unexploited flowers. The distinct metabolic fingerprints revealed that sugar concentration of the study insects was affected by the quality of flowers offered, whatever the species of the previous competitor (bumblebee or hoverfly). The exploitation of the flowers by a previous competitor is very likely to have reduced sugar amounts as nectar cannot be replenished by the plants within minutes after the insect's visit (Fig. SI.C). In this case, secondary visitors were certainly offered lower quality floral resources, with a lower possibility of sugar ingestion.

Table 1 .

 1 Metabolites quantified in hoverflies (E. balteatus) and parasitoids (A. colemani), organized by metabolite families. Letters indicate the presence of the metabolite in hoverflies (H), parasitoids (P) or both (H+P).

	1

Table 2 :

 2 Description of recorded behaviours for hoverflies (E. balteatus) and parasitoids (A. colemani) during the choice test.

	Observed

behaviour (on a given patch) Definition Type

	Feeding	Individual has either its tongue (hoverfly) or mandibles (parasitoid) in the corolla for more than 2 seconds
	Foraging	Individual probes the patch with its tongue (hoverfly) or antenna (parasitoid)	Duration
	Immobility	Individual stays immobile, is grooming itself or is moving on the inflorescence without foraging
	First landing First feeding	Identity of the first patch the individual lands on Identity of the first patch the individual feeds on	Binary
	Entrance/Exit Changing	Individual enters or exits a patch Individual moves from a flower to another on the same patch	Occurrence


Petr Švec
email: petr.svec2@upce.cz

Hélène Cattey

Zdeňka Růžičková

Josef Holub

Aleš Růžička

Laurent Plasseraud

Triorganotin( iv ) cation

come

Introduction

For more than two decades, organotin(IV) compounds are known to promote the chemical transformation of carbon dioxide into chemicals with higher value-added. Thus, since the preliminary work of Kizlink described during the nineties and focusing on the linear organic carbonates, 1 numerous studies have also reported the efficiently of tin complexes for the direct synthesis of 2-oxazolidones, 2 cyclic organic carbonates, 3 and carbamates. 4 In the past, our group has acquired significant structural and spectroscopic knowledge, particularly using high pressure NMR conditions, 5 on the organometallic tin(IV) species involved in some of these CO2-based reactions. It emerges from these previous studies that the nature of ligands bound to tin atoms greatly influences the robustness and reactivity of precursors. Recently, we also reported the synthesis and the activity evaluation of (n-Bu2Sn)2O(CO3), an organotin carbonate, for the direct carbonation of various primary alcohols. This study showed the robustness and versatility of this type of species, and highlighted the relationship between (n-Bu2Sn)2O(CO3) and the deca-tin complex, (n-Bu2SnO)6[(n-Bu2SnOCH3)2(CO3)]2. 6 In this quest we have already shown that the incorporation of the 2-(N,N-dimethylaminomethyl)phenyl-as a C,N-chelating ligand (L CN ) within the structure of various organotin(IV) species leads to the elongation of the Sn-X bond (X = any substituent) which is mutually trans to the N→Sn dative bond. This phenomenon, known as the trans-effect, enables the use of selected C,N-chelated triorganotin(IV) halides as very efficient halogenating agents via the SN1 mechanism. 7 Structurally related organotin(IV) complexes were studied as promising transesterification catalysts 8 or as potential catalysts within the direct synthesis of DMC from methanol and CO2 by us earlier as well. 9 It was found that increase of the ionic nature of the catalytically active species leads to significant improvement of the yield of DMC. Thus the non-ionic (L CN (n-Bu)2Sn)2O provided the nDMC/nSn(cat) ratio of 0.75 while L CN (n-Bu)2SnOTf (for which we propose at least partially ionic character) gave the nDMC/nSn(cat) ratio of 1. 15. As a continuation of our work within this field of chemistry, we now report on the facile synthesis, structural characterization and catalytic activity studies of two novel C,Nchelated organotin(IV) monocarba-closo-dodecaborates.

Results and Discussion

Synthesis

The reaction of L CN R2SnCl (R = n-Bu (1) and Ph (2)) with one equivalent of AgCB11H12 (Ag . 3) in THF gave the desired ionic organotin(IV) species [L CN (n-Bu)2Sn] + [CB11H12] -( 4) and [L CN Ph2Sn] + [CB11H12] -(5), respectively, in a very high yield (85 and 88 %, respectively) after the work-up (Scheme 1). Despite all effort, we were not successful in obtaining single crystals of either 4 or 5. Both compounds are presumably reactive towards water and should be stored under an inert atmosphere (the instability issues are further discussed below in the NMR spectroscopy characterization paragraph). The unprecedented reactivity of other target species (i.e. [L CN

(n-Bu)Sn] 2+ [CB11H12]2 -, [(L CN )2Sn] 2+ [CB11H12]2 -and [L CN Sn] 3+ [CB11H12]3 -)
towards the THF (which started to polymerize) thwarted the isolation of these compounds. The use of a different solvent within the synthesis is not possible because of solubility issues.

NMR spectroscopy investigation of 4, 4a and 5

The 1 H NMR spectra of 4 and 5 (recorded in THF-d8) display somewhat broadened signals with appropriate multiplicity and integral intensity due to the n-Bu or Ph and L CN moieties that are bound to the central tin atom. The resonances of aromatic protons (H( 6)) are accompanied by characteristic tin satellites with reasonable 119 Sn-1 H coupling constants of 61 and 60 Hz, respectively, which is in line other triorganotin compounds bearing the L CN substituent. 10 A characteristic set of broad resonances (similar to that recorder for the starting Ag . 3 in THF-d8) attributable to the CB11H12 anion is observed in the region of 2.19 to 1.05 ppm for both compounds. In the case of 4, some of these signals are overlapped by resonances of the n-Bu moieties and residual THF signal by coincidence.

11 B{ 1 H} NMR spectra of 4 and 5 exhibit a typical pattern of 3 signals at ca. -7.4, -13.9 and -17.0 ppm, respectively, in a 1:5:5 ratio which is usually observed for the parent CB11H12 anion. 11 The dichotomy between 1 H and 11 B{ 1 H}chemical shifts may be explained on the basis of weak CB11H12 cage-tin interactions in THF-d8 as discussed similarly in the literature for Ag . 3. 12 All signals in the 13 C NMR spectra of 4 and 5 are in perfect agreement with expected patterns for respective compounds.

All relevant carbon resonances of the L CN and n-Bu or Ph substituents are accompanied by tin satellites with appropriate n J( 117/119 Sn, 13 C) coupling constants (see Experimental for detailed assignment of all resonances and the n J( 117/119 Sn, 13 C) values). 10 Quite broad resonance of the Ccage is found at 51.5 ppm for both 4 and 5. 11b The most valuable data regarding the structure (i.e. the vicinity of the tin atom) of 4 and 5 in solution simply arose from the 119 Sn NMR spectra. We assume the presence of triorganotin(IV) cations of the type [L CN R2Sn] + while the [CB11H12] -moieties act as very weekly coordinating anions making thus these species of ionic nature in solution. This conclusion is based on the observed 119 Sn NMR chemical shift value of 35.2 ppm (in THF-d8) found for 4, which is shifted to higher frequencies when compared to starting 1 (δ( 119 Sn) = -57.4 ppm in THF-d8). Similar change of chemical shift value towards higher frequencies was reported earlier for purely ionic [L CN (n-Bu)2Sn] + [Ti2Cl9] -(broad resonance at 185.6 ppm in C6D6) 13 or L CN (n-Bu)2SnOTf (δ( 119 Sn) = -1.0 ppm in C6D6) where we postulated a partial dissociation of the latter complex. 9b The same trend is observed for the 119 Sn NMR chemical shift value in the case of 5 (δ( 119 Sn) = -111.9 ppm in THF-d8) which is shifted by ca. 66 ppm to higher frequencies with respect to starting 2 (δ( 119 Sn) = -178 ppm in THF-d8). In general, the ionization phenomenon has been described for structurally related N,C,Nor O,C,O-chelated organotin(IV) compounds as well. 14 Scheme 2: Hydrolysis of 5.

The expected instability of 4 and 5 towards water was studied by NMR techniques, too. The hydrolysis of 5 led to the formation of corresponding hexaorganodistannoxane (L CN Ph2Sn)2O (Scheme 2) which was identified on the basis of its 1 H, 13 C and 119 Sn NMR spectra. 15 The second presumed decomposition product, [H3O] + [CB11H12] -, could be thus plausibly formed as demonstrated by us earlier. 16 Surprisingly, we observed the expected hexaorganodistannoxane (L CN (n-Bu)2Sn)2O only as a very minor decomposition product 14 within the hydrolysis of 4 while an unknown species (δ( 119 Sn) = -43.1 ppm in THF-d8) bearing presumably the L CN (n-Bu)2Sn fragment was identified by the multinuclear NMR spectroscopy as the major one. Further NMR investigation showed that the unknown species may be an adduct of L CN (n-Bu)2SnOH and [H3O] + [CB11H12] -complex which may arise due to the dihydrogen bonding and/or interaction via hydrogen bonding of either nitrogen atom of the pendant arm or oxygen atom of the OH group. All these possible interactions lead to the decrease of the electron density at the central tin atom which is reflected in the observed downfield shift in the 119 Sn NMR spectrum. Furthermore, this species exhibits reasonable signals in 1 H and 11 B(see the Experimental part). Unfortunately, all attempts to grow single crystals failed and thus no sc-XRD analysis could be performed. On the other hand, our hypothesis is further supported by the comparison of its multinuclear NMR spectra to that of an adduct of L CN (n-Bu)2SnOH and B(C6F5)3 (δ( 119 Sn) = -19.3 ppm in C6D6) which was isolated as a product of decomposition of the original L CN (n-Bu)2SnH . B(C6F5)3 species (Scheme 3). 17 Thus, L CN (n-Bu)2SnO(H) . B(C6F5)3 (4a, Fig. 1) exhibits one set of somewhat broadened signals attributable to L CN and n-Bu moieties and a very broad signal of the bridging OH unit at 4.03 ppm in its 1 H NMR spectrum measured in C6D6. Similarly, quite broad resonances are found for L CN and n-Bu substituents in the 13 C NMR spectrum in which a characteristic spectral pattern consisting of doublets of multiplets arises due to the presence of pentafluorophenyl moieties. Very broad resonance at 14.2 ppm is observed in the 11 B{ 1 H} NMR spectrum of 4a. Such chemical shift value is consistent with that reported for compounds of the type L . BR3. 18 Finally, the above mentioned 119 Sn NMR chemical shift of -19.3 ppm clearly reflects the presence of the L CN (n-Bu)2SnOH within the adduct.

Solid-state structures of 4a, 6 and 7

Compound 4a consists of the nearly perfectly trigonal bipyramidal triorganotin(IV) fragment where the carbon substituents are located in the equatorial plane and intramolecularly coordinated nitrogen atom from the pendant amino group and oxygen atom of the O(H) group found in the axial positions (Fig. 1). Very strong coordination of the nitrogen group (Sn1-N1 = 2.414(2) Å), when compared to other triorganotin(IV) species bearing the same ligand (~2.50 Å), 10b,d,19 is most probably the reason of Sn-O distance elongation (Sn1-O1 = 2.3010(17) Å) in comparison to standard bonds of the tin atom to the oxygen bridge being < 2.15 Å. Similar type of elongation is observed in the case of the B-O separation where the interatomic distance O1-B1 of 1.520(3) Å is usually attributed to the donor-acceptor connection than for a covalent bond (~1.35 Å) or the only SnO(H)•••B fragment containing structure reported within the Cambridge Structural Database which is the diorganotin(IV) complex with two phenylboronato(1-) ligands (B-O = 1.482 Å). 20 On the other hand, the B1-O1-Sn1 angle of 145.92( 14)° found in 4a is about 25° larger than reported in the literature. 8), C1-Sn1-C14 122.94 (10), C1-Sn1-C10 120.77(9), C10-Sn1-O1 94.20( 8), C10-Sn1-N1 93.82( 8), B1-O1-Sn1 145.92 (14).

The structure of 6 is composed of trimethylbenzylammonium cation and 1-carba-closo-dodecaborate anion (Fig. S2). Two structurally related compounds with quarternized nitrogen atom and CB11H12 -anion have been already reported in the literature. These include the methylimidazolium 21 and nbutylpyridinium 22 cations. Significant parameters for both reported compounds are very close to that of 6. The identity of 6 has been further confirmed by the multinuclear NMR spectroscopy. Thus the multinuclear NMR spectra of 6 are in absolute agreement with the structure established by the sc-XRD techniques (see the Experimental part for detailed assignment of all resonances).

Very rich structural chemistry of organotin(IV) oxides, hydroxides, alkoxides and carbonates is dominated by aggregation of species to higher oligomers via oxygen bridgesmore than 330 structures reported up to now. 23 Such aggregation is mostly pronounced in cases of diorganotin(IV) alkoxides, where a ladder-or stairs-like structures 24 formed by a four-membered Sn-O-Sn-O rings are mutually interconnected to tetra-, hexa-or octanuclear arrangements. [START_REF] Kundu | Examples of tetranuclear species: a)[END_REF] In cases of diorganotin(IV) carbonates, alkoxy-or oxo-carbonates, the mononuclear arrangements were reported for complexes containing ligands with two adjacent donor arms, 26 dinuclear species for ligands with one additional donor, 27 trinuclear ones for compounds containing bulkier ligands such as t-Bu, 28 while decanuclear 29 and polymeric 30 aggregates contain smaller benzyl or methyl groups. Immediate neutral carbonato decanuclear mixed mono-and diorganotin cluster[(t-Bu)2Sn(OH)OSnR(OH)2OC(OSn(t-Bu)2OH)2(O)SnR(OH)(H2O)]2, here R is 2,6-Mes2C6H3, was isolated from the reaction mixture of organotin oxides and hydroxides which were bubbled with CO2, and has a U-shape structure due to the presence of bulky ligands. 31 [(n-Bu)20Sn10O2(OMe)6(CO3)2] 2+. 2[CB11H12] -complex ( 7), which crystallized from a residual methanolic solution recovered from the reactor after a catalytic run using 4 as a catalyst, has a hybrid structure with the central tetranuclear 4-4-4 Sn-O-Sn-O tricyclic moiety which interconnects two trinuclear 4-4 bicyclic fragments via two carbonato ligands (Fig. 2). The almost co-planar arrangement of all the rings further consists of ten di-n-butyltin(IV), four oxo and six methoxy moieties. The overall 2+ charge is compensated by two CB11H12 - clusters used for stabilization of organotin and other main group metal species in the ionic form. [START_REF] Kašná | Examples of organotin species: a)[END_REF] To the best of our knowledge, there are no cationic species reported in above mentioned series of stannoxane, tin(IV) alkoxide or carbonate complexes. There are four different types of the tin atom in 7, where all of them being five-coordinate with the shape of a deformed trigonal bipyramid with the largest deviation from the ideal angles found for O6-Sn4-O8 (149.3(3) °) and O6-Sn4-O8i (144.8(3) °), respectively. These as well as other values found for interatomic distances and angles are in line with previously published work within the series of similar di-or triorganotin(IV) oxo-compounds mentioned vide supra. 8), Sn1-O2 2.140 (10), Sn1-O3 2.117 (10), Sn2-O1 2.024(9), Sn2-O4 2.106(11), Sn2-O2 2.165 (10), Sn5-O7 2.037(7), Sn5-O7a 2.131( 8), Sn5-O8 2.179(9), Sn3-O1 2.048(9), Sn3-O5 2.158(9), Sn3-O3 2.199(9), Sn4-O7 1.999( 8), Sn4-O6 2.178 (10), Sn4-O8 2.219(9).

In general, the formation of 7 during the catalytic run may be seen as a proof of a presence of a presumably key catalytically active species and/or intermediate within the direct synthesis of DMC from CO2 and methanol. Thus, the presence of several carbonate and methoxy ligands in the skeleton of 7, two and six respectively, can be directly perceived as a reminiscence of the formation of DMC. Interestingly, and from a structural point of view, the composition of 7 shows strong similarities to the deca-tin complex, (n-Bu2SnO)6[(n-Bu2SnOCH3)2(CO3)]2, 29a isolated under similar reaction conditions (mixture of CO2/CH3OH, P 200 bar, T = 150 ° C, using n-Bu2Sn(OCH3)2 as tin precursor) and whose the implication has recently been underlined for the direct carbonation of primary alcohols. 6 Therefore, these polynuclear compounds could even be qualified as dormant species. However and at the present stage of our knowledge, the mechanism leading to the formation of 7 from 4 remains still indeterminate. Based on previous studies, several hypotheses and leads can be advanced:

(i)

The central tetranuclear moiety can be reasonably constructed by the condensation of n-Bu2SnO and n-Bu2Sn(OCH3)2. 33 The resulting distannoxane could then in turn react with CO2 to lead to a complex bearing two terminal hemicarbonate ligands and whose the structure has already been described. 34 (ii) The trinuclear carbonato moieties may be derived from the condensation of n-Bu2Sn(OCH3)2 with (n-Bu2Sn)2OCO3 which can be prepared from n-Bu2SnO under supercritical CO2 conditions. 35 (iii) Hydrolysis of n-Bu2Sn(OCH3)2 leads to the formation of n-Bu2SnO. From another point of view, one can pinpoint the loss of the L CN ligands within the catalytic run which may be explained by the well-known phenyl group migration process that is feasible under the relatively harsh reaction conditions. [START_REF] Kašná | Examples of organotin species: a)[END_REF] Nevertheless, the 1 H, 11 B and 119 Sn NMR spectra of 7 correspond quite well to the structure established by the sc-XRD analysis (see Experimental part for detailed assignment of all resonances). Under exposure to the ambient air, 7 undergoes a hydrolysis rapidly which can be monitored by FT-IR spectroscopy. Over time several modifications are clearly observed changing the initial IR fingerprint (Fig. 3). In particular, after one hour, a new absorption band of high intensity is observed at 3573 cm 1 (spectrum B, attributed to OH) while the band at 2819 cm 1 (OCH), assigned to the -OCH3 units of 7 (spectrum A), disappeared completely. Thus, we assume the transformation of all -OCH3 moieties in favor of OH groups. A representation of the plausibly resulting species is shown in Scheme 4. This hypothesis is experimentally corroborated by the elemental analysis carried out on crystals of 7, subjected several hours to ambient air. The results support satisfactorily the suggested formula (% found: C = 34.90; H = 7.31. Calcd. for C84H210O16B22Sn10 (2901.49): C = 34.77; H = 7.29). However, the hydrolysis causes the irreversible deterioration of the crystals. Thus, and despite several attempts, it was not possible to confirm by sc-XRD analysis the structure of the hydrolysis product.

Evaluation of DMC synthesis

Both ionic organotin(IV) species 4 and 5 were employed as potential catalysts or catalyst precursors within the direct synthesis of DMC from methanol and carbon dioxide (Table 1). Surprisingly, only 4 (or better say its active form 7) turned out to be catalytically active within such synthesis which was proved by the stoichiometry of nDMC/nSn(cat) being 1.5 (the nSn(cat) represents the amount of starting 4 in moles used in a catalytic run). The reproducibility of the carbonation reaction was satisfactorily verified by repeating the procedure four times, and with three distinct batches of precursor. 37 The reaction was also tested using undistilled and undried methanol leading to a decrease in precursor activity. A DMC: Sn molar ratio of 0.9 was then recorded. On the other hand, only traces of DMC were detected by GC techniques when 5 was used. The inactivity of 5 may be regarded to the presumably expected loss of both L CN and/or Ph substituents during the catalytic run which may be explained by the above mentioned phenyl group migration. 38 This loss of organic groups was confirmed by NMR spectroscopy as well as sc-XRD analysis of the by-product 6. Unfortunately, however, the nature of the tin-based residue still remains in doubt.

Conclusions

We have expanded the family of known (C,N-chelated) organotin(IV) complexes that are able to catalyze the direct synthesis of DMC from methanol and carbon dioxide. Further investigations involving the possibility of reusing the catalytically active species recovered from the reactor in subsequent runs are in progress now. In general, an increase of an ionic character of the catalytically active organotin(IV) species leads to higher yield of the desired DMC as discussed vide supra. This was also demonstrated in this paper since the use of the ionic catalyst precursor 4 enabled obtaining of the highest nDMC/nSn(cat) ratio of 1.5 among all organotin(IV)-based catalysts used by us so far. From this point of view, presumably better results regarding the yield of DMC (i.e. significant increase of the nDMC/nSn(cat) ratio) may be achieved by the use of mono-and/or dialkyltin(IV) cationic species (with appropriate counter anion(s)) which synthesis is an subject of our current interest.

Experimental section

General remarks

All reactions were carried out under an inert atmosphere of argon using standard Schlenk techniques. 1 39 , L CN (n-Bu)2SnH, 13 2 40 and Ag . 3 41 were prepared according to published procedures. All reagents (B(C6F5)3) and solvents (methanol, hexane, THF, methanol-d4, C6D6 and THF-d8) were obtained from commercial sources (Sigma-Aldrich or VWR). THF was dried with the help of solvent purification system PureSolv MD 7 supplied by Innovative Technology, Inc., degassed and then stored under an argon atmosphere over a potassium mirror. C6D6 and THF-d8 were distilled from sodium benzophenone ketyl prior to use.

NMR spectroscopy

The NMR spectra were recorded from solutions in THF-d8, C6D6 or methanol-d4 on a Bruker Ascend TM 500 spectrometer (equipped with Z-gradient 5mm Prodigy cryoprobe) at frequencies 500. Positive chemical shift values denote shifts to the higher frequencies relative to the standards. 119 Sn NMR spectra were measured using the inverse gated-decoupling mode. The unambiguous attribution of the resonances in the 1 H and 13 C{ 1 H} NMR spectra was provided by standard 2D NMR techniques. See also the supplementary data file for the NMR numbering of prepared compounds (Fig. S1) and graphical presentation of their NMR spectra.

Crystallography

The X-ray data for colorless crystals of 4a and 7, respectively, were obtained at 150 K using Oxford Cryostream lowtemperature device on a Nonius KappaCCD diffractometer with MoK radiation ( = 0.71073 Å), a graphite monochromator, and the  and  scan mode. Data reductions were performed with DENZO-SMN. 42 The absorption was corrected by integration methods. [START_REF] Coppens | Crystallographic Computing[END_REF] Structures were solved by direct methods (Sir92) [START_REF] Altomare | [END_REF] and refined by full matrix least-square based on F 2 (SHELXL-97). [START_REF] Sheldrick | SHELXL-97[END_REF] The data for structure of 6 were collected on a Bruker APEX-II CCD diffractometer at 150 K using Oxford Cryostream lowtemperature device with MoK radiation ( = 0.71073 Å), the TRIUMPH curved crystal monochromator, and the  and  scan mode. Data reductions were performed with SAINT v8.34A program package. The absorption was corrected by SADABS-2014/5. Structures were solved by ShelXT and refined by XL [START_REF]Bruker V8.34A: SADABS, SAINT, APEX2[END_REF] and Olex2 software. 47 Hydrogen atoms were mostly localized on a difference Fourier map, however to ensure uniformity of the treatment of the crystal, all hydrogen atoms were recalculated into idealized positions (riding model) and assigned temperature factors Hiso(H) = 1.2 Ueq(pivot atom) or of 1.5Ueq for the methyl moiety with C-H = 0.96, 0.97, 1.1 and 0.93 Å for methyl, methylene, and hydrogen atoms in BH, CH groups in carborane cages and aromatic rings moiety, respectively. Hydrogen atom of the OH group in 4a was placed according to a maximum found on the Fourier difference electron density map. Carbon atoms in the carborane cages were assigned to the maxima with shortest separations to another five maxima in the icosahedron. Crystals of 7 were of a poor quality, several disordered n-butyl groups were found within this structure having no influence on the composition of the model. Some of them were split into two positions. Remaining ones were treated with ISOR instruction.

Crystallographic data for structural analysis have been deposited with the Cambridge Crystallographic Data Centre (no. 1590298 (6), 1590299 (7) and 1590300 (4a)). Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EY, UK (fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or www: http:// www.ccdc.cam.ac.uk).

IR spectroscopy

The FT-IR spectra were recorded at ambient temperature on a Bruker Vector 22 equipped with a Specac Golden Gate TM ATR device (Specac Ltd., Orpington, UK) in the wave number range of 4000-500 cm -1 (twelve scans at a spectral resolution of 4 cm - 1 ).

Synthesis

Preparation of [L CN (n-Bu)2Sn] + [CB11H12] -(4)

The synthesis was carried out under the exclusion of light. L CN (n-Bu)2SnCl (805 mg, 2.00 mmol) was dissolved in THF (20 mL) and solution of AgCB11H12 (502 mg, 2.00 mmol) in THF (15 mL) was added dropwise. AgCl precipitated immediately. The reaction mixture was filtered and the filtration cake was washed with THF (2x 10 mL). THF filtrates were combined and the volatiles were removed in vacuo. The oily residue was washed with hexane (15-20 mL) in order to induce precipitation of the title compound. After filtration, the off-white solid was dried in vacuo giving pure 4. Yield 867 mg (85%). M. p. 74-75 °C. 1 H NMR (THF-d8, 295 K, ppm): 7.60 (d, 1H, H(6), 3 J( 1 H, 1 H) = 6.4 Hz, 3 J( 119 Sn, 1 H) = 61 Hz); 7. 43-7.30 (m, 3H, H(3-5)); 3.94 (s, 2H, NCH2); 2.59 (s, 6H, N(CH3)2); 2.12 (br, 1H, CcageH); 1.72-1.62 (br, 4H, -CH2); 1.60-1.50 (br m, 4H, -CH2); 1.41 (m, 4H, γ-CH2); 0.90 (t, 6H, -CH3, 3 J( 1 H, 1 H) = 7.3 Hz); broad BH resonances with overall integral intensity of 11 display a typical pattern ranging from 2.05 to 1.05 ppm and some of them are overlapped by resonances of the n-Bu moieties and residual THF signal by coincidence. 11 B{ 1 H} NMR (THF-d8, 295 K, ppm): -7.4 (br s, 1B, B( 12)); -13.9 (br s, 5B, B(7-11)); -17.0 (br s, 5B, B(2-6)). 13 C{ 1 H} NMR (THF-d8, 295 K, ppm): 144.2 (C(2), 2 J( 119/117 Sn, 13 C) = 33Hz); 138.7 (C(1), 1 J( 119/117 Sn, 13 C) = 618/590 Hz); 137.4 (C(6), 2 J( 119/117 Sn, 13 C) = 42 Hz); 131.2 (C( 4)); 129.3 (C(5), 3 J( 119/117 Sn, 13 C) = 60 Hz); 128.4 (C(3), 3 J( 119/117 Sn, 13 C) = 51 Hz); 66.4 (CH2N, n J( 119/117 Sn, 13 C) = 26 Hz); 51.5 (br, CcageH); 46.3 (NMe2); 29.0 (-C, 2 J( 119/117 Sn, 13 C) = 30 Hz); 27.8 (γ-C, 3 J( 119/117 Sn, 13 C) = 91 Hz); 17.4 (-C, 1 J( 119/117 Sn, 13 C) = 452/433 Hz); 13.9 (-C). 119 Sn{ 1 H} NMR (THF-d8, 295 K, ppm): 35.2 (br). Elemental analysis (%): found: C, 42.7; H, 8.5; N, 2.5. Calcd. for C18H42NB11Sn (510.14): C, 42.38; H, 8.30; N, 2.75.

Hydrolysis of 4

The sealed NMR tube containing the THF-d8 solution of 4 was opened in the air and several drops of water were added. After staying overnight at ambient temperature, the NMR spectra were recorder again. 1 H NMR (THF-d8, 295 K, ppm): 7.70 (br d, 1H, H(6), 3 J( 1 H, 1 H) = 7.0 Hz Hz, 3 J( 119 Sn, 1 H) ≈ 62 Hz); 7. 32-7.23 (br m, 3H, H(3-5)); 4.08 (br, 1H, OH); 3.79 (s, 2H, NCH2); 2.45 (s, 6H, N(CH3)2); 2.08 (br, 1H, CcageH); 1.61 (br, 4H, -CH2); 1. 38-1.32 (br m, 8H, -and γ-CH2); 0.90 (t, 6H, -CH3, 3 J( 1 H, 1 H) = 7.1 Hz); broad BH resonances with overall integral intensity of 11 display a typical pattern ranging from 2.05 to 1.05 ppm and some of them are overlapped by resonances of the n-Bu moieties and residual THF signal by coincidence. 11 B{ 1 H} NMR (THF-d8, 295 K, ppm): -7.5 (br s, 1B, B( 12)); -13.8 (br s, 5B, B(7-11)); - 16.8 (br s, 5B, B(2-6)). 119 Sn{ 1 H} NMR (THF-d8, 295 K, ppm): -43.1.

Preparation of L CN (n-Bu)2SnOH . B(C6F5)3 (4a) L CN (n-Bu)2SnH (368 mg, 1.00 mmol) was reacted with (C6F5)3B (95%, 539 mg, 1.00 mmol) in benzene (20 mL) at ambient temperature overnight providing presumably [L CN (n-Bu)2Sn] + [(C6F5)3BH] -(not isolated). Upon its contact with moist air overnight, the title compound was isolated as colorless crystals in high yield after crystallization from diethyl ether/hexane mixture of solvents. M.p. 145-146 °C. 1 H NMR (C6D6, 295 K, ppm): 7.57 (d, 1H, H(6), 3 J( 1 H, 1 H) = 6.6 Hz, 3 J( 119 Sn, 1 H) = 62 Hz); 6.98 (m, 2H, H(4,5)); 6.63 (d, 1H, H(3), 3 J( 1 H, 1 H) = 6.6 Hz); 4.03 (br, 1H, OH); 2.62 (s, 2H, NCH2); 1.31 (s, 6H, N(CH3)2); 1.25 (br, 4H, -CH2); 1.18 (br m, 4H, -CH2); 1.11 (br, 2H, γ-CH2); 0.98 (br, 2H, γ-CH2); 0.84 (t, 6H, -CH3, 3 J( 1 H, 1 H) = 7.3 Hz). 11 B{ 1 H} NMR (C6D6, 295 K, ppm): 14.2 (br). 13 C{ 1 H} NMR (C6D6, 295 K, ppm): 148.8 (d of m, C6F5, 1 J( 19 F, 13 C) = 252 Hz); 145.0 (br d, C6F5, 1 J( 19 F, 13 C) = 261 Hz); 142.2 (br, C(2), 2 J( 119/117 Sn, 13 C) could not be read); 138.8 (d of m, C6F5, 1 J( 19 F, 13 C) = 252 Hz); 137.0 (br, C(1), 1 J( 119/117 Sn, 13 C) could not be read); 135.4 (br, C( 6)); 131.2 (C( 4)); 127.0 (br, C(3); 64.4 (br, CH2N); 113.8 (br, C6F5); 44.4 (br, NMe2); 28.5 (br, -C); 26.8 (br, γ-C); 16.4 (br, -C, 1 J( 119/117 Sn, 13 C) could not be read); 13.1 (-C); resonance of the C( 4) is overlapped by the signal of C6D6 by coincidence. 119 Sn{ 1 H} NMR (C6D6, 295 K, ppm): -19.3.

Preparation of [L CN Ph2Sn] + [CB11H12] -(5)

5 was prepared according to the procedure described for 4 using L CN Ph2SnCl (885 mg, 2.00 mmol) and AgCB11H12 (502 mg, 2.00 mmol). Off-white powder. Yield 968 mg (88% 12)); -13.8 (br s, 5B, B(7-11)); -16.9 (br s, 5B, B(2-6)). 13 C{ 1 H} NMR (THF-d8, 295 K, ppm): 144.9 (C(2), 2 J( 119/117 Sn, 13 C) = 38 Hz); 138.0 (C( 6), 2 J( 119/117 Sn, 13 C) = 49 Hz); 137.1 (o-Ph, 2 J( 119/117 Sn, 13 C) = 47 Hz); 136.3 (br, i-Ph, 1 J( 119/117 Sn, 13 C) could not be read); 133.9 (br, C(1), 1 J( 119/117 Sn, 13 C) could not be read); 132.4 (br, C(4)); 132.3 (br, p-Ph); 130.9 (m-Ph, 3 J( 119/117 Sn, 13 C) = 71 Hz); 130.2 (C(5), 3 J( 119/117 Sn, 13 C) could not be read); 129.1 (C(3), 3 J( 119/117 Sn, 13 C) = 60 Hz); 65.8 (CH2N, n J( 119/117 Sn, 13 C) = 30 Hz); 51.5 (br, CcageH); 46.6 (NMe2). 119 Sn{ 1 H} NMR (THF-d8, 295 K, ppm): -111.9. Elemental analysis (%): found: C, 48.4; H, 6.4; N, 2.4. Calcd. for C22H34NB11Sn (550.12): C, 48.03; H, 6.23; N, 2.55. 6) Single crystals of 6 were obtained from a methanolic solution of the reaction mixture which was collected from the reactor after the catalytic run where 5 was employed as a catalyst. M.p. 220-222 °C. 1 H NMR (methanol-d4, 295 K, ppm): 7.60-7.50 (m, 5H, Harom.); 4.52 (s, 2H, CH2N); 3.11 (s, 6H, NMe2); 2.19 (br, 1H, CcageH); unresolved BH resonances with overall integral intensity of 11 display a typical pattern ranging from 2.05 to 1.05. 11 B{ 1 H} NMR (THF-d8, 295 K, ppm): -7.5 (br s, 1B, B( 12)); -13.8 (br s, 5B, B(7-11)); - 16.6 (br s, 5B, B(2-6)). 13 C{ 1 H} NMR (methanol-d4, 295 K, ppm): 134.0 (o-Carom.); 132.1 (p-Carom.); 130.4 (m-Carom.); 129.0 (i-Carom.); 70.6 (pseudo t, CH2N, 1 J( 14 N, 13 C) = 3 Hz); 53.1 (pseudo t, NMe2, 1 J( 14 N, 13 C) = 4 Hz); 52.2 (br, CcageH). Elemental analysis (%): found: C, 45.4; H, 10.0; N, 4.5. Calcd. for C11H28NB11 (293.26): C, 45.05; H, 9.62; N, 4.77.

Isolation of [BnNMe3] + [CB11H12] -(
Isolation of [(n-Bu)20Sn10O2(OMe)6( CO3)2] 2+. [CB11H12]2 -( 7) Single ingle crystals of 7 were grown from a methanolic solution which was recovered from the reactor after a catalytic run using 4 as a catalyst. M. p. > 220 °C (dec.). 1 H NMR (THF-d8, 295 K, ppm): 2.72 (br, 18H, OCH3); 2.10 (br, 2H, CcageH); 1.65 (br m, 20H, -CH2); 1.49-1.38 (m, 40H, -and γ-CH2); 0.95 (t, 60H, -CH3, 3 J( 1 H, 1 H) = 7.0 Hz); broad BH resonances with overall integral intensity of 22 display a typical pattern ranging from 2.05 to 1.05 ppm and some of them are overlapped by resonances of the n-Bu moieties and residual THF signal by coincidence. 11 B{ 1 H} NMR (THF-d8, 295 K, ppm): -7.6 (br s, 2B, B( 12)); -13.9 (br s, 10B, B(7-11)); -17.0 (br s, 10B, B(2-6)). 119 Sn{ 1 H} NMR (THF-d8, 295 K, ppm): -164.3 (s, 2Sn); -177.2 (s, 2Sn); -207. 6 (s, 4Sn), -211.8 (s, 2Sn). IR (ATR, selected bands, cm - 1 ): 2956 ν(CH), 2924 ν(CH), 2857 ν(CH), 2819 (OCH), 2535 ν(BH), 1461, 1426, 1409, 1377, 1048, 1019, 964, 870, 836, 670, 619, 580. Elemental analysis found: C, 35.84; H, 7.48. Calcd. for C90H222B22O16Sn10 (2985.65): C, 36.21; H, 7.49.

High-pressure experiments

Safety Warning: Experiments involving pressurized gases can be hazardous and must only be conducted with suitable equipment and following appropriate safety considerations. [START_REF] Isaacs | Safety Procedures for High-Pressure Systems[END_REF] General procedure for reactions of methanol with carbon dioxide: The reaction was carried out in a 125 mL stainless steel reactor equipped with a magnetic stirrer. The reactor was purged with argon and a 20 mL solution of the appropriate C,Nchelated organotin(IV) species (4 and 5) in anhydrous methanol was introduced by syringe. Then, CO2 was admitted to the desired amount (see Table 1). The reaction temperature (150 °C) was controlled by an internal thermocouple. After a reaction time of 24 hours the reactor was cooled down to 0°C, the pressure was gently released and the liquid phase was transferred to a Schlenk tube. Trap-to-trap distillation under vacuum at ambient temperature allowed separation of volatile compounds that were quantitatively analyzed by GC (DEC external standard, Fisons 8000, J&W Scientific DB-WAX 30 m capillary column, FID detector). Tin-based residue was firstly characterized by IR and multinuclear NMR spectroscopy.

Scheme 1 :

 1 Scheme 1: Synthesis of triorganotin(IV) catalysts 4 and 5 (R = n-Bu or Ph).

Scheme 3 :

 3 Scheme 3: Plausible formation of 4a as a model compound with a Sn-O(H)•••B linkage.

Fig. 1 :

 1 Fig. 1: Molecular structure of 4a (ORTEP presentation, 50% probability level). Hydrogen atoms, except of the OH group, and the n-hexane solvent molecule are omitted for clarity. Selected interatomic distances [Å] and angles [°]: Sn1-O1 2.3010(17), Sn1-N1 2.414(2), Sn1-C14 2.137(2), Sn1-C1 2.126(2), Sn1-C10 2.138(2), O1-B1 1.520(3), O1-Sn1-N1 169.29(7), C14-Sn1-O1 85.46(8), C14-Sn1-N1 97.33(9), C14-Sn1-C10 116.19(9), C1-Sn1-O1 93.46(8), C1-Sn1-N1 76.33(8), C1-Sn1-C14 122.94(10), C1-Sn1-C10 120.77(9), C10-Sn1-O1 94.20(8), C10-Sn1-N1 93.82(8), B1-O1-Sn1 145.92(14).

Fig. 2 :

 2 Fig. 2: Molecular structure of 7 (ORTEP presentation, 30% probability level). Hydrogen atoms and two CB11H12 -anions are omitted for clarity. Selected interatomic distances [Å]: Sn1-O1 2.080(8), Sn1-O2 2.140(10), Sn1-O3 2.117(10), Sn2-O1 2.024(9), Sn2-O4 2.106(11), Sn2-O2 2.165(10), Sn5-O7 2.037(7), Sn5-O7a 2.131(8), Sn5-O8 2.179(9), Sn3-

Fig. 3 :

 3 Fig. 3: FT-IR spectra of 7 (A) and its evolution after one hour of exposure to ambient air (B).

Scheme 4 :

 4 Scheme 4: Possible molecular representation of the hydrolysis product of 7 (R = n-Bu).

  OEt2 (δ( 11 B) = 0.0 ppm). The values of13 C chemical shifts were calibrated to signals of THF-d8 (δ( 13 C) = 67.57 ppm), C6D6 (δ( 13 C) = 128.4 ppm) or methanol-d4 (δ( 13 C) = 49.1 ppm). The 119 Sn chemical shift values are referred to external neat tetramethylstannane (δ( 119 Sn) = 0.0 ppm).

Table 1 .

 1 Summary of results of direct synthesis of DMC from CO2 and methanol using compounds 4 and 5 as catalyst precursor (P(CO2) = 200 bar, T = 150 °C, reaction time 24 hours).

	Complex	Solvent	CO2 used (g)	nDMC (mmol) a	nSn(cat) (mmol)	nDMC/nSn(cat)
		MeOH
	4		37.5	1.7	1.10	1.5
		(20 mL)
		MeOH
	5		32.3	traces	1.17	-
		(20 mL)
	a Determined by GC using diethyl carbonate (DEC) as external
	standard.

  13 MHz for 1 H, 160.48 MHz for 11 B{ 1 H}, 125.76 MHz for 13 C{ 1 H} and 186.50 MHz for 119 Sn{ 1 H} at 295 K. The solutions were obtained by dissolving of approximately 40-60 mg of each compound in 0.6 ml of deuterated solvent. The values of 1 H chemical shifts were calibrated to residual signals

	of THF-d8 (δ( 1 H) = 3.58 ppm), C6D6 (δ( 1 H) = 7.16 ppm) or
	methanol-d4 (δ( 1 H) = 3.31 ppm). The 11 B chemical shift values
	are referred to external BF3 .

Acknowledgement

The Czech authors would like to thank the Czech Science Foundation (project 16-01618S) for the financial support of this work. The French authors gratefully acknowledge the Centre National de la Recherche Scientifique (CNRS, France) and the University of Bourgogne Franche-Comté (Dijon, France).