Ruth Cebolla

Alberto Urbaneja

Joan Van Baaren

Dr Alejandro Tena
email: atena@ivia.es

Negative effect of global warming on biological control is mitigated by direct competition between sympatric parasitoids

Keywords: Aphytis, Aonidiella aurantii, citrus, interspecific competition, displacement

Parasitoids are among the most important and successful groups of natural enemies used in the biological control of insect pests. In most systems, several parasitoid species can parasitize the same pest. The coexistence of parasitoids in agroecosystems and their efficacy as biological control agents may be disrupted by global warming. An increase of approximately 3°C is predicted by the end of the twenty-first century in the Mediterranean basin (IPCC, 2014). In this context, we compared the present and future performance of two sympatric parasitoids of the genus Aphytis (Hymenoptera: Aphelinidae), which control the armoured scale Aonidiella aurantii (Hemiptera: Diaspididae) in Mediterranean citrus, either alone or in combination. The net reproductive rate (R0) of the introduced Aphytis melinus DeBach was higher than that of its competitor, the native Aphytis chrysomphali (Mercet), under current conditions. The two parasitoids responded differently to higher temperature and competition. The R0 of A. chrysomphali decreased by 50% when both parasitoids competed in the same patch, but was not affected by the temperature increase. The R0 of A. melinus decreased approximately 40% with the increase in temperature because the proportion of females was reduced.

However, the presence of A. chrysomphali competing in the same patch mitigated the negative effect of the increase in temperature on A. melinus (R0 decreased by only 20%). Overall, our results suggest that global warming will have a negative effect on the biological control of A. aurantii and that this effect will be higher in areas, such as southern Spain, where A. melinus has displaced A. chrysomphali.

INTRODUCTION

Due to global warming, average temperatures have risen by approximately 0.8°C since the early twentieth century, and a further increase of 3°C is predicted by the end of the twenty-first century for summer temperatures in the Mediterranean basin (IPCC, 2014). Because insects are ectotherms, their physiology, behaviour and fitness are directly affected by ambient temperature variation [START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF]. The impact of global warming is likely to be less predictable in higher trophic levels than in lower trophic levels because the former also depend on the capacity of the lower trophic level to adapt to these changes [START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF][START_REF] Van Baaren | Consequences of Climate Change for Aphid-Based Multi-trophic Systems[END_REF]. That impact is observed for parasitoids in which immature individuals feed and develop in (endoparasitoids) or on (ectoparasitoids) hosts. Parasitoids are the most important and successful group of natural enemies used in the biological control of insect pests [START_REF] Godfray | Parasitoids. Behavioral and Evolutionary Ecology, Monographs in behavior and ecology[END_REF][START_REF] Jervis | Insects as natural enemies: A practical perspective[END_REF], and their efficacy could be disrupted by changes in environmental conditions [START_REF] Van Baaren | Consequences of Climate Change for Aphid-Based Multi-trophic Systems[END_REF].

An increase in temperature can affect host-parasitoid relationships, mostly because they may have different thermal preferences (developmental or phenological), which can affect the temporal or geographical synchronization of the suitable instar/stage of the host with the adult parasitoids [START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF]. For parasitoids, an increase in temperature can affect their morphology (body size, wing size, wing loading), fecundity, longevity, dispersal capacity, metabolism rate, trade-offs between lifehistory traits, capacity to locate and evaluate host quality and the capacity of the larvae to evade or overcome the host immune response [START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF][START_REF] Moiroux | Local adaptations of life-history traits of a Drosophila parasitoid, Leptopilina boulardi: does climate drive evolution?[END_REF][START_REF] Vayssade | The response of life-history traits to a new species in the community: A story of Drosophila parasitoids from the Rhône and Saône valleys[END_REF][START_REF] Vuarin | Geographic variations of life history traits and potential trade-offs in different populations of the parasitoid Leptopilina heterotoma[END_REF][START_REF] Van Baaren | Consequences of Climate Change for Aphid-Based Multi-trophic Systems[END_REF]. Indeed, an increase in temperature may induce a number of physiological changes, the cost of which may be expressed by a reduction in reproductive output, decrease in growth of immature stages and in lifespan and/or changes in mating behaviour [START_REF] Angilletta | Thermal Adaptation: A Theoretical and Empirical Synthesis[END_REF][START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF][START_REF] Omer | Demography of the leafminer parasitoid Ganaspidium utilis Beardsley (Hymenoptera: Eucoilidae) at different temperatures[END_REF][START_REF] Řežucha | Heat induced male sterility in Drosophila melanogaster: adaptive genetic variations among geographic populations and role of the Y chromosome[END_REF]. Understanding these characteristics is crucial for using parasitoids as biological control agents.

Because of interspecific differences in thermal responses among parasitoid species [START_REF] Berg | Adapt or disperse: Understanding species persistence in a changing world[END_REF], global warming can also have a major influence on interspecific competition between species [START_REF] Northfield | Coevolution and the effects of climate change on interacting species[END_REF]. As such, global warming will have consequences for parasitoid species distributions, community compositions, and ecosystem services, i.e., biological control [START_REF] Bale | Herbivory in global climate change research: Direct effects of rising temperature on insect herbivores[END_REF][START_REF] Hance | Impact of extreme temperatures on parasitoids in a climate change perspective[END_REF][START_REF] Northfield | Coevolution and the effects of climate change on interacting species[END_REF][START_REF] Tougeron | Intraspecific maternal competition induces summer diapause in insect parasitoids[END_REF].

In this context, we investigated the influence of the expected temperature increase on the efficacy and competition of parasitoids of the genus Aphytis (Hymenoptera: Aphelinidae), which are the most successful and widespread biological control agents of Aonidiella aurantii (Hemiptera: Diaspididae) in citrus [START_REF] Debach | Biological control by natural enemies[END_REF][START_REF] Forster | The role of natural enemies of California red scale in an IPM program in California citrus[END_REF][START_REF] Murdoch | Host suppression and stability in a parasitoidhost system: experimental demonstration[END_REF][START_REF] Pekas | Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids[END_REF].

These specialist parasitoids can reduce their shared host to levels nearly 200 times below the average density observed in their absence [START_REF] Debach | Biological control of coccids by introduced natural enemies[END_REF], suggesting strong resource competition between parasitoid species because they parasitized the same host stages and niches [START_REF] Borer | Parasitoid coexistence: linking spatial field patterns with mechanism[END_REF][START_REF] Pekas | Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids[END_REF]. Species of the genus Aphytis represent one of the best-known cases of competitive displacement in insects [START_REF] Luck | Host selection and egg allocation behaviour by Aphytis melinus and A. lignanensis: Comparison of two facultatively gregarious parasitoids[END_REF][START_REF] Luck | A Darwinian view of host selection and its practical implications[END_REF][START_REF] Luck | Competitive exclusion of Aphytis lingnanensis by A. melinus: potential role of host size[END_REF][START_REF] Pekas | Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids[END_REF][START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF]. In the Mediterranean basin, A. aurantii became a key citrus pest at the end of the last century, and it was rapidly parasitized by the native parasitoid Aphytis chrysomphali (Mercet). Later, its coevolved parasitoid Aphytis melinus DeBach was introduced into a classical biological control programme, and it has displaced A. chrysomphali in some areas, whereas both species coexist in others [START_REF] Boyero | Displacement of Aphytis chrysomphali by Aphytis melinus, parasitoids of the California red scale, in the Iberian Peninsula[END_REF][START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF]. Both parasitoids are present in the field from February to November showing higher parasitism levels between August and November (highest in September) and lower in February and March [START_REF] Pekas | Influence of host size on parasitism by Aphytis chrysomphali and A. melinus (Hymenoptera: Aphelinidae) in Mediterranean populations of California red scale Aonidiella aurantii (Hemiptera: Diaspididae)[END_REF]. Although A. chrysomphali reproduces parthenogenetically and produces only females [START_REF] Pina | Mercet) (Hymenoptera : Aphelinidae)[END_REF][START_REF] Cebolla | Does host quality dictate the outcome of interference competition between sympatric parasitoids? Effects on their coexistence[END_REF], A. melinus is considered to be a superior competitor in the field because it has a higher capacity for dispersion [START_REF] Mclaren | A behavioural study of Aphytis chrysomphali (Mercet) and Aphytis melinus DeBach (Hymenoptera: Aphelinidae) parasitic on California red scale, Aonidiella aurantii[END_REF] and is better adapted to dry and hot climates where citrus is cultivated (Abdelrahman, 1974a;[START_REF] Rosen | Species of Aphytis of the world (Hymenoptera: Aphelinidae)[END_REF]. Consequently, the relative proportion of A. melinus is higher during the warm months, and the abundance of A. chrysomphali increases from south to north, being higher in the cooler northern areas. This alteration in parasitoid dominance could be one of the reasons why the more efficient parasitoid A. melinus has not completely displaced A. chrysomphali in most Valencia citrus orchards [START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF]. Therefore, we hypothesize that the superiority of A. melinus will be accentuated when the temperature increases due to global warming, possibly leading to the extinction of the weaker competitor. To test this hypothesis, we performed laboratory experiments to evaluate how the increase in temperature in summer, when A. chrysomphali is a weaker competitor, will affect i) the fecundity and parasitism rate of both parasitoids when they exploit hosts alone or in competition and ii) the influence of temperature and competition on their efficacy as the biological control of A. aurantii by comparing their net reproductive rates (R0) and host-induced mortality.

MATERIALS AND METHODS

Abiotic conditions and treatments

The

Insects

The phytophagous host, Aonidiella aurantii, was reared on lemons from a laboratory colony at the Instituto Valenciano de Investigaciones Agrarias, IVIA (Moncada, Valencia, Spain). This colony was initiated in 1999 from scales collected in citrus fields in Alzira (Valencia, Spain) and renewed every 2-3 years with field-collected scales. Approximately 2/3 of the surface of each lemon was covered with red paraffin around the mid-section to retard its desiccation. The red paraffin was prepared with a mixture of 1 kg of paraffin pearls (Parafina USP Perlas; Guinama S.L., Alboraya, Spain) and 1 g of red pigment (Sudan III; Panreac Química S.A., Castellar del Vallés, Spain). The remaining surface (aprox. 24-cm 2 area) of the lemons was infested by exposing them to gravid female scales of the A. aurantii colony for 48 h at 27 ± 1°C at 70 ± 5% RH and LD 14:10. Once infested, lemons were kept in climate chambers (SANYO MLR-350; Sanyo, Japan) at the two temperature conditions described above until female scales reached the third nymphal instar (19-25 days), which was later used for rearing the parasitoids and for the experiments (Treatments 2,3 and 4) or until females become gravid (43-50 days) for the fecundity investigation (Treatment 1).

Aphytis melinus and A. chrysomphali are facultative gregarious ectoparasitoids [START_REF] Rosen | Species of Aphytis of the world (Hymenoptera: Aphelinidae)[END_REF]. Females of both species mature eggs throughout their adult life (synovigenic) and lay between 2 and 6 eggs per day [START_REF] Heimpel | Adult feeding and lifetime reproductive success in the parasitoid Aphytis melinus[END_REF]Casas et al., 2000;[START_REF] Tena | Sugar provisioning maximizes the biocontrol service of parasitoids[END_REF]. These species are also idiobibionts (i.e., the host is paralyzed and arrests development once parasitized) and feed on the haemolymph of hosts which they do not use to lay eggs. Individuals of both species were obtained by exposing third-instar A. aurantii on lemons to parasitism by insectary-reared adult wasps.

The colonies of A. melinus and A. chrysomphali were initiated in 2008 and 2013, respectively, from A.

aurantii scales collected in citrus fields located in the Valencia region (Valencia, Spain). Both colonies are renewed yearly with field-collected parasitoids.

For this experiment, five adults of each species were transferred to rearing cages containing third-instar A. aurantii on lemons reared in the two abiotic conditions (described above) and were maintained in climatic chambers at these two abiotic conditions to obtain parasitoids. Between 10-12 days later, scales were observed under binoculars and late-stage pupae of both parasitoids were removed from parasitized scales. Pupae were held in crystal vials 8 mm in diameter and 35 mm long tapped with a cotton plug and with a drop of honey on the wall. At emergence, parasitoids were held in these vials for one day to obtain mated females of A. melinus [A. chrysomphali reproduces parthenogenetically [START_REF] Gottlieb | Phylogenetic analysis of parthenogenesisinducing Wolbachia in the genus Aphytis (Hymenoptera: Aphelinidae)[END_REF]]. Next, parasitoids were sexed, and females were isolated in vials (same as above). One A. aurantii female was introduced daily to let them feed on the host until they were used 2-3 days later [START_REF] Heimpel | Adult feeding and lifetime reproductive success in the parasitoid Aphytis melinus[END_REF]. Vials were stored in a climatic chamber at the two different abiotic conditions until they were used in the experiment (Treatments 2, 3 and 4).

Experimental microcosm and measures

For all treatments, the experimental microcosm was composed of a polystyrene plastic box (10 × 14×14 cm) with a lateral hole (4 × 9 cm) covered with muslin. One lemon infested with A. aurantii was introduced in the box. An acrylic cylinder (5 cm diameter) was used to hold the lemons. We used a dissecting microscope to select 40 scales from the lemon surface, and we removed the rest using an entomological pin and a paper towel moistened with water. The selected scales were 0.85 ± 0.05 mm 2 .

To estimate scale sizes, photos of the scales were taken with a Leica EC 3 3.1 megapixel digital colour camera (Leica Microsystemps GmbH, Spain), and the images were processed with Leica LAS EZ imaging software for Windows (Leica Microsystems GmbH, Spain).

Effect of competition and a temperature increase on parasitoid fitness and biological control potential

For treatments 1, 2 and 3, parasitoids were introduced in the box and remained in contact with hosts for 72 hours. Aphytis parasitoids parasitize between two to five hosts per day and per female [START_REF] Heimpel | Adult feeding and lifetime reproductive success in the parasitoid Aphytis melinus[END_REF][START_REF] Pina | Mercet) (Hymenoptera : Aphelinidae)[END_REF]Cebolla et al., submitted). Therefore, microcosms contained hosts ad libitum in all the treatments. A drop of honey was added to the inside wall as a food source.

Microcosms were held in climatic chambers at one of the two abiotic conditions. Seven days after parasitoid removal, scale covers were carefully removed with an entomological needle under a binocular lens. The numbers of unparasitized (turgent), parasitized and dead hosts were counted. We used these data to evaluate i) the number of parasitized and ii) the dead hosts per patch. The number of dead was the sum of parasitized and hosts dead by unknown reasons (caused by the parasitoids or natural death of hosts). Aphytis parasitoids cause the death of their host when they host-feed and when they probe the host with their ovipositor even when they reject the host [START_REF] Cebolla | Does host quality dictate the outcome of interference competition between sympatric parasitoids? Effects on their coexistence[END_REF].

The parasitoid pupae of each parasitized host were measured and then transferred to crystal vials as described above. Between one and three pupae were obtained per host. To measure the effect of temperature and parasitoid competition on parasitoid size (measured as the length of the pupae), only pupae of females that developed in solitary were considered because sex and brood size can affect pupae size (Salt, 1940;Abdelrahman, 1974b). At emergence, parasitoids were identified and sexed. Aphytis chrysomphali pupae are identified by the presence of a longitudinal black line on the mesosternum which is not present in A. melinus [START_REF] Rosen | Species of Aphytis of the world (Hymenoptera: Aphelinidae)[END_REF]. Progeny production was calculated as the number of adults of each species that emerged per patch. Secondary sex ratio was calculated as the proportion of males of each species per host and patch.

Effect of an increase in temperature on host fecundity

To determine the effect of an increase in temperature on host fecundity (Treatment 4), we used the same methodology described by [START_REF] Vanaclocha | Life history parameters and scale-cover surface area of Aonidiella aurantii are altered in a mating disruption environment: Implications for biological control[END_REF]. In detail, infested lemons were kept in climatic chambers at the two abiotic conditions. Before the female scales began to produce crawlers (~40 days), four scales per lemon were isolated with a double-sided sticky plastic ring (3M Scotch®;

CergyPontoise Cedex, France) to trap the crawlers. Sticky plastic rings were replaced twice a week and the crawlers were counted under binoculars. The total number of progeny was calculated as the sum of each ring per female.

Effect of an increase in temperature on parasitoid net reproductive rate (R0)

The net reproductive rate (R0) during 72 hours was compared between the two parasitoid species in treatments 1, 2 (without competition) and 3 (with competition). R0 was calculated as R0 = ∑ l x • m x . R0 represents the mean number of female offspring produced by each female [START_REF] Birch | The Intrinsic Rate of Natural Increase of an Insect Population[END_REF][START_REF] Carey | Applied demography for biologists[END_REF], where x is the age class, l x is the probability of survival till class x, and m x is the fecundity of class x. Values of R0 < 1 indicate a declining population, R0 > 1 an increasing population, and R0 = 1 a stable population [START_REF] Carey | Applied demography for biologists[END_REF]. To facilitate comparison of the demographic parameters, we calculated the standard errors (SE) of the demographic parameters at each temperature using a jackknife algorithm described by [START_REF] Meyer | Estimating uncertainty in population growth rates: jackknife vs. bootstrap techniques[END_REF]. The jackknife analysis method removes one observation at a time from the original dataset and recalculates the statistic of interest from the truncated data. The values of R0 per female and per temperature were calculated in Microsoft Excel and its numerical solver. The method can estimate R0 values with their respective jackknife variances.

Statistical analyses

Proportional and count data were analysed with generalized linear models (GLMs). Initially, we assumed a Poisson error variance for count data (number of parasitized hosts and number of progeny per patch) and a binomial error variance for proportional data (sex ratio and hosts killed per patch).

We assessed the assumed error structures by a heterogeneity factor equal to the residual deviance divided by the residual degrees of freedom. If we detected an over-or underdispersion, we reevaluated the significance of the explanatory variables using an F test after rescaling the statistical model by a Pearson's chi-square divided by the residual degrees of freedom [START_REF] Crawley | The R Book[END_REF]. We present the means of untransformed proportion and count data (in preference to less intuitive statistics such as the back-transformed means of logit-transformed data).

We compared pupa size of the offspring, host fecundity and R o using ANOVAs. The normality assumption was assessed using Shapiro's test, and the homoscedasticity assumption was assessed with the Levene test. All data analyses were performed with the R freeware statistical package (http://www.R-project.org/).

RESULTS

3.1

Effect of competition and an increase in temperature on parasitoids fitness and biological control potential

Number of parasitized hosts

The number of hosts parasitized by A. melinus females was independent of the increase in temperature (F 1, 83 = 0.34; P = 0.56) and the presence of their competitor, A. chrysomphali (F 1, 83 = 0.31; P = 0.58) (Fig. 1). The interaction between temperature and competition was not significant (F 1, 82 = 0.31; P = 0.58).

The number of hosts parasitized by A. chrysomphali females was independent of temperature (F 1, 87 = 0.15; P = 0.70) but it decreased with the presence of A. melinus (interspecific competition) (F 1, 87 = 13.92; P < 0.001) (Fig. 1). The interaction between temperature and competition was not significant (F 1, 86 = 0.74; P = 0.79).

Progeny production

The amount of progeny emerging from the hosts parasitized by A. melinus was independent of temperature (F 1, 83 = 0.044; P = 0.83) and competition (F 1, 83 = 0.55; P = 0.46) (Fig. 2). The interaction between temperature and competition was not significant (F 1, 82 = 0.041; P = 0.84).

The amount of progeny emerging from the hosts parasitized by A. chrysomphali was independent of temperature (F 1, 87 = 0.012; P = 0.91) but it decreased in the presence of A. melinus (competition) (F 1, 87 = 13.86; P < 0.001) (Fig. 2). The interaction between temperature and competition was not significant (F 1, 86 = 0.33; P = 0.56).

Parasitoid size

The pupae size of A. melinus and A. chrysomphali offspring was not affected by the increase in temperature (A. melinus: F 1, 46 = 2.93; P = 0.094; A. chrysomphali: F 1, 41 = 0.26; P = 0.61) or competition (A. melinus: F 1, 46 = 0.31; P = 0.58; A. chrysomphali: F 1, 41 = 0.044; P = 0.83) (Fig. 3).

The interaction between temperature and competition was not significant in either parasitoid species (A. melinus: F 1, 45 = 0.032; P = 0.86; A. chrysomphali: F 1, 40 = 1.54; P = 0.22).

Sex ratio

The secondary sex ratio (proportion of males out of the total emerging wasps) of A. melinus was affected by temperature (F 1, 65 = 5.37; P = 0.024). The proportion of males increased with temperature. However, sex ratio was independent of the presence of the competitor, A. chrysomphali (F 1, 65 = 1.83; P = 0.18) (Fig. 4). The interaction between temperature and competition was not significant (F 1, 64 = 0.049; P = 0.83). As expected, all emerging A. chrysomphali were females.

Host mortality

In the absence of competition, the number of A. aurantii hosts killed by A. melinus and A.

chrysomphali was not affected by the increase in temperature (A. melinus: F 1, 40 = 0.24; P = 0.63; A.

chrysomphali: F 1, 44 = 0.028; P = 0.87). The same phenomenon occurred when both parasitoids were competing (F 1, 86 = 0.18; P = 0.68) (Fig. 5).

Effect of an increase in temperature on host fecundity

Aonidiella aurantii fecundity, measured as the total number of crawlers, was not affected by an increase in temperature (20-26°C = 224.82 ± 11.99 crawlers; 23-29°C = 242.29 ± 23.37 crawlers; F 1, 35 = 0.48; P = 0.49).

Effect of an increase in temperature on parasitoid net reproductive rate (R0)

The net reproductive rate (R0) of A. melinus was affected by the interaction between temperature and competition (F 1, 88 = 279.73; P < 0.0001) (Table 1), indicating that R0 increased with competition when there is a high temperature but it decreased with competition at a low temperature.

This finding showed that the negative effect of a temperature increase is reduced in the presence of an inferior competitor.

The net reproductive rate (R0) of A. chrysomphali was affected by the interaction between temperature and competition (F 1, 91 = 50.69; P < 0.0001) (Table 1), indicating that R0 decreased with the increase in temperature when there was no competition but it increased with the increase in temperature when there was competition. This showed that the negative effect of a temperature increase is absent in the presence of a superior competitor.

DISCUSSION

The presence of the introduced parasitoid Aphytis melinus reduced the efficacy of the native Aphytis chrysomphali as a biological control agent of the California red scale (Aonidiella aurantii) by approximately 50% in the present conditions. The potential of Aphytis parasitoids was measured using the net reproductive rate (R0), which represents the mean number of female offspring produced by each female, considering also the probability of survival of the female. The reduction in net reproductive rate was mainly due to the reduction of scales successfully parasitized by A.

chrysomphali females when they searched in the same patch as A. melinus females compared to patches without competition. Several biological traits described previously in both parasitoids can explain the reduction in the number of scales successfully parasitized by A. chrysomphali when it shared the patch with its competitor. Aphytis melinus females tend to kill A. chrysomphali eggs (i.e., ovicide) before laying their own egg when they are locating a host already parasitized by A.

chrysomphali (Cebolla et al., 2017a, b). Moreover, A. melinus larvae seem to be more aggressive than those of A. chrysomphali (Cebolla et al., 2017a, b). Therefore, A. melinus might have killed some of the progeny of A. chrysomphali, reducing its R0. Another non-mutually exclusive reason that may explain the reduction of A. chrysomphali R0 is that females of this species might avoid using patches where A. melinus are searching. However, this hypothesis needs to be tested. Overall, the 50% reduction of A. chrysomphali R0 when it searches in the same patch as A. melinus represents another result that explains the displacement of A. chrysomphali by A. melinus in southern Spain [START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF][START_REF] Boyero | Displacement of Aphytis chrysomphali by Aphytis melinus, parasitoids of the California red scale, in the Iberian Peninsula[END_REF]. Other reasons are the lower searching capacity and tolerance to hot and dry climates of A. chrysomphali (Abdelrahman, 1974a, b;[START_REF] Mclaren | A behavioural study of Aphytis chrysomphali (Mercet) and Aphytis melinus DeBach (Hymenoptera: Aphelinidae) parasitic on California red scale, Aonidiella aurantii[END_REF]. Both parasitoids, however, coexist in eastern Spain where the proportion of A. chrysomphali increases with latitude and colder temperatures [START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF][START_REF] Pekas | Influence of host size on parasitism by Aphytis chrysomphali and A. melinus (Hymenoptera: Aphelinidae) in Mediterranean populations of California red scale Aonidiella aurantii (Hemiptera: Diaspididae)[END_REF][START_REF] Pekas | Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids[END_REF]. Under this scenario, we expected that the predicted increase in temperature under global warming, especially in the summer, would negatively affect the net reproductive rate of A. chrysomphali, accelerating its displacement by A. melinus. Our results, however, showed that the net reproductive rate of A. chrysomphali was not affected by the expected increase of temperature.

The expected increase in temperature in summer will hinder the efficacy of A. melinus as a biological control agent of A. aurantii, as the R0 of this parasitoid was reduced by half. The reduction of A. melinus R0 when temperature increased was mostly due to the reduction in the proportion of females (i.e., increase of the secondary sex ratio proportion). This result is in accordance with previous studies of hymenopteran parasitoids, which suggest that the proportion of males increases with temperature [START_REF] King | Offspring sex ratios in parasitoid wasps[END_REF]. The biological reasons behind the change in sex ratio are less clear. Two hypotheses might explain the increase in A. melinus sex ratio with temperature. First, mothers might consider high temperatures unfavourable for the development of their offspring and thus may have intentionally allocated sons, as suggested by [START_REF] Force | Fecundity, reproductive rates, and innate capacity for increase of three parasites of Therioaphis maculata (Buckton)[END_REF] and [START_REF] Moiroux | Sex ratio variations with temperature in an egg parasitoid: Behavioural adjustment and physiological constraint[END_REF].

Second, the high temperatures used in this assay might also decrease the ability of A. melinus to mate, as was previously observed in parasitoids exposed to hot thermal stresses [START_REF] Abram | Behavioural effects of temperature on ectothermic animals: unifying thermal physiology and behavioural plasticity[END_REF][START_REF] Jørgensen | Heat tolerance and the effect of mild heat stress on reproductive characters in Drosophila buzzatii males[END_REF][START_REF] Krebs | Effects of exposure to short-term heat stress on fitness components in Drosophila melanogaster[END_REF][START_REF] Patton | The effect of thermal stress on the mating behavior of three Drosophila species[END_REF][START_REF] Řežucha | Heat induced male sterility in Drosophila melanogaster: adaptive genetic variations among geographic populations and role of the Y chromosome[END_REF][START_REF] Roux | How does heat shock affect the life history traits of adults and progeny of the aphid parasitoid Aphidius avenae (Hymenoptera: Aphidiidae)? Bull[END_REF][START_REF] Sisodia | Effect of exposure to short-term heat stress on survival and fecundity in Drosophila ananassae[END_REF][START_REF] Wilkes | Environmental causes of variation in the sex ratio of an arrhenotokous insect, Dahlbominus fuliginosus (Nees) (Hymenoptera: Eulophidae)[END_REF]. In our assay, adult parasitoids were paired in small vials under the two selected abiotic conditions before the experiments started. Two out of the 24 couples exposed to the temperatures expected for 2035 did not produce females, whereas all couples produced at least one female under the current summer temperatures. This result suggests that mating was negatively affected by the increase in temperature but can, together with other traits, explain the increase in sex ratio. Conversely, we do not expect that host size and/or the infection of A.

melinus by Wolbachia affected the sex ratio of A. melinus because we used hosts of similar size in the experiment (see section 2.3 in Materials and Methods) and temperatures below 30°C do not completely cure A. melinus of the infection [START_REF] Vasquez | Discovery of a CI-inducing Wolbachia and its associated fitness costs in the biological control agent Aphytis melinus DeBach ( Hymenoptera : Aphelinidae )[END_REF]. The curation of females might have produced incompatible crosses and production of males by cured females [START_REF] Vasquez | Discovery of a CI-inducing Wolbachia and its associated fitness costs in the biological control agent Aphytis melinus DeBach ( Hymenoptera : Aphelinidae )[END_REF].

The presence of A. chrysomphali did not reduce A. melinus R0 when females of both species shared the same patch. Additionally, the competition between both parasitoids mitigated the negative effect of the increase in temperature on A. melinus. Under the summer temperatures expected for the end of the twenty-first century, the introduced parasitoid had higher R0 when females competed with A. chrysomphali than when they searched alone in a patch. Generally, high temperatures affect the metabolic rate of insects and can diminish their locomotion speed and activity level [START_REF] Vogt | Effects of temperature and season on foraging activity of red imported fire ants (Hymenoptera: Formicidae) in Oklahoma[END_REF][START_REF] Pachauri | Climate Change 2014: Synthesis report. Contribution of working groups I, II and III to the Fifth assessment report of the Intergovernmental Panel on Climate Change, Core Writing Team[END_REF], increasing, in the case of the hymenopteran parasitoids, the handling time per oviposition [START_REF] Langer | Oviposition, flight and walking capacity at low temperatures of four aphid parasitoid species (Hymenoptera : Aphidiinae)[END_REF][START_REF] Wu | Temperature influences the handling efficiency of an aphid parasitoid through body size-mediated effects[END_REF]. The presence of a competitor could, however, stimulate A. melinus to complete oviposition faster as occurs in the solitary egg parasitoid Enoggera nassaui (Griault) (Hymenoptera: Pteromalidae) when it shares a patch with competitors (Mansfield, 2016). Another possibility is that the presence of a competitor provoked an aggressive behaviour in A.

melinus. There are numerous studies linking female-female competition and aggressiveness in diverse animal taxa [START_REF] Stockley | Female competition and aggression: interdisciplinary perspectives[END_REF]. This aggressiveness could be reflected in direct aggressive behaviours towards the competing parasitoid, which would explain the reduction of A.

chrysomphali R0 when competing together but could also be reflected in an increase in parasitism that could compensate for the diminution of the R0 consequence of the temperature increase.

Overall, our study highlights the importance of considering competition to predict the consequences of global warming for biological control. Our results, however, have to be interpreted with care, since these are based on small microcosms (cages) and, therefore, the effect of global warming on the dispersion capacity of both parasitoids has not been considered. Further research could use larger cages or flying studies under different temperatures to include their dispersion capacity and to confirm the results. Nevertheless, our data strongly suggests that the expected increase in temperature in the Mediterranean basin will negatively affect the introduced parasitoid A. melinus and, consequently, the biological control of A. aurantii, one of the main citrus pests (Tena and Garcia-Marí, 2011). Interestingly, in southern Spain and other areas where A. melinus has already displaced the native A. chrysomphali, the effect of global warming will be higher than in eastern Spain and other areas where both parasitoids coexist. This is because A. chrysomphali R0 will not be affected by the increase in temperature and competition will mitigate the negative effect of the increase in temperature on A. melinus. Several non-excluding hypotheses have been proposed to explain their coexistence in eastern Spain. These hypotheses include the presence of alternative hosts and climate, conditional patch partitioning and local weather conditions [START_REF] Pina | Mercet) (Hymenoptera : Aphelinidae)[END_REF][START_REF] Sorribas | Parasitoid competitive displacement and coexistence in citrus agroecosystems: linking species distribution with climate[END_REF][START_REF] Pekas | Host size and spatiotemporal patterns mediate the coexistence of specialist parasitoids[END_REF]Cebolla et al., 2017a). If the native is finally displaced in this area, a decline in the biological control of A. aurantii can also be expected. Data are presented as the mean ± SE, obtained by the jackknife method. Different lowercase letters denote significant differences between temperatures within a treatment (without competition and with competition) and parasitoid species at P < 0.05. Different uppercase letters denote significant differences between treatments (with and without competition) within a temperature and parasitoid species at P < 0.05.

Figure legend

Fig. 1 .Fig. 2 .

 12 Fig. 1. Effect of temperature (20-26°C or 23-29°C) and interspecific competition on the number of hosts parasitized by the parasitoids Aphytis melinus and A. chrysomphali during 72 hours. The different temperatures represent the mean temperature of summer from 2009 to 2014 in the Mediterranean basin and the temperature predicted by the end of the twenty-first century by the IPCC. Different letters above columns denote significant differences between treatments (with and without competition) within a parasitoid species and temperature at P < 0.05.

Fig. 3 .

 3 Fig. 3. Effect of temperature (20-26°C or 23-29°C) and interspecific competition on the pupa size of the parasitoids Aphytis melinus and A. chrysomphali. The different temperatures represent the mean temperature of summer from 2009 to 2014 in the Mediterranean basin and the temperature predicted by the end of the twentyfirst century by the IPCC).

Fig. 4 .

 4 Fig. 4. Effect of temperature (20-26°C or 23-29°C) and interspecific competition on the sex ratio of the parasitoid Aphytis melinus. Presented as the mean proportion of males of A. melinus (± SE). The different temperatures represent the mean temperature of summer from 2009 to 2014 in the Mediterranean basin and the temperature predicted by the end of the twenty-first century by the IPCC. Different letters above columns denote significant differences between temperatures within the same competition treatment at P < 0.05.

Fig. 5 .

 5 Fig. 5. Effect of parasitoid species (Aphytis melinus or A. chrysomphali), temperature (20-26°C or 23-29°C) and parasitoid competition on the mortality of their common host, Aonidiella aurantii. Presented as the mean number of A. aurantii killed (± SE) by the parasitoids during 72 hours. The different temperatures represent the mean temperature of summer from 2009 to 2014 in the Mediterranean basin and the temperature predicted by the end of the twenty-first century by the IPCC.

Fig

  Fig. 1.

Table 1 .

 1 Net reproductive rate (R0) of the parasitoids Aphytis melinus and A. chrysomphali at two temperatures, with and without interspecific competition.

	R0

Acknowledgements

The authors would like to thank P. Bru (IVIA) for technical assistance with experiments. R. Cebolla received a pre-doctoral fellowship from the Spanish Ministry of Science and Innovation. A. Tena received postdoctoral fellowships from INIA and IVIA. This work was partially funded through the Spanish Ministry of Science and Innovation (AGL2011-30538-C03-02).

Highlights

Control of Aonidiella aurantii by two parasitoids was measured under two temperature regimes Aphytis chrysomphali R 0 was decreased by 50% in competition, but was unaffected by higher temperature A. melinus R 0 decreased 40% at higher temperature because the proportion of females was reduced A. chrysomphali presence partially mitigated the effect of higher temperature on A. melinus


Pedro Mateus

Barbara Wicher

Yann Ferrand

Ivan Huc

Alkali and alkaline earth metal ion

come

1

Alkali and alkaline earth metal ion binding by a foldamer capsule: selective recognition of magnesium hydrate †

Pedro Mateus, a Barbara Wicher, b Yann Ferrand a and Ivan Huc* a a Univ. Bordeaux and CNRS, CBMN (UMR 5248), IECB, 2 rue Robert Escarpit, F-33600 Pessac, France. E-mail: i.huc@iecb.ubordeaux.fr. b Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland. Alkali and alkaline earth metal ion binding by an aromatic oligoamide foldamer was shown to induce its folding into a helical capsule. CD and NMR titrations revealed tight and selective binding of Mg 2+ . Crystallographic studies demonstrated that, depending on the metal, binding may involve the first or second coordination spheres of the metal hydrates.

Aromatic helical foldamers and related macrocycles have emerged as versatile bioinspired containers for molecular recognition. [1][2][START_REF] Berl | Neutral guest recognition[END_REF][START_REF] Prince | Cation recognition[END_REF][5] When foldamer helices possess reduced diameters at both extremities they form capsules that sequester guest molecules and isolate them from the solvent. 3g-k,5f In sequences based on aza-aromatic oligoamides, the helix inner wall is composed of numerous hydrogen bond donors and acceptors that may give rise to tight, selective, and diastereoselective binding of polyhydroxylated guests such as monosaccharides, 3k and organic hydroxy-acids. 3i,j We have recently introduced sequence 1 (Fig. 1) whose central pyz-pyrpyz segment was shown to bind to transition metal ions Cu(I), Cu(II) and Ag(I) in such a way that the metal sits on the cavity wall and that part of its coordination sphere remains available to contribute to the recognition of an organic guest. 6 Metal ion binding was also shown to drive helical folding: in the absence of metal ion, the pyz-pyr-pyz segment exists in an anti-anti conformation favoured by repulsion between endocyclic nitrogen atoms (Fig. 1c) that confers the oligomer with an extended conformation in which the two hemi-capsules are not facing each other (Fig. 1a, centre).

Here we report the binding of alkali and alkaline-earth metal ions by sequence 1. The outer s-electron shell of these metal ions does not promote an affinity of pyridine-based ligands as large as that of transition metals. We nevertheless find that Na + , K + , Mg 2+ , Ca 2+ and Ba 2+ all bind to 1 and induce its folding in a helix; outstanding affinity and selectivity for Mg 2+ was observed. The hydration sphere of the metals is essential to the binding process to such an extent that, in the case of Mg 2+ , Ca 2+ and Ba 2+ binding does not involve the pyzpyr-pyz segment as a first coordination sphere ligand. Instead, the first coordination sphere water molecules undergo second coordination sphere interactions with the capsule that are so strong that they overcome helix folding in the case of barium nona-hydrate, a guest too large to fit in the cavity. Titrations of 1 (1 mM) with Na + , K + , Mg 2+ , Ca 2+ and Ba 2+ in CDCl3/CD3CN (6:4 vol/vol) were monitored by 1 H NMR. In each case, a new set of sharp signals emerged indicating the formation of a metal complex in slow exchange on the NMR timescale with uncomplexed 1 (Fig. 2a and Figs. S6-10 in the ESI). Saturation was reached at 1 equiv. of metal ion, showing that binding is too strong (Ka > 10 5 M -1 ) to be accurately determined by this technique. Complexes with Na + , K + , Mg 2+ and Ca 2+ display six amide NH signals consistent with the conservation of the average C2 symmetry of the helix. In contrast, the Ba 2+ complex displays many more signals that span over a wider chemical shift range, indicating a loss of symmetry of the complex and the possible coexistence of several binding modes. Signals found at lower fields (> 12 ppm) mark weaker ring current effects associated with aromatic stacking and thus a possible perturbation of the helical structure. Further titrations were monitored by CD spectroscopy using 2 (0.03 mM) in CDCl3/CD3CN (6:4 vol/vol). This sequence is equipped with terminal (1S)-(-)-camphanyl groups (Fig. 1b) that quantitatively induce P helicity. 7 The Q3PN2 segments of 2 should thus both be P-helical regardless of the conformation of the central pyz-pyr-pyz and give rise to a positive CD band in the 350-400 nm region. 8 Yet the anti-anti to syn-syn conformational change induced by the metal ion was found to be associated with a substantial increase of the molar ellipticity (Fig. 2b,c conformer also contributes to the P-helicity. Saturation was again reached near 1 equiv. of guest. It was nevertheless possible to accurately determine Ka values for Ba 2+ and K + . For Na + , Mg 2+ and Ca 2+ Ka values are so high that they cannot be calculated directly, but they could be determined in 1 H NMR competition experiments (Figs. S11-13 in the ESI). The results, compiled in Table 1, showed the binding trend Ba 2+ < K + << Ca 2+ ≈ Na + << Mg 2+ . Quite remarkably the capsule is extremely selective for Mg 2+ , displaying a 450 fold selectivity over the second best bound guest Na + .

A 1 H NMR competition experiment was set up to better demonstrate selective Mg 2+ binding (Fig. 3). A sample of sequence 1 was prepared in CDCl3/CD3CN (6:4 vol/vol) mixture in an NMR tube to which Ba 2+ and K + (1 equiv. each) were added (Fig. 3a). The spectrum then clearly shows preferential binding of K + over Ba 2+ . Upon adding Ca 2+ (1 equiv.) to the same NMR tube, the spectrum shows that this metal ion displaces Ba 2+ and most of K + (Fig. 3b). The further addition of Na + (1 equiv.) led to the disappearance of 1⊃K + and to the coexistence of 1⊃Na + and 1⊃Ca 2+ which have similar stability (Fig. 3c). Finally, Mg 2+ (1 equiv.) quantitatively displaced all other metal ions to produce 1⊃Mg 2+ (Fig. 3d).

For most applications, discrimination from Ca 2+ is the major difficulty in the design of Mg 2+ ionophores. 9 The selective binding of magnesium over calcium by capsule 1 (K Mg /K Ca = 740) is thus remarkable. Among the numerous examples of Mg 2+ ionophores reported in the literature, 10 it appears that only one surpasses capsule 1 and does not bind calcium. 11 Also noteworthy is the fact that 1 prefers Mg 2+ over transition metals Cu + and Zn 2+ . (Figs. S14 and S15 in the ESI). Crystallographic studies were undertaken to gain further insights into the recognition modes (Fig. 4). Single crystals suitable for X-ray diffraction were obtained by slow diffusion of hexane into solutions of the 1⊃Na+, 1⊃K+, 1⊃Ca2+ and 1⊃Ba2+ complexes. Despite the stability of 1⊃Mg2+, crystals were not obtained under these conditions and attempts using different magnesium salts or solvent systems also failed. The crystal structures of 1⊃Na+ and 1⊃K+ (Fig. 4a,b), revealed tridentate coordination of the pyz-pyr-pyz unit to the metal ions (see also Figs. S16 and S17 in the ESI). Sodium displays shorter nitrogen-metal distances than potassium (2.586( 8), 2.625( 8) and 2.701( 8) Å for Na+ and 2.747(9), 3.031(10) and 3.119( 8) Å for K+) consistent with its higher association constant. In both cases the metal ions keep part of their water of solvation (two water molecules in the case of Na+ and three in the case of K+). These coordinated water molecules establish hydrogen bonds with endocyclic nitrogen atoms of the capsule inner wall, i.e. second coordination sphere interactions, contributing to stabilize the complex and the helical structure. Additional water molecules occupy the remaining space and hydrogen bond to both the capsule and the complex. Interestingly, the number and positions of these water molecules are similar for 1⊃Na + and 1⊃K + .

The crystal structure of 1⊃Ca 2+ (Fig. 4c) showed an encapsulated calcium ion fully surrounded by water molecules. Here the cation is bound through second-sphere coordination only, with the seven water molecules of its hydration shell establishing hydrogen bonds with the capsule. One water molecule was found to hydrogen bond to the pyz-pyr-pyz unit and to occupy the position where metal ions are found when they directly coordinate to nitrogen atoms (see Fig. S18). This water presumably stabilizes the syn-syn conformation, and contributes to helical folding. In the absence of structure, we can only speculate that Mg 2+ is bound in way similar to Ca 2+ .

The crystal structure of the 1⊃Ba 2+ complex revealed an unexpected unfolding pattern with one terminal Q3 cap flipped away from the helix, allowing the Ba 2+ -triflate ion pair to protrude out of the cavity (Fig 4d). Capsule opening occurs most likely because the hydrated barium is too voluminous to fit in the closed cavity. The local disruption of the helix to accommodate Ba 2+ must have a high enthalpic price and illustrates the avidity of the capsule inner wall for these metal hydrates. Nevertheless, the conformational penalty results in weaker binding for Ba 2+ . As for Ca 2+ , Ba 2+ does not directly coordinate to the pyz-pyr-pyz unit but interacts through a water molecule (Fig. S19). Binding is thus mostly through second sphere coordination but for one carbonyl oxygen atom of a Q-P amide bond that coordinates directly to the metal. A triflate completes the first coordination sphere. It is the only anion observed coordinating the metal ion among all four structures, indicating that in most cases, negative charges are borne by water molecules.

The prime role of second-sphere coordination, i.e. hydrogen bonds, to encapsulate Ca 2+ and Ba 2+ called for additional experiments to tests the stability of these complexes in presence of a solvent that competes for hydrogen bonding. Adding 5% DMSO-d6 (vol/vol) to the complexes leads to release of Mg 2+ , Ca 2+ and Ba 2+ , while Na + and K + remain bound even in the presence of 30% DMSO-d6. This is fully consistent with the binding modes found in the crystal structures. Only when first-sphere coordination to pyzpyr-pyz is involved does the complex resist the presence of DMSO. In contrast, the binding mode of the metal hydrates of Mg 2+ , Ca 2+ and Ba 2+ resembles that of organic hydroxy acids and of monosaccharides. 3i-k This investigation thus expands the scope of molecular recognition by aromatic helical foldamers, in particular with respect to metal binding via second-sphere coordination and with respect to the reported high selectivity for magnesium.

This work was supported by the European Union (H2020-MSCA-IF-2015-707071 -RAMSES, postdoctoral fellowship to PM).

Notes and references

1 Reviews: (a) H. Juwarker, J.-m. Suk and K-S. Jeong, Chem. Soc. Rev., 2009, 38, 3316; (b) I. Saraogia and A. D. Hamilton, Chem. Soc. Rev., 2009, 38, 1726; (c)

Fig. 1

 1 Fig. 1 (a) Schematic representation of metal-induced folding modes of a helicalcapsule: first-sphere coordination (left) and second-sphere coordination (right). (b) Formula of aromatic oligoamide sequences 1 and 2. Q, N, P, pyz and pyr stand for 8-amino-2-quinolinecarboxylic acid, 7-amino-1,8-naphthyridine-2-carboxylic acid, 2,6-diaminopyridine, pyridazine and pyridine monomers, respectively. (c) Preferred conformations of pyz-pyr-pyz in the free form and in the presence of a guest.

  and Figs. S1-5 in the ESI) as the syn-syn

Fig. 2

 2 Fig. 2 (a) Excerpts from the 400 MHz 1 H NMR spectra showing the amide resonances of capsule 1 at 1 mM in CDCl3/CD3CN (6:4 vol/vol) upon binding metal ions. (b) CD spectra recorded at 298 K for the binding of Na + by capsule 2 in CHCl3/MeCN (6:4 vol/vol). [2]initial = 0.030 mM, [NaOTf]titrant = 0.80 mM; (c) Plot of ∆ε vs equiv. of Na + at 350 nm. Ka > 10 7 M -1 .

Fig. 3

 3 Fig. 3 Part of the 1 H NMR spectrum (400 MHz) at 298 K of a sample of capsule 1 (1 mM) in CDCl3/CD3CN (6:4 vol/vol) showing the respective amide resonances change upon sequentially adding each metal ion to the same sample: (a) Ba(OTf)2:KOTf 1 equiv. each; (b) plus one equiv. Ca(OTf)2; (c) plus one equiv. NaOTf (d) plus one equiv. Mg(OTf)2. Signals of the 1⊃Ba 2+ ¸ 1⊃K + , 1⊃Ca 2+ , 1⊃Na + and 1⊃Mg 2+ complexes are marked with filled circle, empty circle, filled square, empty square and filled triangle, respectively. Peaks corresponding to the 1⊃Na+complex are denoted as grey stars. Note: Peaks corresponding to the 1⊃Na+ complex are present in spectra (a) and (b) even though no Na+ was added, as this metal ion is a well-known, ubiquitous, contaminant of, for instance, glassware.

Fig. 4

 4 Fig. 4 Solid-state structures of complexes formed between capsule P-1 and (a) [Na(H2O)2] + ; (b) [K(H2O)3] + ; (c) [Ca(H2O)7] 2+ and (d) [Ba(H2O)7] 2+ . Top view of the complexes of P-1 with (e) [Na(H2O)2] + ; (f) [K(H2O)3] + ; (g) [Ca(H2O)7] 2+ and (h) [Ba(H2O)7] 2+ , showing the heterocycles that interact with the metal ions and water molecules. Dashed lines indicate hydrogen bonds. Hydrogen bonding distances can be found in the Supplementary Tables S3, S6, S9 and S12. (i) Numbering of the units of sequence 1 used in this figure. Isobutoxy side chains and cavity-excluded solvent molecules and counterions are omitted for clarity. The capsule backbone is represented in sticks. Sodium, potassium, calcium and barium atoms are represented in pink, purple, yellow and orange scaled balls, respectively. Oxygen atoms of water molecules are depicted as red balls. Blue ball in (d) and (h) represents a carbonyl oxygen atom.

Table 1

 1 Thermodynamic data for metal ion binding.

		Na + a	K + b	Mg 2+ a	Ca 2+ a	Ba 2+ b
	log Ka	8.51(5)	6.08(1)	11.16(5)	8.29(5)	5.53(1)
	Rion /Å c	1.09	1.50	0.76	1.12	1.48
	dion-water /Å c	2.43	2.84	2.10	2.46	2.82
	V /Å 3 d	60	96	39	62	93

a Determined by competition titration by 1 H NMR (400 MHz) at 298 K and in CDCl3/CD3CN (6:4 vol/vol) using capsule 1; b determined by CD at 298 K and in CHCl3/MeCN (6:4 vol/vol) using capsule 2; c from ref. 12; d calculated using dionwater as the radius of the sphere.

  B.-B. Ni, Q. Yan, Y.Ma and D. Zhao, Coord. Chem. Rev., 2010, 254, 954; (d) K.

Yamato, M. Kline and B. Gong, Chem. Commun., 2012, 48, 12142

; (e) D.-W. Zhang, X. Zhao and Z.-T.

Li, Acc. Chem. Res., 2014, 47, 1961.


Pedro Mateus

Barbara Wicher

Yann Ferrand
email: y.ferrand@cbmn.u-bordeaux.fr.

Ivan Huc
email: ivan.huc@cup.lmu.de

Carbohydrate binding through first-and second-sphere coordination within aromatic oligoamide metallofoldamers

published or not. The documents may come

The design of molecular receptors for the recognition of a given complex target guest remains a challenging task. Early approaches aimed at building macro(poly)cyclic architectures 1 or self-assembled capsules 2 designed to surround their substrates and establish multiple complementary interactions through pre-organized convergent functionalities. However, the design and synthesis of molecular receptors is frequently hampered by a lack of precise knowledge about the conformational features of both receptors and substrates and by hurdles associated with the synthesis of elaborate and relatively large structures that are difficult to further modify and optimize.

Nature has selected folding as the means to create well defined cavities with atomically precise arrays of chemical groups in space that achieve high binding affinity and selectivity. Taking this as inspiration, chemists have developed oligomeric sequences that also rely on folding to produce well defined architectures in solutioni.e. foldamers. 3 In particular, helical structures may possess a cavity suitable for the recognition of cations, 4 anions 5 or neutral molecules. 6 Furthermore, foldamer helices have been designed in such way that they possess a reduced diameter at each extremity and a wide diameter at the centre thereby forming capsules that enclose guest molecules and isolate them from the surrounding medium (Fig. 1a, right). 7 In aza-aromatic oligoamide capsules, numerous hydrogen bond donors and acceptors decorate the helix inner wall and promote tight, selective, and diastereoselective binding of polyhydroxylated guests such as organic hydroxy-acids 7c,g and monosaccharides. 7f In an effort to extend the potential for tight and selective guest binding of aromatic oligoamide-based capsules, we have recently described aromatic oligoamide capsule oligomer 1 (Fig. 1b). In this sequence, the central pyz-pyr-pyz segment coordinates transition metal ions 8 Cu + , Cu 2+ and Ag + as well as alkali metals Na + and K + . 9 In all cases the metal sits on the cavity wall leaving part of its coordination sphere occupied solely by solvent molecules, and thus available to bind a guest (Fig. 1a). Coordination bonds to the guest may indeed enhance binding affinity, while the capsule shell gives rise to shape and size selectivity. Here we show that the metal complexes of sequences 1 and 2 can be used for the binding of complex chiral guests such as carbohydrates by taking advantage of first-and/or second-sphere coordination.

The inner volume of the helical capsule 1 is relatively modest and may be suitable for small carbohydrates. Titrations of 1, 1-Na + and 1-K + with four-carbon carbohydrate D-threitol 3 at 298K in CHCl3/DMSO (9:1 vol/vol) were monitored by circular dichroism (CD) spectroscopy. In the absence of metal ion, sequence 1 was found not to bind D-3 (Fig. 2a). This was attributed to the fact that when no metal is present the pyz-pyr-pyz segment exists in an anti-anti conformation favoured by repulsion between endocyclic nitrogen atoms that confers the oligomer with an extended conformation instead of the capsular shape. 8 Coordination of Na + and K + induces folding of sequence 1 into a capsular shape by favouring a syn-syn conformation of the pyz-pyr-pyz monomer. 9 We then found that capsule 1-Na + binds to D-3, although with a modest affinity (Ka = 145 M -1 ) and with poor diastereoselectivity as evidenced by the small induced CD signal observed (Fig. 2b). In contrast, 1-K + binds the same guest almost 3 times stronger (Ka = 400 M -1 ) and shows much higher diastereoselectivity (Fig. 2c) with a negative band suggestive of a preferred M handedness. 10 A 1 H NMR titration of 1-K + with D/L-3 at 298K in CDCl3/DMSO-d6 (9:1 vol/vol) gave consistent results (Ka = 370 M -1 ). Only one set of host-guest complex signals were observed, which confirms the quantitative diastereoselectivity of the association (Fig. S8 in the ESI). For comparison, a 1 H NMR titration of 1-K + with a slightly larger carbohydrate, xylitol (4), revealed a lower binding (Ka = 150 M -1 , see Fig. S9 in the ESI), presumably because 4 cannot properly fit inside the cavity. Binding studies using the Cu 2+ complex of 1 showed that 1-Cu 2+ binds D-3 almost 14 times stronger than 1-K + (Ka = 5500 M -1 ) and adopts the opposite helix handedness (Fig. 2d).

Next, we sought to obtain structural information on the complexes. Single crystals suitable for X-ray diffraction analysis were grown by slow diffusion of hexane into solutions of 1-K + ⊃D/L-3 and 1-Cu 2+ ⊃D/L-3. S3 andS6. The crystal structure of 1-K + ⊃D/L-3 (Fig. 3a and3c) validated (i) the complete encapsulation of the guest; (ii) the position of the metal ion on the cavity wall via tridendate coordination to pyz-pyr-pyz (see also Fig. S10 in the ESI); and (iii) the handedness preference indicated by CD (D-3 is bound by the M helix). The structure also revealed bidentate coordination of the two central hydroxy groups of the guest. Furthermore, hydrogen bonds are established between the guest's peripheral hydroxy groups and endocyclic nitrogen atoms of the cavity walls. The volume fraction of the cavity occupied by the guest was calculated using SURFNET 11 and reaches 68% (Table 1), well above the common benchmark of 55%. 12 Interestingly, the volume of the cavity in the 1-K + ⊃D/L-3 complex is slightly smaller than when the cavity of 1-K + is occupied solely by water molecules 9 (139 versus 148 Å 3 , see Table 1) which means that there is a contraction of the foldamer structure to better accommodate the guest.

The crystal structure of 1-Cu 2+ ⊃ D/L-3 (Fig. 3b and3c) also corroborated CD data and showed an opposite handedness preference (D-3 is bound by the P helix). The Cu(II) centre displays a square-pyramidal stereochemistry with three nitrogen groups of the pyz-pyr-pyz monomer and an oxygen atom of a water molecule defining the basal plane. An oxygen atom of a carbonyl group of quinoline monomer occupies the apical position, which creates an angle between quinoline monomers that leads to a conformation in which the capsule is slightly open in one of the extremities. This is reflected in the larger cavity volume found for 1-Cu 2+ ⊃D/L-3 (165 Å 3 ) compared with the cavity of 1-Cu 2+ (144 Å 3 ) only occupied by an acetonitrile molecule (Table 1). 8 It is not clear whether the coordination of the main chain amide carbonyl is the cause or the consequence of the distortion and volume enlargement of the capsule to accommodate the guest. The two events are likely to be synergistic. Interestingly, the guest is not directly coordinated to the metal ion but instead is bound through second-sphere coordination in which a coordinated water molecule bridges the metal and the guest. This result was surprising, taking into account that 1-Cu 2+ binds D-3 almost 14 times stronger than 1-K + , where direct coordination is in effect. This indicates that Cu 2+ polarizes the water molecule in such way that it becomes a very strong hydrogen bond donor. Second-sphere interactions involve two hydroxyl groups in positions 1 and 3 of the threitol molecule. Consequently, the guest is not centrally located causing the deformation of the capsule cavity.

In order to target larger carbohydrates, new sequence 2 was prepared (Fig. 1b). It features two additional H monomers that almost double the binding cavity volume with respect to 1 (Table 1). These monomers present a hydrazide carbonyl group towards the binding cavity as a good hydrogen acceptor. 7f CD titrations at 298K in CHCl3/DMSO (8:2 vol/vol) showed that 2-Cu 2+ can bind various monosaccharide guests in solution (see Figs. S4-S7 in the ESI) and revealed a preference D-mannose 5 (Ka = 450 M -1 ) over D-glucose 6, D-galactose 7 and D-fructose 8 (Ka of 270, 100 and 40 M -1 , respectively). An Xray crystal structure of 2-Cu 2+ ⊃D/L-5 was eventually obtained (Fig. 4). The Cu(II) centre in 2-Cu 2+ ⊃D/L-5 again adopts a square pyramidal geometry, being bound by three nitrogen groups of the pyz-pyr-pyz monomer and by two oxygen atoms of two water molecules. The water molecule in the apical position is located outside the cavity and establishes a hydrogen bond with a carbonyl oxygen of a quinoline monomer, while the second water molecule is encapsulated and points towards the interior of the cavity. As in the case of 1-Cu 2+ ⊃D/L-3, the metal ion does not bind directly to the guest, but instead solvent molecules (a water and a methanol molecules) bridge the metal and guest. Clearly the cavity is too large to efficiently bind D-5, which occupies only 43% of the total cavity volume (Table 1). This agrees with the moderate affinities found for the monosaccharide guests. Interestingly, although D-5 exists predominantly (97%) in the α-pyranose form in CHCl3/DMSO 8:2 (vol/vol) solution, 7f it assumes the β-pyranose form in the 2-Cu 2+ ⊃D/L-5 complex. First-sphere metal coordination to assist molecular recognition has been exemplified before. 6n,13 Secondcoordination sphere interactions between Cu 2+ hydrates and carbohydrate guests is less common and is to be related to that previously observed for the binding of Ca 2+ and Mg 2+ hydrates within 1. 9 It points at the strong benefits of polarizing hydrogen bonds through metal ions and also invites to considering stronger hydrogen bond donors and acceptors such as acidic phenols or pyridine N-oxides as main chain features of the foldamer backbones. The three crystal structures described here also constitute important milestones in the challenging area of sugar recognition by synthetic receptors, 6n,14 a field that had until recently 9f,15 suffered from a complete lack of solid state evidence.

This work was supported by the European Union (H2020-MSCA-IF-2015-707071 -RAMSES, postdoctoral fellowship to PM) and benefited from the facilities and expertise of the Biophysical and Structural Chemistry platform at IECB, CNRS UMS3033, INSERM US001, Bordeaux University, France. We thank Dr. B. Kauffmann for assistance with crystallographic data collection.

Notes and references

Fig. 1

 1 Fig. 1 (a) Principle of helical-capsule folding upon metal coordination followed by metal-assisted guest encapsulation. (b) Oligoamide sequences 1 and 2. Note that amide orientation with respect to the sequence is inverted at each of the diamine and diacid sites. The two terminal Q units have an 8-nitro group instead of an 8-amino function. (c) Letter and colour codes of the amino acid, diamine, and diacid monomers. (d) Formulae of guest molecules: D-threitol, D-3; xylitol, 4; D-mannopyranose, D-5; Dglucopyranose, D-6; D-galactopyranose, D-7; D-fructopyranose, D-8. The monosaccharides are depicted in their pyranose form although in solution they exist as mixtures of α/β-pyranoses and α/β-furanoses.

Fig. 2

 2 Fig. 2 Induced CD spectra upon binding of D-3 by: (a) 1, (b) 1-Na + ; (c) 1-K + ; (d) 1-Cu 2+ at 298 K, [host] = 60 µM, 9:1 (vol/vol) CHCl3/DMSO. Red-coloured spectra in (a-d) denotes the molar ellipticity of the hosts in the presence of D-3 at the concentrations of 21, 19, 32 and 2 mM, respectively.

Fig. 3

 3 Fig. 3 Solid-state structures of the association between D-3 and complexes M-1-K + (a) and P-1-Cu 2+ (b). Top views of M-1-K + ⊃D-3 (c) and P-1-Cu 2+ ⊃D-3 (d), showing the heterocycles that interact with the guest molecules. Dashed blue lines indicate hydrogen bonds. Details of these hydrogen bonds (distances, angles) can be found in the Supplementary TablesS3 and S6. (e) Numbering of the monomers of sequence 1 used in this figure. Isobutoxy side chains, cavity-excluded solvent molecules and counterions are omitted for clarity. The capsule backbone is represented in sticks. Potassium and copper atoms are represented in purple and bronze scaled balls, respectively.

  Fig. 3 Solid-state structures of the association between D-3 and complexes M-1-K + (a) and P-1-Cu 2+ (b). Top views of M-1-K + ⊃D-3 (c) and P-1-Cu 2+ ⊃D-3 (d), showing the heterocycles that interact with the guest molecules. Dashed blue lines indicate hydrogen bonds. Details of these hydrogen bonds (distances, angles) can be found in the Supplementary TablesS3 and S6. (e) Numbering of the monomers of sequence 1 used in this figure. Isobutoxy side chains, cavity-excluded solvent molecules and counterions are omitted for clarity. The capsule backbone is represented in sticks. Potassium and copper atoms are represented in purple and bronze scaled balls, respectively.

a

  Volume of capsule cavities determined using SURFNET v1.4 (see SI). b Packing coefficients defined here as the ratio of the guest volume to the host cavity volume. Volumes of guests D-3 and D-5 were found to be 94 and 128 Å 3 , respectively. c From ref. 9. In this case the cavity of 1-K + is occupied by water molecules. d From ref. 8. In this case the cavity of 1-Cu 2+ is occupied by an acetonitrile molecule. e Predicted capsule structure obtained by molecular modelling (MMFFs force field) using Maestro v6.5.

Fig. 4

 4 Fig. 4 (a) Side view of the solid-state structure of the complex formed between capsule M-2-Cu 2+ and β-D-mannopyranose 5; (b) top view showing the heterocycles that interact with the guest. Dashed blue lines indicate hydrogen bonds. Hydrogen bonding distances and angles can be found in the Supplementary Table S9. (c) Numbering of the monomers of sequence 2 used in this figure. Isobutoxy side chains and cavity-excluded solvent molecules and counterions are omitted for clarity. The capsule backbone is represented in sticks. Copper atoms are represented bronze scaled balls.

Table 1

 1 Calculated cavity volume.