Amandine André

Nathalie Wojtowicz

Kaatio Touré

Didier Stien

Véronique Eparvier

New acorane sesquiterpenes isolated from the endophytic fungus Colletotrichum gloeosporioides SNB-GSS07

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

Endophytes are a group of microorganisms, mostly bacteria and fungi, which reside inside all plant species. [START_REF] Kusari | Chemical Ecology of Endophytic Fungi: Origins of Secondary Metabolites[END_REF] These microorganisms contribute to the defense and resilience of their host. [START_REF] Aschehoug | Fungal endophyte increases the allelopathic effects of an invasive forb[END_REF] They can be viewed as a rich source of new compounds with original biological activities. [START_REF] Jia | A Friendly Relationship between Endophytic Fungi and Medicinal Plants: A Systematic Review[END_REF] Colletotrichum gloeosporioides is a fungus from the Glomerellaceae family. It is distributed worldwide, and has been described as a phytopathogene or an endophyte. [START_REF] Jia | A Friendly Relationship between Endophytic Fungi and Medicinal Plants: A Systematic Review[END_REF][START_REF] Meyer | The structure of gloeosporone, a novel germination self-inhibitor from conidia of Collectotrichum gloeosporioides[END_REF] This fungal species can be responsible for the anthracnose disease in tropical fruitbearing plants like mango and papaya, but has also been isolated as an endophyte of many healthy plants. A large diversity of secondary metabolites such as gloeosporone, [START_REF] Meyer | The structure of gloeosporone, a novel germination self-inhibitor from conidia of Collectotrichum gloeosporioides[END_REF] ferricrocin, [START_REF] Ohra | Production of the Phytotoxic Metabolite, Ferricrocin, by the Fungus Colletotrichum gloeosporioides[END_REF] colletotric acid, [START_REF] Zou | Metabolites of Colletotrichum gloeosporioides, an Endophytic Fungus in Artemisia mongolica[END_REF] 2-phenylethyl-1H-indol-3-yl-acetate, [START_REF] Chapla | Antifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic Fungus from Michelia champaca[END_REF] 2-(4hydroxyphenyl)acetic acid, [START_REF] Chapla | Antifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic Fungus from Michelia champaca[END_REF] 2-(2-hydroxyphenyl)acetic acid, [START_REF] Chapla | Antifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic Fungus from Michelia champaca[END_REF] or 4-hydroxybenzamide have been isolated from C. gloeosporioides strains. [START_REF] Chapla | Antifungal Compounds Produced by Colletotrichum gloeosporioides, an Endophytic Fungus from Michelia champaca[END_REF] Our group has been investigating for a few years non-cytotoxic fungal extracts looking for new anti-glycation agents. The ethyl acetate extract of C. gloeosporioides SNB-GSS07, which had been isolated from the leaves of Sabicea cinerea, 8 exhibited interesting activity. Thorough chemical investigation of this extract, obtained from a solid culture of C. gloeosporioides strain, led to the isolation of four acoranes (1-4) that had not been described before in the literature. The 1 H-1 H COSY spectrum showed two coupling systems leading to the description of two partial structures (Figure 2). On one side the sequence of correlations H14, H4, H3, H2, H1, H11, H12/H13, allowed building the C1-C4 partial structure, substituted in C1 by an isopropyl group, in C3 by a hydroxyl, and in C4 by a methyl. On the other side, the H10 oxygenated methyne correlated with the olefinic proton H9 that, in turn, correlated with H15. In HMBC (Figure 2), protons H14, H4, H2, H1, H11 correlated to C5, and so did protons H10, H9 and H6b. Furthermore, H6a and H6b correlated to the ketone C7.

Overall it was possible to identify a C5 spiro ring system with a cyclopentane ring on one side and a 4-hydroxy-2-methyl-cyclohex-2-en-1-one moiety on the other side.

Compound 1 was an acorane type sesquiterpenoid. Its planar structure was identical to that of 3,10-dihydroxyacoronene. [START_REF] Wen | A new acorane sesquiterpene from Lysionotus pauciflorus[END_REF] Eventually, it was possible to find out differences in the relative configuration as established by analysis of the ROESY spectrum (Figure 3).

First, the relative configuration at C1, C5 and C10 can be determined based on the sequence of correlations H1-H6b-H10-H11/12. The model presented here was built based on Feng's 3,10-dihydroxyacoronene crystallographic data. [START_REF] Feng | New sesquiterpenoids from the rhizomes of Acorus tatarinowii[END_REF] It shows H6b and H10 in pseudo-axial position with the cyclohexenone ring in a half chair conformation.

This observation is consistent with the experimental ROE measurements. The correlation between H6a and H4 proved the relative configuration at C4, and the sequence of ROE correlations H14-H3-H2b-H11 confirmed the relative configuration at C-3.

The absolute configuration was obtained by comparison of the CD spectrum with the one recorded by Feng. [START_REF] Snatzke | Circulardichroismus-IX[END_REF] Both spectra exhibited a negative Cotton effect at 320 nm (Figure S2). This wavelength corresponds to the n- * transition of the --unsaturated ketone. In the 7-oxo-acoranes series, the sign of the Cotton effect depends on the absolute configuration of the spiro carbon, which was therefore identical in 3,10dihydroxyacoronene and in 1. The octant rule would also provide a negative sign for the Cotton effect. [START_REF] Snatzke | Circulardichroismus-IX[END_REF] These data demonstrated that the absolute configuration of compound 1 was 1R,3S,4S,5S,10R. Compound 1 was new to Science and was named after acorenone (3S,10R)-1,4-epi-3,10-dihydroxyacorenone. 12 S2, Figure S1). The relative configuration of 2 was determined based on the NOESY experiment. Briefly, the coupling of H6a with H1 and H4 along with the coupling of H6b with H13 allowed us to define the relative configuration of carbons C1, C4 and C5 and the relative position of H6a and H6b. Then, the correlations between H10a and both H14 and H3, and the coupling of H10b with H11, H12 and H13 confirmed the established relative configuration and allowed us to establish the relative configuration of carbon C3.

Overall, the relative configuration was the same as for compound 1. As regards absolute configuration, the CD spectrum again showed a negative Cotton effect at 320 nm which indicated that the absolute configuration at C-5 was identical to that of 3,10dihydroxyacoronene and 1 (Figure S2). Therefore, due to the Cahn-Ingold-Prelog priority rules, the absolute configuration of 2 was 1R,3S,4S,5R. This new acorane sesquiterpene was named (3S)-1,4-epi-3-hydroxyacorenone.

Compound 3 HRESIMS analysis indicated a molecular formula of C15H24O3 (m/z 253.1803 for [M+H] + ), as for compound 1. The planar structure of 3 was deduced from interpretation of 1D and 2D NMR experiments (Table 1, Table S3). The C1-C4 partial structure was identical to that of 1 (Figure 4). However, on the other side of the molecule, the methylene protons H6 correlated to an oxygenated methine H7, which in turn correlated with H15. In HMBC, protons H2a, H4, H11 and H14 correlated to C5, and so did protons H6, H7and H9. Moreover, H1, H4, H6 and H15 all correlated to ketone in C10, and H9, H6, H7 correlated to the oxygenated carbon C7. Overall, it was found that 3 was an isomer of compound 1 with a carbonyl group on C10 and a hydroxyl on C7.

The relative configuration of 3 was determined by analysis of the ROESY spectrum (Figure 4). The relative configuration of the stereogenic centers C1, C4 and C5 was established based on correlations between based on correlations between H6a and H4 on one side on the cyclohexenone, and H6b and H12 on the other side. The configuration at C7 was defined by the correlations of H7 with H6b and H11. Lastly, NOE coupling between H2b and both H3 and H14, along with a coupling between H14 and H3, indicated that these protons were on the same side of the cyclopentane ring, therefore confirming the relative stereochemistry at C3 as represented in Figure 5. The relative stereochemistry was identical to that of compounds 1 and 2, and it is highly unlikely that the absolute configuration was the opposite based on biosynthetic considerations.

Hence, the absolute configuration of 3 was 1S,3S,4S,5S,7R. Compound 3 was given the trivial name (3S,7R)-1,4-epi-3,7-dihydroxyacoren-10-one. The molecular formula of compound 4 was determined to be C15H24O2 by HRESIMS (m/z 237.1836 for [M+H] + ), indicating one fewer hydroxyl group compared to 3. The planar structure of 4 was determined using 1D and 2D NMR analysis, and by comparison to data of compound 3 (Table 1, Table S4). The main difference was in the cyclopentane moiety, with one methylene (C 32.7, H 1.81/1.63) instead of a methine. The COSY and HMBC correlations confirmed the loss of the hydroxyl group in position C3 (see also Figure S1). The relative configuration of 4 was determined by the analysis of the NOESY spectrum. Briefly, couplings between H4 and H6b, and between H6a and H1/H12 defined the stereocenters C1, C4 and C5. Then correlations of H7 with H6a and H11 established the relative configuration in C7. The relative configuration was identical to those of compounds 1-3, and biosynthetic considerations would provide the absolute stereochemistry as 1R,4R,5R,7R. This new compound was named (7R)-1,4-epi-7hydroxyacoren-10-one.

Other compounds isolated in the ethyl acetate extract of SNB-GSS07 were Sumiki's acid (5), [START_REF] Matsui | Identification of a New Natural Vasorelaxatant Compound, (+)-Osbeckic Acid, from Rutin-free Tartary Buckwheat Extract[END_REF] acetyl Sumiki's acid (6), 14 convolvulopyrone (7), [START_REF] Tsantrizos | Phytotoxic metabolites of Phomopsisconvolvulus, a host-specific pathogen of field bindweed[END_REF] p-hydroxybenzaldehyde (8), [START_REF] Zhao | A new 9,10-dihydrophenanthrene from Dendrobium moniliforme[END_REF] 4-hydroxyphenylacetic acid (9), [START_REF] Milne | Iodide-catalyzed reductions: development of a synthesis of phenylacetic acids[END_REF] indole-3-carboxylic acid (10), [START_REF] Sun | Chemical Constituents of Marine Sponge Halichondria sp. from South China Sea[END_REF] and indole-3carboxaldehyde (11). [START_REF] Zhang | Antioxidants and αglucosidase inhibitors from Ipomoea batatas leaves identified by bioassay-guided approach and structure-activity relationships[END_REF] Compounds 5-11 were identified by comparison of their spectral data with those reported in the literature.

In conclusion, four new acorane sesquiterpenes were isolated from the endophytic fungi Colletotrichum gloeosporioides SNB-GSS07. Their structures and absolute configurations were determined by mass spectrometry, NMR and circular dichroïsm. To our knowledge, this is the first time that acorane type sesquiterpenes have been isolated from endophytic sources. Literature indicates isolations of these kinds of structures from various plants, but never from associated microorganisms.

Figure 1 . 4 Compound 1

 141 Figure 1. Structure of compounds 1-4

Figure 2 . 1 Figure 3 .

 213 Figure 2. Key COSY (bold lines) and HMBC (dashed arrows) correlations of compounds 1

Figure 4 .

 4 Figure 4. (a) Key COSY (bold lines) and HMBC (dashed arrows) correlations of compound 3 ; (b) Key

Table

  1, TableS1). 1 H and[START_REF] Matsui | Identification of a New Natural Vasorelaxatant Compound, (+)-Osbeckic Acid, from Rutin-free Tartary Buckwheat Extract[END_REF] C NMR spectra, along with HSQC

	experiment indicated the presence of four methyls (C 15.2, H 1.71; C 17.6, H 0.92; C
	22.5, H 0.97; C 24.7, H 1.06), two methylenes (C 39.1, H 1.62/2.13 ; C 51.7, H
	2.08/2.95), four methines among which two were oxygenated (C 30.2, H 1.87; C 69.7,
	H 4.79; C 79.0, H 3.99; C 152.7, H 6.61), and three quaternary carbons (one aliphatic
	carbon at C 57.8, one olefinic carbon at C 135.2, one α,β-unsaturated carbonyl moiety
	at C 202.1).

Table 1 .

 1 The molecular formula of compound 2 was determined to be C15H24O2 by HRESIMS (m/z 237.1845 for [M+H] + ), indicating one fewer oxygen atom compared to 1. The planar structure of 2 was deduced by interpretation of 1D and 2D NMR experiments (Table1,

			1		2		3		4
	Pos.	C	H, m (J in	C	H, m (J Hz)	C	H, m (J in Hz)	C	H, m (J in Hz)
	1	55.9, CH 1.55, br. q (9.0)	55.6, CH 1.61, ddd (10.5, 8.7, 6.1)	57.7, CH 2.13, m	61.9, CH 1.87, m
	2	39.1, CH2 a: 1.62, ddd (13.1, 9.5,
		2.2)
		b: 2.13, br. q (11.6)

[START_REF] Kusari | Chemical Ecology of Endophytic Fungi: Origins of Secondary Metabolites[END_REF]

H and

[START_REF] Matsui | Identification of a New Natural Vasorelaxatant Compound, (+)-Osbeckic Acid, from Rutin-free Tartary Buckwheat Extract[END_REF]

C NMR spectroscopic data of compounds 1, 2, 3 and 4 in CD 3 OD

Acknowledgements

This work has benefited from a CIFRE grant managed by the Association Nationale de la Recherche et de la Technologie, France (ANRT), and from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche, France (CEBA, ref. ANR-10-LABX-25-01). The authors are very grateful to Thiago M. Casella for collecting and isolating the endopohyte, and to Joanna Wdzieczak-Bakala for her valuable help during this work.


M Zakardjian

Hervé Jourdan

Violette Le Féon

Benoît Geslin

Marie Zakardji An

Assessing the impact of alien bees on native ones

published or not. The documents may come   L'archive ouverte pluridisciplinaire

A species introduced outside of its native range, voluntarily or not, is defined as an alien species [START_REF] Blackburn | A proposed unified framework for biological invasions[END_REF]. These alien species may become invasive if their establishment and spread negatively affect biodiversity, ecosystem functioning , and/or the economy [START_REF] Diagne | High and rising economic costs of biological invasions worldwide[END_REF]. Biological invasions are among the most important threats to biodiversity worldwide [START_REF] Kumschick | Ecological impacts of alien species: quantification, scope, caveats, and recommendations[END_REF]. Frequentl y pointed out as the second cause of species extinction (Bellard et al., 2016a), the magnitude of negative effects of invasive alien species on biodiversity is nevertheles s still regularly debated in the literature [START_REF] Courchamp | Invasion biology: specific problems and possible solutions[END_REF]. With the exponential rise in global trade, the number of alien species has increased in the past few years [START_REF] Bellard | Major drivers of invasion risks throughout the world[END_REF], and projections forecast a continuous growth in the number of introductions in the following 30 years [START_REF] Seebens | No saturation in the accumulation of alien species worldwide[END_REF][START_REF] Seebens | Projecting the continental accumulation of alien species through to 2050[END_REF]. Among alien species, insects have been less studied [START_REF] Kenis | Ecological effects of invasive alien insects[END_REF] despite the importance of their reported negative effects on biodiversity componen ts , especially the ones exerted by social insects such as termites or ants [START_REF] Bertelsmeier | Globalization and the anthropogenic spread of invasive social insects[END_REF]. Notably, this is the case for bees, be they social or solitary, managed or wild [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF][START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Russo | The managed-to-invasive species continuum in social and solitary bees and impacts on native bee conservation[END_REF]. Indeed, bees are generally wellperceived by the public [START_REF] Sumner | Why we love bees and hate wasps[END_REF].mostly because of their important role in the reproduction of wild [START_REF] Ollerton | How many flowering plants are pollinated by animals?[END_REF] and cultivated plants [START_REF] Klein | Importance of pollinators in changing landscapes for world crops[END_REF][START_REF] Gill | Protecting an ecosystem service[END_REF]. Therefore, because of this beneficial role, little attention is paid when an alien bee species enters a new ecosystem.

However, to date, [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF] recorded 80 bee species introduced out of their native range (Fig. 1). Among these, Apis mellifera (L.) and Bombus terrestris (L.) are the two species that have been the focus of attention [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Mallinger | Do managed bees have negative effects on wild bees?: a systematic review of the literature[END_REF][START_REF] Goulson | Effects of introduced bees on native ecosystems[END_REF]. These two social and domesticated species have mostly been introduced either to produce honey (for Apis mellifera) or to improve pollination services to crops [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF], sometimes doing more harm than good [START_REF] Aizen | When mutualism goes bad: density-dependent impacts of introduced bees on plant reproduction[END_REF][START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF][START_REF] Sáez | Extremely frequent bee visits increase pollen deposition but reduce drupelet set in raspberry[END_REF]. Of course, not all 80 alien species described by [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF] have become invasive, but the literature report many examples of deleterious consequences of the introduction of bees outside their native range [START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF][START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF][START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Mallinger | Do managed bees have negative effects on wild bees?: a systematic review of the literature[END_REF][START_REF] Traveset | Mutualistic interactions and biological invasions[END_REF][START_REF] Goulson | Effects of introduced bees on native ecosystems[END_REF].

Hence, alien bees can directly or indirectly affect native ecosystems and therefore become invasive [START_REF] Dafni | Bombus terrestris, pollinator, invasive and pest: an assessment of problems associated with its widespread introductions for commercial purposes[END_REF]Russo et al., 2021, Fig. 2). They can compete with native bees for the acquisition of food resources (nectar and pollen) or nesting sites potentially affecting local populations [START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF][START_REF] Hung | Non-native honey bees disproportionately dominate the most abundant floral resources in a biodiversity hotspot[END_REF][START_REF] Ollerton | Pollinator diversity: distribution, ecological function, and conservation[END_REF][START_REF] Schaffer | Competition for nectar between introduced honeybees and native North American bees and ants[END_REF]. Alien bees can transport parasites and diseases, which can, in turn, affect native populations Figure 2 Positive and negative interactions (respectively green and red links) between alien and native species may disrupt native plant-pollinator systems. First, alien bees may compete for floral resources and/or nesting sites, spread parasites and pathogens and hybridize with native bees (A). Then, alien bees may also not be efficient pollinators of some native plants (e.g. due to morphological mismatch) and could thus alter their reproductive success (B). Second, alien plants may compete with native ones for soil resources (C) and corner all or part of their pollinators (D). Finally, it seems that alien plants tend to promo te the presence of alien bees and in some cases alien bees visit preferentially those plant species. Thus, alien species may promote the establishment and spread of each other, potentially leading to invasional meltdowns (E). All these interactions may lead to a local decrease of abundance and richness of native species. [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF][START_REF] Goulson | Bee declines driven by combined stress from parasites, pesticides, and lack of flowers[END_REF][START_REF] Graystock | The Trojan hives: pollinator pathogens, imported and distributed in bumblebee colonies[END_REF]. Alien bees can also disrupt plant-pollinator networks by robbing flowers, therefore decreasing their reproductive success [START_REF] Dafni | Bombus terrestris, pollinator, invasive and pest: an assessment of problems associated with its widespread introductions for commercial purposes[END_REF][START_REF] Dohzono | Alien bumble bee affects native plant reproduction through interactions with native bumble bees[END_REF], modifying the amount or the nature of the pollen deposited [START_REF] Agüero | Impact of invasive bees on plant-pollinator interactions and reproductive success of plant species in mixed Nothofagus antarctica Forests[END_REF], or by reinforcing the pollination of particular plant species [START_REF] Sanguinetti | Invasive bees promote high reproductive success in Andean orchids[END_REF]. In some cases, alien bees can even interact preferentially with alien or invasive plants, potentiall y enhancing their spread and negative impacts, a phenomenon called invasiona l meltdown [START_REF] Zakardjian | Effects of urbanization on plant-pollinator interactions in the tropics: an experimental approach using exotic plants[END_REF][START_REF] Traveset | Mutualistic interactions and biological invasions[END_REF][START_REF] Beavon | Invasional meltdown: pollination of the invasive liana Passiflora tripartita var. mollissima (Passifloraceae) in New Zealand[END_REF].

Despite these reported effects, the impacts of alien bees on native ecosystem s are little considered on a global scale. For example, despite the devastating consequences of multiple introductions of Bombus terrestris worldwide (e.g. [START_REF] Acosta | Worldwide alien invasion: a methodological approach to forecast the potential spread of a highly invasive pollinator[END_REF], the commercial trade of Bombus species outside of their native range continues [START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF] to the point that the international trade of bees, mostly bumblebees, is nowadays an important conservation issue for global biodiversity [START_REF] Sutherland | A horizon scan of global conservation issues for 2016[END_REF]. Therefore, it is important and timely to assess the impact of alien bees on native pollinators. This chapter focuses on the direct and indirect impacts of alien bees on native pollinators. We will not explore the effect of alien bees on plant communities or plant-pollinator networks to focus solely on their effect on native bees. Also, becaus e we focus on alien bees, we will not provide examples of competitive interactions between wild and domestic bees in the native range of domestic bees. In the second part, we will focus on a case study, the invasion of the alien bee Megachile sculpturali s in North America and Europe. We will then delineate some of the main shortcoming s and directions for future studies and guide land managers to prevent future introductions.

2 Assessment of key issues related to the introduction of alien bees

1 Direct interactions: interference competition

Interference competition is a direct interaction, generally by aggressive contact, through which a resource that an individual should have used is made available by and for another individual [START_REF] Case | Interference competition and niche theory[END_REF]. Among alien bees, interference competition may be used to acquire food resources, nesting sites, or defend mating territories. Although rare, interference competitions between alien and native bees have been reported in other literature.

In Yucatan (Mexico), the Africanised honey bee (Apis mellifera scutellata) has aggressively displaced stingless bees from floral and water resources. In this region, some native stingless bees appear to have declined from anthropogenic pressures . Interference competition by honey bees could have compound effects on the most vulnerable native species [START_REF] Cairns | Bee populations, forest disturbance, and Africanization in Mexico[END_REF]. In Indiana (US), honey bees have also been observed to steal pollen loads directly from the body parts of several native bees such as Bombus impatiens, Megachile montivaga, and Melissodes despons a [START_REF] Jean | Quantifying a rare event: pollen theft by honey bees from bumble bees and other bees (Apoidea: Apidae, Megachilidae) foraging at flowers[END_REF]. As not all pollen loads were removed and those events remain rare, this kind of physical interaction is unlikely to severely impact native bees' fitness.

Anthidium manicatum, the most widespread unmanaged alien bee species worldwide [START_REF] Strange | Global invasion by Anthidium manicatum (Linnaeus) (Hymenoptera: Megachilidae): assessing potential distribution in North America and beyond[END_REF], uses interference competition to defend floral territories and thus assuring mating opportunities [START_REF] Starks | Condition-based alternative reproductive tactics in the woolcarder bee, Anthidium manicatum[END_REF][START_REF] Severinghaus | The reproductive Behavior of Anthidium manicatum (Hymenoptera: Megachilidae) and the Significance of Size for Territorial Males[END_REF]. In New Zealand, Anthidium manicatum aggressively interferes with native Lasioglossum spp. [START_REF] Soper | Assessing the impact of an introduced bee, Anthidium manicatum, on pollinator communities in New Zealand[END_REF]. However, in this study, native bees were less contacted than conspecific individuals and two other alien species, Apis mellifera and Bombus spp. Native Lasioglossum spp. shared fewer floral resources than Apis mellifera and Bombus spp. with Anthidium manicatum , thus reducing the probability of interference events [START_REF] Case | Interference competition and niche theory[END_REF].

Although the impact of these interactions was not tested, the authors concluded that Anthidium manicatum posed a minor threat to the native bee fauna of New Zealand.

In North America, Megachile spp. in an urban area experienced reduced visitation frequencies in the presence of Anthidium manicatum [START_REF] Taggar | Competition between a native and introduced pollinator in unmanaged urban meadows[END_REF].

Unfortunately, in this study, alien and native Megachile spp. were not discriminated, and it was therefore complicated to state whether this decrease was due to interference or exploitative competition. A native bee, Bombus impatiens, was shown to avoid floral enclosures where Anthidium manicatum was present. Even if the authors did not discriminate between interference and exploitative competiti on to explain their results, no effect was shown on the colony fitness of the native bumblebee [START_REF] Graham | Anthidium manicatum, an invasive bee, excludes a native bumble bee, Bombus impatiens, from floral resources[END_REF].

In Europe and North America, several events of aggressive behaviour have been documented for Megachile sculpturalis. This species is likely to interfere with native species with similar nesting habits (see the case study below).

Physical interaction events appear to be uncommon between alien and native bees.

Thus, interference competition is unlikely to have a deleterious impact on native bee populations. However, such behaviours could increase the potential of a species to become established or spread if coupled with other characteristics such as a high capability of exploitative competition for floral resources and nesting sites.

2 Exploitative competition

Alien bees may overlap with native bees in floral resource use (Fig. 3) and/ or nesting sites. Exploitative competition occurs when shared resources are limiting and implies negative impacts on the fitness of native bees.

1 Exploitative competition for floral resources

Managed eusocial bees, such as Apis mellifera and Bombus terrestris, have a strong competitive potential over native wild bees. Indeed, the number of workers per managed colony compared to solitary bees and the regular renewal of populations by beekeepers (when needed) make them prone to exploit efficiently floral resources with potential impacts on native bees. Their impacts on native and invaded areas have already been reviewed by several authors (e.g. [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF][START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF][START_REF] Mallinger | Do managed bees have negative effects on wild bees?: a systematic review of the literature[END_REF][START_REF] Osterman | Global trends in the number and diversity of managed pollinator species[END_REF][START_REF] Russo | The managed-to-invasive species continuum in social and solitary bees and impacts on native bee conservation[END_REF][START_REF] Vanbergen | Risks to pollinators and pollination from invasive alien species[END_REF], but we present here some examples of exploitative competition. A 2-year experiment conducted in Western Australia showed that the monolectic resource used by a native bee, Hylaeus alcyoneus, was also used by honey bees for pollen and nectar provision. This resource overlap appears to explain the decrease in native bee fecundity (measured as the number of nests constructed) in sites with increasing honey bee density [START_REF] Paini | Commercial honey bees (Apis mellifera) reduce the fecundity of an Australian native bee (Hylaeus alcyoneus)[END_REF]. In this case, the monolectic behaviour of the native bee likely made it more vulnerable to exploitative competition exerted by honey bees. Another negative impact of honey bees on native bees was demonstrated in Tenerife (Canary Islands), where the number of pollinator species and that of visits by some native bees were lower after the setup of beehives [START_REF] Valido | Honey bees disrupt the structure and functionality of plant-pollinator networks[END_REF]. In this case, exploitative competition from honey bees had a negative impact on the richness and abundance of generalist native bees, with implications for pollination ecosystem services and plant reproducti ve success.

Conversely, some studies did not find any evidence of a detrimental effect of the exploitative competition exerted by alien honey bees on native bees. For example, the experimental increase in honey bees in a Patagonian Forest ecosystem did not affect the number of visits made by native pollinators but rather induced exploitati ve competition with two other invasive alien species (i.e. Bombus ruderatus and Bombus terrestris; [START_REF] Agüero | Impact of invasive bees on plant-pollinator interactions and reproductive success of plant species in mixed Nothofagus antarctica Forests[END_REF]. However, in this study, native pollinators were studied as a group, and contrasting responses between pollinator species may not have been highlighted. In an urban ecosystem in Montreal (Canada), an increase in honey bee capture rate did not affect the abundance of wild bees [START_REF] Mccune | Response of wild bee communities to beekeeping, urbanization, and flower availability[END_REF]. The authors expected a negative effect and hypothesised that the abundance of floral resources and the very low density of beehives compared with other cities (e.g. see [START_REF] Ropars | Wild pollinator activities negatively related to honey bee colony densities in urban context[END_REF]) mitigated competition. In addition, in the Ogasawara islands (Japan), Apis mellifera appears to have a competitive advantag e over native bees [START_REF] Kato | Impact of introduced honey bees, Apis mellifera, upon native bee communities in the Bonin (Ogasawara) Islands[END_REF], but the decline of native pollinators is mostly due to an invasive predator [START_REF] Abe | Why have endemic pollinators declined on the Ogasawara Islands?[END_REF]. Finally, in Ontario (Canada), within an agroecosystem (Cucurbita spp.), Willis Chan and Raine (2021) found no negative impact of the alien bee Apis mellifera on the wild oligolectic hoary squash bees (Eucera Peponapis pruinosa) foraging on Cucurbita spp., likely because of an intraday phenological difference in bees' activities (honey bees were foraging later than hoary squash bees).

Another managed eusocial bee, Bombus terrestris, has been massively introduced worldwide and is known to have caused disastrous impacts on native bee populations (see below, e.g. [START_REF] Acosta | Worldwide alien invasion: a methodological approach to forecast the potential spread of a highly invasive pollinator[END_REF][START_REF] Aizen | Invasive mutualists erode native pollination webs[END_REF][START_REF] Aizen | When mutualism goes bad: density-dependent impacts of introduced bees on plant reproduction[END_REF][START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF][START_REF] Morales | Invasive mutualisms and the structure of plant-pollinator interactions in the temperate forests of north-west Patagonia, Argentina[END_REF][START_REF] Morales | Rapid ecological replacement of a native bumble bee by invasive species[END_REF]. In Japan, Bombus terrestris may be an efficient competitor for floral resources over some native bumblebees. For example, an experiment demonstrated that Bombus terrestris harvested more pollen per unit time and per unit body mass than the native Bombus ignitus [START_REF] Inoue | Is Bombus terrestris (Hymenoptera: Apidae) a stronger competitor against native Japanese species? A comparison of foraging efficiency[END_REF].

However, the evidence of exploitative competition effects on native bees is unclear.

Indeed, an experiment failed to detect a negative impact of Bombus terrestris on two native Japanese bumblebees (Bombus ardens and Bombus hypocrita; Nagamitsu et al., 2007a). Nevertheless, field removal of Bombus terrestris queens from an urba n area led to an increased number of these two native bumblebee queens among five native bee species captured in the study area [START_REF] Nagamitsu | Competitive effects of the exotic Bombus terrestris on native bumble bees revealed by a field removal experiment[END_REF]. Bombus ardens and B. hypocrita are likely to overlap for floral resource use with Bombus terrestris due to their tongue length (Nagamitsu et al., 2007). Thus, although the effect of competition for nesting sites could not be discriminated, the release of exploitative competition for floral resources was a major hypothesis to explain these results.

Corroborating this hypothesis, Bombus terrestris may be responsible for the increase in robbed flowers of a native plant (Corydalis ambigua) and the subsequent decrease in visitation by B. ardens [START_REF] Dohzono | Alien bumble bee affects native plant reproduction through interactions with native bumble bees[END_REF].

However, in an alpine-montane ecosystem in New Zealand, alien bees mostly composed of Bombus terrestris did not overlap with native bees for floral resource use [START_REF] Iwasaki | Floral usage partitioning and competition between social (Apis mellifera, Bombus terrestris) and solitary bees in New Zealand: niche partitioning via floral preferences?[END_REF]. Indeed, Bombus terrestris preferentially visited alien plants when native bees were mostly observed on native ones, suggesting a low potential for exploitative competition for floral resources. In addition, 3-years monitoring conducted in Japan did not show any evidence of an impact of shared plant resources between Bombus terrestris and the native species B. pseudobaicalensis [START_REF] Inoue | Displacement of Japanese native bumblebees by the recently introduced Bombus terrestris (L.) (Hymenoptera: Apidae)[END_REF].

Notably, the introduction and invasion of B. terrestris and B. ruderatus in South

America had notorious negative impacts on native ecosystems, including the decline and local extinction of B. dahlbomii, a Patagonian native bumblebee [START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF][START_REF] Geslin | New records reveal rapid geographic expansion of Bombus terrestris Linnaeus, 1758 (Hymenoptera: Apidae), an invasive species in Argentina[END_REF][START_REF] Morales | Rapid ecological replacement of a native bumble bee by invasive species[END_REF][START_REF] Morales | Introducción de abejorros (Bombus) no nativos: causas, consecuencias ecológicas y perspectivas[END_REF]. In particular, B. ruderatus overlaps with B. dahlbomii for floral resources [START_REF] Aizen | Invasive mutualists erode native pollination webs[END_REF][START_REF] Morales | Invasive mutualisms and the structure of plant-pollinator interactions in the temperate forests of north-west Patagonia, Argentina[END_REF]. However, [START_REF] Aizen | Comparative nectar-foraging behaviors and efficiencies of an alien and a native bumble bee[END_REF] found no evidence of exploitative competition for floral resources between these two species, and the spread of pathogens is a favoured hypothesis to explain the decline of B. dahlbomii (see Section 2.3).

When first recorded in a new environment, alien solitary bees generally raise concerns about potential negative impacts on native species. Unfortunately, for most of these species, field studies following their detection rarely test for their effect on the fitness of native bee populations. However, a recent study analysing historical pan trap records in the US showed that the arrival of two alien bees, Osmia cornifrons, and especially Osmia taurus, was followed by a decline in six native Osmia spp. [START_REF] Lecroy | Decline of six native mason bee species following the arrival of an exotic congener[END_REF]. Several factors have been hypothesised to explain this result, including exploitative competition, either for floral resources or nesting sites.

Another study conducted in the US showed that urban sites with a high density of alien bees were associated with a lower abundance of native bees [START_REF] Fitch | Does urbanization favour exotic bee species? Implications for the conservation of native bees in cities[END_REF].

Once again, exploitative competition for floral resources, nesting sites, or spillover of pathogens were all hypothesised to explain these observations.

The effects of wild alien bees on native bees are also context-dependent. A study conducted in an insular system in the Southwest Pacific showed that the presence of alien bees did not affect the number of visits realised by native ones [START_REF] Zakardjian | Effects of urbanization on plant-pollinator interactions in the tropics: an experimental approach using exotic plants[END_REF]. However, this result was explained by the generalist foraging behaviour of some insular native bees. In Brazil, a study conducted in an urban area found no impact from two alien bee species (Apis mellifera and Anthidium manicatum) on the structure of the native bee community [START_REF] Graf | Ecological impact and population status of nonnative bees in a Brazilian urban environment[END_REF], suggesting that no exploitative competition occurred. The authors proposed two hypotheses to explain this result. First, it is possible that these two alien bees, which were introduced a few decades ago, had an impact at the beginning of their establishment. Second, the urba n environment may not be suitable for the species that are the most vulnerable to competition.

Most studies estimate niche overlap and the displacement of visits without measuring the impact of competition on native bee fitness. However, these metrics still reveal an effect of the competitive pressure exerted by alien bees on native ones.

Mixed responses of native bees to exploitative competition by alien ones reveal that competitive pressure is context-dependent, varying according to the ecosystem (number of floral resources), the capacity of native bees to adapt their foraging preferences, and the characteristics of alien bees, such as their level of generalism and their reproductive success.

2 Exploitative competition for nesting sites

Bombus terrestris has the potential to compete with native bumblebees for nesting resources. In Japan, 3 years of monitoring studied the abundance of three native bumblebees, along with the abundance of Bombus terrestris queens. The results showed that a native species using the same nesting resources as Bombus terrestris declined with an increase in Bombus terrestris queens, suggesting exploitati ve competition for nesting sites between these two species [START_REF] Inoue | Displacement of Japanese native bumblebees by the recently introduced Bombus terrestris (L.) (Hymenoptera: Apidae)[END_REF].

Concerning wild alien bees, Megachile sculpturalis has been reported to be a potential competitor for nesting resources (see above). In Marseille (France), near its first detection point, the presence of Megachile sculpturalis in bee hotels reduced the probability of occupation by native Osmia spp. [START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF].

3 Apparent competition (pathogens spillover)

Parasites and pathogens may be transferred among bee species when infected individuals are abundant and share floral resources or nesting sites with other species. Thus, bees are ideal candidates for the transmission and dispersion of pathogens.

In New York State (US), the presence of the deformed wing virus (DWV; Fig. 4) in some native Melandrena spp. was linked to honey bee abundance [START_REF] Murray | Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny[END_REF]. Even if there was no correlation with two other pathogens (black queen cell virus and sacbrood virus), these results indicate that honey bees may be a source of infection for solitary native bees, potentially increasing viral incidence for at least DWV. Still in North America, wild pollinators found close to beehives with honey bees infected with Israeli acute paralysis virus also presented this virus, contrary to wild pollinators captured close to healthy beehives [START_REF] Singh | RNA viruses in hymenopteran pollinators: evidence of inter-Taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species[END_REF]. Similarly, [START_REF] Alger | RNA virus spillover from managed honey bees (Apis mellifera) to wild bumblebees (Bombus spp.)[END_REF] found a strong prevalence of DWV in wild bumblebees foraging close to apiaries, whereas no DWV was found in bumblebees in the absence of honey bees.

In Argentina, Apicystis bombi, a parasite that especially infects bumble-bees, was possibly co-introduced with Bombus terrestris or B. ruderatus [START_REF] Arbetman | Alien parasite hitchhikes to Patagonia on invasive bumblebee[END_REF][START_REF] Plischuk | Apicystis bombi (Apicomplexa: Neogregarinorida) parasitizing Apis mellifera and Bombus terrestris (Hymenoptera: Apidae) in Argentina[END_REF] and is now also present in Colombia [START_REF] Gamboa | Bee pathogens found in Bombus atratus from Colombia: a case study[END_REF] and Patagonia [START_REF] Arbetman | Alien parasite hitchhikes to Patagonia on invasive bumblebee[END_REF]. In Patagonia, it appears that the alien B. ruderatus and the native B. dahlbomii were free of this parasite before the invasion of Bombus terrestris [START_REF] Arbetman | Alien parasite hitchhikes to Patagonia on invasive bumblebee[END_REF]. Thus, A. bombi likely arrived with and spilt over from Bombus terrestris to these two bumblebee species. The severe decline of B. dahlbomii is potentially linked to this parasite spillover [START_REF] Arbetman | Alien parasite hitchhikes to Patagonia on invasive bumblebee[END_REF].

4 Mating interference

In Korea, laboratory experiments demonstrated that interspecific hybridisation may happen between Bombus terrestris and two native bumblebees (B. ignitus and B. hypocrita). Males of the two native species mated with Bombus terrestris queens [START_REF] Yoon | Interspecific hybridization of the Korean native bumblebee Bombus hypocrita sapporoensia and the European bumblebee B. terrestris[END_REF][START_REF] Yoon | Interspecific hybridization of the bumblebees Bombus ignitus and B. terrestris[END_REF]. Thus, Bombus terrestris may interfere with native bumblebees for mating males, with potential negative impacts on native bees' fitness. In addition, during this experiment, Bombus terrestris males mated with native bumblebee queens, highlighting a risk of genetic contamination.

Case study: Megachile sculpturalis

1 History of introduction and spread

Megachile (Callomegachile) sculpturalis Smith (Hymenoptera: Megachilidae) -(Msc) is a wild bee species native to Southeast Asia (Korea, China, Taiwan, Hong Kong, and Japan; Fig. 5). We chose this species as a case study because it was the first alien bee to enter the European continent in 2008 [START_REF] Vereecken | Premières données sur la présence de l'abeille asiatique Megachile (Callomegachile) sculpturalis SMITH (Hymenoptera, Megachilidae) en Europe[END_REF], and its sprea d and impacts have been carefully followed since (Le Féon et al., 2018, 2021). Before arriving in Europe, however, Msc colonised the North American continent. The first mention of Msc outside of its native range dates from 1994 in North Carolina (US; [START_REF] Magnum | First records of Megachile (Callomegachile) sculpturalis Smith (Hymenop-tera: Megachilidae) in the Continental United States[END_REF]. Since then, the species has rapidly been detected in other US states, up to Texas in 2011 [START_REF] Parys | The Giant resin Bee, Megachile sculpturalis Smith: new Distributional Records for the Mid-and Gulf-south USA[END_REF][START_REF] Hinojosa-Díaz | The giant resin bee making its way west: first record in Kansas (Hymenoptera: Megachilidae)[END_REF]. Besides the US, Msc has also expanded its range to Canada, especially in Ontario [START_REF] Paiero | The giant resin bee, Megachile sculpturalis Smith, and other newly introduced and newly recorded native Megachlidae and Andrenidae (Apoidea) from ontario[END_REF].

In Europe, Msc was first detected in Allauch, a small city close to Marseille (France; [START_REF] Vereecken | Premières données sur la présence de l'abeille asiatique Megachile (Callomegachile) sculpturalis SMITH (Hymenoptera, Megachilidae) en Europe[END_REF]. Between 2008 and 2021, Msc has remarkably extended its range in France, with a reported presence in 259 different cities, with the most northern location being approximately 800 km from Marseille, in the northeast of Paris (near Reims, Le Féon et al., 2021). Msc now occupies approximately half of France, from the Atlantic coast to the Alps (Le Féon et al., 2018, 2021;Le Féon and Geslin, 2018).

Besides France, Msc has also spread to many European countries, starting with Italy in 2009 [START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of Europe[END_REF], Switzerland in 2010 [START_REF] Amiet | Die Blattschneiderbiene Megachile sculpturalis Smith, 1853 (Hymenoptera, Apidae) nun auch in der Schweiz[END_REF], Germany [START_REF] Westrich | Megachile sculpturalis Smith 1853 (Hymenoptera, Apidae), a new species for the bee fauna of Germany, now north of the Alps[END_REF], and Hungary in 2015 [START_REF] Kovàcs | Megachile sculpturalis Smith, 1853. In: Hungary (Hymenoptera, Megachilidae)[END_REF], Slovenia in 2016 [START_REF] Gogala | First record of Megachile sculpturalis smith in Slovenia (hymenoptera: Megachilidae)[END_REF], Austria [START_REF] Westrich | Faszination Wildbienen, Forschungsprojekte: Megachile sculpturalis[END_REF] and Serbia (Ćetković et al., 2020) in 2017, Spain [START_REF] Ortiz-Sánchez | Megachile (Callomegachile) sculpturalis Smith, 1853, nueva especie para la fauna ibérica (Hymenoptera, Megachilidae)[END_REF] and Crimean peninsula [START_REF] Ivanov | First record of the invasive giant resin bee Megachile (callomegachile) sculpturalis smith, 1853 (hymenoptera: Megachilidae) in the crimea[END_REF] in 2018, Croatia (inaturalist.org) and Lichtenstein (Lanner et al., 2020a) in 2019, and finally in Bosnia-Herzegovina in 2020 (inaturalist.org; Fig. 6). Megachile sculpturalis is therefore now widespread in Europe and occupies several habitats from very urbanised ones such as Marseille, Lyon, or Vienna [START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF][START_REF] Lanner | Dispersal patterns of an introduced wild bee, Megachile sculpturalis Smith, 1853 (Hymenoptera: Megachilidae) in European alpine countries[END_REF][START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF], to montane habitats (up to 1846 m above sea level; Le Féon and Geslin, 2018).

The spread of this species is remarkably fast even if similar dispersal rates have been reported for other invasive hymenopterans such as Bombus terrestris in South America [START_REF] Schmid-Hempel | The invasion of southern South America by imported bumblebees and associated parasites[END_REF] or Vespa velutina in Europe [START_REF] Robinet | Rapid spread of the invasive yellow -legged hornet in France: the role of human-mediated dispersal and the effects of control measures[END_REF]. One of the hypotheses explaining this fast spread [START_REF] Aguado | Megachile (Callomegachile) sculpturalis Smith, 1853 (Apoidea: Megachilidae): a new exotic species in the Iberian Peninsula, and some notes about its biology[END_REF][START_REF] Amiet | Die Blattschneiderbiene Megachile sculpturalis Smith, 1853 (Hymenoptera, Apidae) nun auch in der Schweiz[END_REF][START_REF] Ćetković | Project: monitoring the spread of sculptured resin bee (Megachile sculpturalis) in Serbia. Centar za biologiju pčela[END_REF][START_REF] Gogala | First record of Megachile sculpturalis smith in Slovenia (hymenoptera: Megachilidae)[END_REF][START_REF] Ivanov | First record of the invasive giant resin bee Megachile (callomegachile) sculpturalis smith, 1853 (hymenoptera: Megachilidae) in the crimea[END_REF]Kovács, 2015;[START_REF] Lanner | Dispersal patterns of an introduced wild bee, Megachile sculpturalis Smith, 1853 (Hymenoptera: Megachilidae) in European alpine countries[END_REF][START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of Europe[END_REF][START_REF] Vereecken | Premières données sur la présence de l'abeille asiatique Megachile (Callomegachile) sculpturalis SMITH (Hymenoptera, Megachilidae) en Europe[END_REF][START_REF] Westrich | Megachile sculpturalis Smith 1853 (Hymenoptera, Apidae), a new species for the bee fauna of Germany, now north of the Alps[END_REF][START_REF] Westrich | Faszination Wildbienen, Forschungsprojekte: Megachile sculpturalis[END_REF]. The homogeneous colouring of national territories can be misleading. For example, concerning the islands, Msc is so far unknown in Corsica, Sicily, and Sardinia. However, it has been recorded on the island of Elba and in Majorca, islands that we have circled in red. Map made with QGIS version 3.14. Base map downloaded from Natural Earth (naturalearthdata.com).

lies in the nesting preferences of Msc (in deadwood and cavities, see below), and Msc may have benefited from the timber trade through human-mediated transportati on (e.g. Le Féon et al., 2018). Using the species distribution model [START_REF] Lanner | On the road: anthropogenic factors drive the invasion risk of a wild solitary bee species[END_REF] recently showed that the spread of Msc is largely explained by anthropogenic factors and particularly facilitated by roads and maritime traffic. Through genetic analysis, [START_REF] Lanner | Evidence for multiple introductions of an invasive wild bee species currently under rapid range expansion in Europe[END_REF] have also emphasised that several introductions may have occurred in Europe, partly explaining the rapid geographical expansion of Msc.

2 The morphology and ecology of Megachile sculpturalis

One of the vernacular names of Megachile sculpturalis is the giant resin bee. This appellation comes from the nesting biology of Msc, which closes its nest and brood cells with resin mainly collected from coniferous trees. Megachile sculpturalis is a large bee species (male 14-19 mm; female 22-27 mm; [START_REF] Batra | Biology of the giant resin bee, Megachile sculpturalis Smith, a conspicuous new immigrant in Maryland[END_REF], with orange to brown hairs on the thorax and infuscated wings (Fig. 5). The head is dark, with large mandibles used by females to carry mud and resin to build their nests (Fig. 7). Females carry pollen for their larvae in their ventral scopa.

Bees forage on flowers to gather pollen and nectar. While nectar is useful as a direct energy source for both adults and larvae, pollen is the principal source of protein and is gathered mostly to feed the larvae. Bees can be a generalist when seeking for nectar but can be more of a specialist when foraging for a particular pollen. Megachile sculpturalis is a generalist species when it comes to nectar, meaning that this species can visit many different plant families. For example, in France, Msc has been observed foraging on 20 different plant species from 8 families, including Asteraceae (i.e. Cirsium arvense), Capriofoliacea (i.e.

Scabiosa columbaria), and Lamiaceae (i.e. Lavendula) (Le Féon et al., 2018). However, for pollen, Msc appears to be more specialist with strong preferences for alien woody species also originating from Asia. In Europe, Msc is known for gathering pollen from alien tree species such as Sophora japonica (Fabaceae; [START_REF] Andrieu-Ponel | A propos du comportement de butinage de Megachile sculpturalis Smith, 1853, en France méditerranéenne (Nîmes et Montpellier) (Hymenoptera, Megachilidae)[END_REF] and collecting nectar from Ligustrum lucidum (native to Asia) and Ligustrum vulgare (native to Europe) (Oleaceae; [START_REF] Aguado | Megachile (Callomegachile) sculpturalis Smith, 1853 (Apoidea: Megachilidae): a new exotic species in the Iberian Peninsula, and some notes about its biology[END_REF].

Megachile sculpturalis nests in pre-existing cavities, mostly in deadwood, bamboo, or reeds. As stated earlier, Msc closes its nest entrances with resin, vegetal fibres, mud, and clay. Female Msc widely uses insect hotels (or bee hotels) to lay eggs, with much evidence in North America (MacIvor and Packer, 2015) and Europe [START_REF] Díaz | Nesting, sex ratio and natural enemies of the Giant Resin Bee in relation to native species in Europe[END_REF][START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF]Le Féon et al., 2018;[START_REF] Fortel | Use of human-made nesting structures by wild bees in an urban environment[END_REF]Quaranta et al., 2014, Fig. 7). This propensity to nest in human-made structures may also be the reason for the rapid spread of Msc in Europe [START_REF] Lanner | On the road: anthropogenic factors drive the invasion risk of a wild solitary bee species[END_REF].

3 Impacts on native bees

The first insight into the potential impacts of Megachile sculpturalis on native fauna was published in 2012 [START_REF] Roulston | Aggressive eviction of the eastern carpenter bee (Xylocopa virginica (Linnaeus)) from its nest by the giant resin bee (Megachile sculpturalis Smith)[END_REF]. The authors reported an aggressive eviction of a native carpenter bee (Xylocopa virginica) from its nest by Msc in Virginia (US). In the same year, [START_REF] Laport | Occupation of active Xylocopa virginica nests by the recently invasive Megachile sculpturalis in upstate New York[END_REF] reported the occupation of a nest of an active X. virginica by Msc. Both observations suggest potential nest competition between Msc and local fauna, together with aggressive territoria l behaviours. Similar observations were obtained a few years ago in Europe, with (a) aggressive evictions, (b) occupation of nests, and (c) emptying of native bee nests, including Osmia bicornis, O. cornuta, and recently, Anthidium septemspinosum (Le Féon et al., 2018, 2021). In 2020, Geslin et al. conducted a study in urban parks of Marseille (France) to assess the impact of Msc on wild bees. They installed 96 bee hotels in 12 parks in the city and explored the relationship between the abundance of Msc and native bees emerging from bee hotels. First, they found that the most abundant bee nesting in bee hotels was Msc, with more than 40% of individuals . Second, they discovered a negative correlation between the presence of native bees and that of Msc within insect hotels, suggesting a negative impact of Msc on local native populations of wild bees. Two recent publications have provided new information on the impact of Msc on wild fauna. First, Lanner et al. (2020a) reported the killing of several Heriades spp. individuals, a French native species, by Msc, thereby documenting another direct impact of the alien bee species on native ones. Finally, [START_REF] Díaz | Nesting, sex ratio and natural enemies of the Giant Resin Bee in relation to native species in Europe[END_REF] documented the occupation by Msc of Osmia cornuta nests without entirely emptying the nests. This means that Msc females laid eggs close to the cavity entrance, blocking this entrance with mud and resin. Osmia cornuta is active in spring, unlike Msc, which is active during summer (from June to September in Europe). Emerging individuals of O. cornuta are therefore blocked by this resin, incapable of exiting the nest, and may ultimately die from starvation or exhaustion.

Taken together, these findings show that Msc could have a deleterious impact on local wild bee populations (Fig. 8). However, the magnitude of these impacts remains poorly understood. For now, it does not appear easy to determine whether Msc could profoundly modify the structure of local bee populations, for example, by causing local extinctions of some species. The extent to which Msc disrupts native pollinators remains to be carefully evaluated, and future research will help us explore this question.

4 Other impacts

To date, there has been no documentation of other potential impacts of Msc on ecosystems or human economy and infrastructures. As stated earlier, Msc appears to preferentially forage for pollen from alien plant species, potentiall y helping their spread. However, there is currently no evidence for such an effect. In addition, [START_REF] Díaz | Nesting, sex ratio and natural enemies of the Giant Resin Bee in relation to native species in Europe[END_REF] showed that Msc could be affected by common, widespread generalist macro parasites without a substantial effect on the emerging success of Msc. At this stage, it appears difficult to determine whether Msc could reinforce parasite populations which could in turn affect native wild bees, and this remains to be explored. Finally, to the best of our knowledge, there is no reported effect of Msc on human economy and infrastructures. Msc is an opportunistic species that nests in pre-existing cavities and does not dig holes in wood by itself, reducing its potential impact on infrastructure.

Regarding the economy, a single example has been reported in the literature regarding the potential positive impact of Msc on agriculture: the pollination of Sunn hemp (Crotalaria juncea L.: Fabaceae) in Florida [START_REF] Hall | Megachile sculpturalis, the Giant resin Bee, overcomes the blossom structure of Sunn Hemp (Crotalaria juncea) that impedes pollination[END_REF]. Msc has been associated with a higher seed set for this cover crop, which contributes to soil nitrogen enhancement [START_REF] Hall | Megachile sculpturalis, the Giant resin Bee, overcomes the blossom structure of Sunn Hemp (Crotalaria juncea) that impedes pollination[END_REF]. Nevertheless, it is worth noting that C.

juncea is an alien plant in the US. Moreover, this species is classified as invasive in some Pacific islands (see [START_REF] Hall | Megachile sculpturalis, the Giant resin Bee, overcomes the blossom structure of Sunn Hemp (Crotalaria juncea) that impedes pollination[END_REF]; therefore, it is difficult to classify the interaction between Msc and C. juncea as positive or negative.

Conclusion

In this chapter, we have presented an overview of the known impacts of alien bees on native ones. As emphasised in the introduction, we voluntarily excluded the other known impacts of alien bees on native ecosystems, such as the disruption of native plant-pollinator networks, the population enhancement of alien plants (invasional meltdown), or the impact on crop production (except for the case study, for a comprehensive review on this subject see [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF]. As shown here, the potential effects exerted by alien bees on native bees are important and could, in extreme cases, be responsible for dramatic population losses of native bees (the case of Bombus dahlbomii in South America; see above). To date, 80 bee species have been recorded outside their native ranges [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF]. The different cases we exposed in this chapter are among the most iconic, with the strongest recorded negative impacts. Overall, however, the global impact of alien bees on native bees and ecosystems is largely unknown. Very few cases of introductions have been carefully studied, and for some of the recorded alien bees, little is known regarding their impacts on native bees and ecosystems. The case of Msc is an exception, being one of the single invasions that is relatively well documented (as is the case of Bombus terrestris in South America, Japan, and Tasmania). Hence, we need to amplify our efforts to document new introductions, the geographic spread of alien bees, and their impacts. The number of new introducti ons is not expected to drop in the coming years [START_REF] Seebens | No saturation in the accumulation of alien species worldwide[END_REF], and we need to anticipate the potentially dramatic consequences of these introductions to avoid them. For example, in 2018, Bortolotti et al. reported the presence of a second alien bee species in continental Europe, Megachile disjunctiformis. This species resembles Msc morphologically (Fig. 9) and has a similar native geographical repartition. To date, M. disjunctiformis has only been detected in Italy and has not yet extended its geographical range to neighbouring countries. However, we believe that this is likely to occur soon. We have little information about the ecology of this new alien species, and the consequences of its introduction should be closely observed in the future.

As stated earlier, the introduction of a new bee species in an ecosystem is often disregarded because of its role in the pollination of plants. However, we shoul d change the way we perceive bee introductions. Like any other introduced species, alien bees can become invasive and affect native pollinators and ecosystem functioning. Introducing a new species into an environment is never a zero-sum game, and such practices must be abolished. Instead, native ecosystems should be preserved, and native bees allowed to thrive. Friendly practices favouring bees, such as the exclusion of pesticides, planting native flowers, and reduced mowing frequency (see [START_REF] Kawahara | Eight simple actions that individuals can take to save insects from global declines[END_REF], should be encouraged. To prevent new invasions, biosecurity between countries must be reinforced and cross-border legal instruments homogenised [START_REF] Diagne | High and rising economic costs of biological invasions worldwide[END_REF][START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF]; see Section 5). 5

Future trends in research

In this section, we have identified five major gaps that should be addressed in future research:

1 Even if some new data have been accumulated since, the first simple recommendation we can provide is to amplify research efforts on alien bees .

We advocate for a new review of bees introduced worldwide 6 years after the one of [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF]. The cases of M. disjunctiformis in Italy [START_REF] Bortolotti | First record of the Asiatic bee Megachile disjunctiformis in Europe[END_REF] or Xylocopa pubescens in the Canary Islands [START_REF] Ruiz | First record of the carpenter bee Xylocopa pubescens (Hymenoptera, Apidae) in the Canary Islands confirmed by DNA barcoding[END_REF] are examples of introductions that occurred after the review of Russo ( 2016).

2 We should estimate the ecological impacts of alien bees more accurately, be they positive or negative. For example, [START_REF] Russo | Positive and negative impacts of non-native bee species around the world[END_REF] determined that 11 out of 80 species could positively affect native ecosystems, but no review on these effects exists to date. Of course, fieldwork and experiments to estimate impacts are costly [START_REF] Goulson | Effects of introduced bees on native ecosystems[END_REF], but they provide valuabl e information for estimating impacts (e.g. see [START_REF] Díaz | Nesting, sex ratio and natural enemies of the Giant Resin Bee in relation to native species in Europe[END_REF][START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF].

3 We need to estimate the agronomic costs of alien bees. In their recent review, [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF] provided convincing examples of the deleterious impacts of alien bees on agroecosystems. For example, the invasion of Bombus terrestris in Argentina affected the production of raspberries [START_REF] Sáez | Extremely frequent bee visits increase pollen deposition but reduce drupelet set in raspberry[END_REF][START_REF] Sáez | Invasive bumble bees reduce nectar availability for honey bees by robbing raspberry flower buds[END_REF][START_REF] Sáez | The costs and benefits of pollinator dependence: empirically-based simulations predict raspberry fruit quality[END_REF].

One underlying mechanism is the over-visitation of raspberry flowers by Bombus terrestris, leading to flower damage, such as style breakage (see [START_REF] Badano | Potential negative effects of exotic honey bees on the diversity of native pollinators and yield of highland coffee plantations[END_REF] for an example on coffee plantations).

4 The economic cost associated with alien bees must be estimated. In additi on to the costs related to agricultural production, it has been suggested that some wood-nesting bees can potentially represent a problem for wooden structures as nest excavation may generate structural weakness in lumber [START_REF] Rust | Nesting biology and foraging ecology of the wood-boring bee Lithurgus Chrysurus (Hymenoptera: Megachilidae)[END_REF]. In their recent review on the global economic costs of invasive species, Diagne et al. ( 2021) estimated a mean annual cost of invasive invertebrates of US$ 8.7 billion from 1970 to 2017. However, this review also highlights the critical need for more research on the econom i c costs of invasive insects.

5 If possible, we should provide stakeholders with management techniques to avoid the spread of alien bees. For example, [START_REF] Geslin | Bee hotels host a high abundance of exotic bees in an urban context[END_REF] showed that Megachile sculpturalis rarely nests in bamboo with a hole diameter < 8 mm.

This observation could help land managers install and build insect hotels to avoid the nesting of Msc.

How to improve biosecurity

Among the new pathways of invasions, the increase of commercial offers for the public of both managed and wild bees is of great concern [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF]. Over the past 30 years, we have seen an exponential development of pollination supplementation practices in order to fight against pollinator declines and to compensate the limitation of pollination service delivered to entomophilous crops and ornamental species which may limit their yields. Since the 1970s, this has led to the first wave of global trade in social bees and bumblebees, initially for greenhous e use. As we have previously seen in this chapter, many reports of escapes of these imported bees into the wild have occurred. More recently, the second wave of the commercial trade of wild bees is emerging. In Europe or in North America, various commercial companies are selling wild solitary bees to promote better pollination service in gardens and field crops, arguing that these bees offer a much greater pollination service than domesticated social bees. Several mason bees and leafcutting bees have been selected for commercial purposes according to their nesting habits in cavities, offering an easy way to ship living cocoons in bamboo twigs.

However, in the US and Canada, this may also promote the spread of some alien bees (e.g. Osmia cornifrons). These new commercial practices bring new pathways for alien pollinators to spread as the business may be done worldwide. For example, by June 2021, a wild bee package shipped from France to Seattle (US) was intercepted in New Caledonia by biosecurity services. Inside the package, we found bamboo twigs containing 23 lived cocoons of Osmia bicornis and two recentl y emerged females (H. Jourdan, pers. obs.). This illustrates the tremendous risk of moving large numbers of alien pollinators to a wide range of regions without any biosecurity rules implemented.

Beyond this specific pollinator trade, there are still other pathways for the involuntary spread of alien pollinators associated with global trade. The rule that the more the volume of traded commodities increases between distant countries, the more the biosecurity risk increases for alien arrivals [START_REF] Hulme | Trade, transport and trouble: managing invasive species pathways in an era of globalization[END_REF], is also valid for pollinators. Mason bees (including resin bees and leaf-cutting bees) and carpenter bees (including hollow stem nesting species) appear more likely to be propagated by human trade.

To deal with these new threats, enforcement of biosecurity regulations and practices against the transfer or arrival of alien bees is urgently needed. According to the Food and Agriculture Organization of the United Nations standards , biosecurity is defined as 'a strategic and integrated approach that encompasses the policy and regulatory frameworks (including instruments and activities) for analysing and managing relevant risks to human, animal and plant life and health, and associated risks to the environment'. It appears critical in the context of biological invasions [START_REF] Hulme | Biosecurity: the changing face of invasion biology[END_REF]. Islands are particularly at risk and deserve special attention [START_REF] Cox | Pollinator extinction in the Pacific Islands[END_REF][START_REF] Moore | Protecting islands from pest invasion: optimal allocation of biosecurity resources between quarantine and surveillance[END_REF]. But since the late 1980s, most of the biosecurity efforts have focused on preventing the spread of parasites and pathogens of managed bees and not alien bees in general.

In recent decades, Australia has appeared as a good example of biosecurity measures enforced to protect native pollinators. Even if it was established mostly for protecting the bee industry with highly enforced regulation and surveillance against the introduction of Varroa destructor, tracheal mites with European (Apis melifera) or Asian (Apis cerana) beehives, it is also focusing on some bee diseases to avoid possible spillover between managed bees and wild bees [START_REF] Heersink | Quantifying the establishment likelihood of invasive alien species introductions through ports with application to honey bees in Australia[END_REF]Robert et al., 2017;[START_REF] Dalmon | Possible spillover of pathogens between bee communities foraging on the same floral resource[END_REF]. These biosecurity efforts rely on a national network of sentinel beehives. Such a sentinel coordinated approach is also applied to the larger Pacific region (New Zealand -despite Varroa installation -New Caledonia, French Polynesia, and to a lesser extent Fiji).

Another alternative solution to reduce the unfortunate trade of alien pollinators relies on the valuing and promotion of local wild pollinators for pollination effectiveness. This implies the conservation and restoration of "green infrastructures" across landscapes that enhance wild bee populations. In Australia, native stingless bees are promoted in a commercial way to enhance pollination service, and troubles in Tasmania linked to the European Bumblebee (Bombus terrestris) have led to research for local alternative pollinators. Thus, native Amegilla spp. were identified as highly efficient pollinators in gardens, especially for Solanaceae [START_REF] Mcquillan | Displacement of Tasmanian native megachilid bees by the recently introduced bumblebee Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae)[END_REF][START_REF] Hogendoorn | Native Australian carpenter bees as a potential alternative to introducing bumble bees for tomato pollination in greenhouses[END_REF][START_REF] Hogendoorn | Foraging behaviour of a blue banded bee, Amegilla chlorocyanea in greenhouses: implications for use as tomato pollinators[END_REF]. But without awareness of the public, a new biosecurity concern may develop, as peopl e may deliberately introduce Amegilla spp. into neighbouring oceanian territories , mostly archipelagos, for agricultural purposes. This has been suspected of Amegilla pulchra in Fiji, French Polynesia, and New Caledonia [START_REF] Groom | Origins and implications of apid bees (Hymentopera: Apidae) in French Polynesia[END_REF][START_REF] Zakardjian | Effects of urbanization on plant-pollinator interactions in the tropics: an experimental approach using exotic plants[END_REF]. However, once introduced, Amegilla spp. may not limit their foraging to valuable crop species and are likely to visit alien plants. If those alien plants are usually poorly visited by native pollinators, the arrival of alien bees such as Amegilla spp. could result in the spread of those plant species.

Finally, there is an emerging corpus of methodolog y and theory to better implement efficient biosecurity measures in a changing world (climate chang e and global trade), especially urgently needed on islands, which could be applied to pollinators [START_REF] Moore | Protecting islands from pest invasion: optimal allocation of biosecurity resources between quarantine and surveillance[END_REF][START_REF] Aizen | Coordinated species importation policies are needed to reduce serious invasions globally: the case of alien bumblebees in South America[END_REF][START_REF] Proesmans | Pathways for novel epidemiology: plant-pollinator-pathogen networks and global change[END_REF]. As pointed out by [START_REF] Hulme | Invasion pathways at a crossroad: policy and research challenges for managing alien species introductions[END_REF], pathways for alien species introducti ons fall into six broad classes: deliberate release; escape from captivity; contaminati on of a commodity; stowaway on a transport vector; via an infrastructure corri dor (without which spread would not be possible) or unaided from already invaded regions. Each of them should be examined carefully in the context of alien pollinators spread, resulting in enforcement of regulation policies and ad hoc surveillance to prevent further biological invasions. More specifically, in the context of safeguarding pollination services, ten general rules have already been promoted as guidelines to improve governmental regulation, including an emphasis on the regulation of movement of managed pollinators and developm ent of long-term monitoring of pollinators and pollination [START_REF] Dicks | Ten policies for pollinators[END_REF]. However, movement regulation should be extended to the wild bee trade and closest links should be developed with scientific research for robust longterm monitoring of pollinators and pollination at a global scale across a variety of landscapes. Hulme (2020) also promotes a "one biosecurity approach" relying on multidisciplina ry input from ecology, social sciences, and economics. Such a global biosecurity approach should be promoted for pollination service, which is critical to ecosystem's maintenance and human well-being in our changing worl d [START_REF] Hulme | One biosecurity: a unified concept to integrate human, animal, plant, and environmental health[END_REF][START_REF] Proesmans | Pathways for novel epidemiology: plant-pollinator-pathogen networks and global change[END_REF].

Where to look for further information

The following articles are the key papers on the impact of alien bee species on native bees:

• Aizen, M. A., Smith-Ramírez, C., Morales, C. L., Vieli, L., Sáez, A., Barahona-Segovia, R. M., Arbetman, M. P., Montalva, J., Garibaldi, L. A., Inouye, D.

W. and Harder, L. D. (2018). Coordinated species importation policies are needed to reduce serious invasions globally: The case of alien bumblebees in South America. J. Appl. Ecol. 1-7.

• Geslin, B., Gauzens, B., Baude, M., Dajoz, I., Fontaine, C., Henry, M., [START_REF] Geslin | Massively introduced managed species and their consequences for plant-pollinator interactions[END_REF]. Massively introduced managed species and their consequences for plant-pollinator interactions. In: Advances in Ecological Research, pp. 147-199. • Goulson, D. (2003). Effects of introduced bees on native ecosystems.

Annu. Rev. Ecol. Evol. Syst. 34, 1-26.

• Mallinger, R. E., Gaines-Day, H. R. and Gratton, C. (2017). Do managed bees have negative effects on wild bees?: A systematic review of the literature. PLoS ONE 12, e0189268.

• Paini, D. R. (2004). Impact of the introduced honey bee (Apis mellifera) (Hymenoptera: Apidae) on native bees: A review. Austral Ecol. 29, 399-407.

• Russo, L. ( 2016). Positive and negative impacts of non-native bee species around the world. Insects 7, 101-103.

For a focus on agricultural production, we recommend:

• Aizen, M. A., Arbetman, M. P., Chacoff, N. P., Chalcoff, V. R., Feinsinger, P., Garibaldi, L. A., Harder, L. D., [START_REF] Aizen | Invasive bees and their impact on agriculture[END_REF]. Invasive bees and their impact on agriculture, 1st ed., Advances in Ecological Research. Elsevier Ltd.

We particularly recommend the work of Dr M. Aizen and his team on this thematic:

• https://sites.google.com/site/marceloaiz en/ hom e.

The open science projects following the spread of Megachile sculpturalis in Europe and in the Balkans can be found here:

• https://oabeilles.net/projets/ observatoire-a beilles-exoti ques.

• https://www.citizen-science.at/ blog/ blogger/lannerj.

• https://srbee.bio.bg.a c.rs/english.

Key journals to read articles on invasion biology are:

• Biological invasions: https://www.springer.com/journal/10530.

• Diversity and Distribution: https://onlinelibrary.wiley.com/journal/14724642.

• NeoBiota: https://neobiota.pensoft.net/.

Other interesting websites:

• https://ipbes.net/invasi ve-alien-species-assessment.

• http://www.issg.org/.

• https://www.gbif.org/fr/dataset/39f 36f10-559b-427f-8c86-2d28afff68ca.

• https://easin.jrc.ec.europa.eu/easi n.

• https://serc.si.edu/research/ resea rch-topics/ biol ogical-invasions.

Some interesting outreaches:

• https://theconversati on.com/ bee-battles-w hy-our-native-pollinators-a re -losing-the-war-40620. • https://theconvers ati on.com/a ussi e-sci entis ts-need-your-hel p-keepi ng -track-of-bees-please-128932.

• https://idtools.org/i d/ bees/exoti c/.

Figure 1

 1 Figure 1 A sample of bee species introduced outside of their native range. (a) Anthidium manicatum (Linnaeus, 1758); Frank Vassen/Flickr/CC BY 2.0. (b) Bombus ruderatus (Fabricius, 1775); Dave Young/Flickr/CC BY 2.0. (c) Megachile disjunctiformis (Cockerell, 1911) (Franziska Maria Luthi). (d) Osmia taurus (Smith, 1873) Benjamin Smith/Wikimedia Commons / CC BY 2.0. (e) Osmia cornifrons (Radoszkowski, 1887) Flickr/CC 1.0. (f) Xylocopa augusti (Lepeletier, 1841) Clinton.sm/Wikimedia Commons/CC BY-SA 4.0.

Figure 3

 3 Figure3A honey bee and a bumblebee sharing the same floral resource (CC 1.0).

Figure 4

 4 Figure 4 Honey bee with deformed wing virus (DWV) and Varroa destructor (CC0 1.0).

Figure 5

 5 Figure 5 Megachile sculpturalis female. Le Collet-de-Dèze, France. Picture: Frank Vassen.

Figure 6 :

 6 Figure 6 : Year of the first record of Megachile sculpturalis in European countries (sources:[START_REF] Aguado | Megachile (Callomegachile) sculpturalis Smith, 1853 (Apoidea: Megachilidae): a new exotic species in the Iberian Peninsula, and some notes about its biology[END_REF][START_REF] Amiet | Die Blattschneiderbiene Megachile sculpturalis Smith, 1853 (Hymenoptera, Apidae) nun auch in der Schweiz[END_REF][START_REF] Ćetković | Project: monitoring the spread of sculptured resin bee (Megachile sculpturalis) in Serbia. Centar za biologiju pčela[END_REF][START_REF] Gogala | First record of Megachile sculpturalis smith in Slovenia (hymenoptera: Megachilidae)[END_REF][START_REF] Ivanov | First record of the invasive giant resin bee Megachile (callomegachile) sculpturalis smith, 1853 (hymenoptera: Megachilidae) in the crimea[END_REF] Kovács, 2015;[START_REF] Lanner | Dispersal patterns of an introduced wild bee, Megachile sculpturalis Smith, 1853 (Hymenoptera: Megachilidae) in European alpine countries[END_REF][START_REF] Quaranta | A new species for the bee fauna of italy: Megachile sculpturalis continues its colonization of Europe[END_REF][START_REF] Vereecken | Premières données sur la présence de l'abeille asiatique Megachile (Callomegachile) sculpturalis SMITH (Hymenoptera, Megachilidae) en Europe[END_REF][START_REF] Westrich | Megachile sculpturalis Smith 1853 (Hymenoptera, Apidae), a new species for the bee fauna of Germany, now north of the Alps[END_REF][START_REF] Westrich | Faszination Wildbienen, Forschungsprojekte: Megachile sculpturalis[END_REF]. The homogeneous colouring of national territories can be misleading. For example, concerning the islands, Msc is so far unknown in Corsica, Sicily, and Sardinia. However, it has been recorded on the island of Elba and in Majorca, islands that we have circled in red. Map made with QGIS version 3.14. Base map downloaded from Natural Earth (naturalearthdata.com).

Figure 7

 7 Figure 7Megachile sculpturalis female closing its nest with tree resin. In the background two more of her nests can be seen. Oberhofen am Thunersee, Switzerland. Picture: Christa Rohrbach.

Figure 8

 8 Figure 8 Megachile sculpturalis female emptying the nest of a female of Anthidium sp. Anglet, France. Picture: Pierre-Jean Vandoorne.

Figure 9

 9 Figure 9 Megachile disjunctiformis. Picture: Franziska Luthi.


Layal Fahed

Madona Khoury

Didier Stien

Naïm Ouaini

Véronique Eparvier

Marc El Beyrouthy

Essential oils composition and antimicrobial activity of six conifers harvested in Lebanon

Keywords: Chemical composition, Antimicrobial activity, Essential oils, Conifers, Lebanon

The chemical composition and antimicrobial activity of the essential oils (EOs) of six conifers harvested in Lebanon, Abies cilicica, Cupressus sempervirens, Juniperus excelsa, Juniperus oxycedrus, Cedrus libani and Cupressus macrocarpa gold crest, were investigated. The EOs were obtained by hydrodistillation using a Clevenger-type apparatus and characterized by GC and GC-MS analyses. A principal components analysis based on Pearson correlation between essential oils chemical analyses was also conducted. The minimum inhibitory concentrations (MICs) of these essentials oils were determined against a range of bacteria and fungi responsible for cutaneous infections in human, using the broth microdilution technique. The EOs showed the most interesting bioactivity on the dermatophytes species (MIC values 32-64 µg/ml). Each of the major compounds of C. macrocarpa as well as an artificial reconstructed EO were tested on Trichophyton rubrum showing a contribution of the minor components to the overall activity.

Introduction

Conifers are woody plants with most of them being evergreen trees. About 630 species of conifers can be found as wild populations in all continents except Antarctica [START_REF]Conifers; Status Survey and Conservation Action Plan[END_REF]. In Lebanon they are represented by ten species belonging to five genera [START_REF] Talhouk | [END_REF]. Cedrus libani A.Rich. , the national emblem of Lebanon, was mentioned in the Old Testament of the Holy Bible over 75 times and in the Epic of Gilgamesh, the oldest recorded legend [START_REF]Conifers; Status Survey and Conservation Action Plan[END_REF].

Its wood was intensively exploited by ancient civilizations in the building of temples, palaces, and boats [3], and its essential oil was used by the Egyptians in the mummification process [START_REF] Wilson | Ancient Near Eastern Texts Relating to the Old Testament[END_REF]. Lebanese conifer species are an important part of the folk medicine systems. Abies cilicica (Antoine & Kotschy) Carrière a plant endemic to Lebanon, Syria and asiatic Turkey is traditionally used in Lebanon for the treatment of rheumatism and lumbago, and is used in Turkey against gastrointestinal diseases, asthma and bronchitis [START_REF] Beyrouthy | [END_REF] [START_REF] Sargin | [END_REF]. Cupressus sempervirens L. f. horizontalis (Mill.) Voss is known as an antiasthmatic, antitussive, antiodontalgic, antiulcer and antirheumatic herbal remedy, while Juniperus species J. excelsa M. Bieb. and J. oxycedrus L. are priced for their antirheumatic and antineuralgic properties [7]. Beside indigenous species, many conifers are introduced in Lebanon generally for ornamental purposes. This is the case of Cupressus macrocarpa Hartw. goldcrest, a plant used popularly in interior landscaping due to its bright color and agreeable lemon fragrance.

Antimicrobial resistance is a global concern requiring urgent actions across all concerned sectors. This phenomenon is accelerated by the excessive and inappropriate use of antimicrobial agents [8].

In an attempt to search for alternatives to conventional antibiotics and antifungals for the treatment of topical, non severe infections, the essential oils of the six abovementioned conifer species were subjected to chemical analysis and evaluation of their antimicrobial activity.

Results and Discussion

Chemical Analysis

The chemical compositions of the six conifers essential oils are presented in table 1.

The yields of the EOs obtained by hydrodistillation were as follows: 0.08% (J. oxycedrus), 0.29% (C. sempervirens), 0.34% (C. libani), 0.68% (C. macrocarpa), 0.93% (A. cilicica) and 1.1% (J. excelsa). GC and GC-MS analyses led to the identification of 91 components accounting for 81.6% (A. cilicica) to 94.3% (C. macrocarpa) of the oils.

*Please insert table 1 here*

The data matrix was used to generate a biplot superimposing principal component analysis (vectors) with a map of the variables (i.e. molecules) (Fig. 1). Loading factors 1 and 2 in PCA described 77.5% of the variability in the dataset. It can be seen that 4 out of 6 EOs clustered in one chemical group, with a high relative proportion of pinene and a lower proportion of -pinene. Two outliers are present in the dataset, A. cilicica and C. macrocarpa EOs. A. cilicica EO contains a significant amount of pinene (8.5%) and is therefore not orthogonal to the group of -pinene rich oils. What makes it peculiar in this dataset is the high proportion of -3-carene, as indicated by the biplot. As regards C. macrocarpa EO, it is orthogonal to the others and its specific descriptors are sabinene, terpinen-4-ol and citronellol. These compounds are either absent or minorly present in the other EOs. *Please insert figure 1 here*

Antimicrobial activity

The results of the antimicrobial activity of the conifers EOs are reported in table 2. EOs with minimal inhibitory concentrations (MICs) of 128 μg/ml and below are generally considered active [9] [10].

S. aureus was significantly sensitive to J. excelsa, J. oxycedrus and C. libani EOs (MIC of 64 μg/ml) and resistant to three others (MIC > 512 μg/ml). According to the MICs values reported in the literature, -pinene, the main component of the active oils, couldn't be responsible for their activities [11] [12]. This goes along with the fact that -pinene is also mainly present in two of the inactive oils. On the other hand, none of the EOs was active against P. aeruginosa, E. coli or C. albicans (MIC ≥ 512 μg/ml).

Conversely, Trichophyton species were sensitive to most of the tested EOs (MICs from 32 to 512 μg/ml).

*Please insert table 2 here*

In order to elucidate the origin of the interesting activity of C. macrocarpa on the dermatophytes (MICs from 32 to 64 μg/ml), the activity of its major components were evaluated individually as well as a reconstructed EO against Trichophyton rubrum (Table 3). The artificial EO was prepared using pure terpenes of the major compounds in the same proportions, whereas the minor ones were replaced by DMSO (25.8%).

Terpinene-4-ol was the most active compound (MIC of 64 μg/ml), suggesting a strong contribution to the antifungal activity of the oil. The other components showed less significant activities (MICs from 256 to 512 μg/ml).

The reconstructed EO was less active than the natural one (256 μg/ml vs 64 μg/ml), therefore the activity of C. macrocarpa EO couldn't be explained by a synergy between its major compounds. These results suggest that the minor constituents have played a significant role in the antimicrobial potential of the oil.

*Please insert table 3 here*

Conclusions

Lebanese conifers essential oils showed interesting antimicrobial activity particularly against S. aureus and Trichophyton species. These EOs should therefore be considered for a possible integration in antimicrobial preparations intended to be used in the treatment of superficial infections.

Experimental Part

Plant Material

Specimens of the six conifers were collected as follows:

-Leaves from Abies cilicica were collected from Fneidek, North Lebanon (34°27'54.48" N 36°13'10.74" E), at approximately 1560 m altitude, in January 2013. Mouterde [START_REF] Mouterde | Nouvelle flore du Liban et de la Syrie[END_REF]. Air drying of the plant materiel was performed in a shady place for two weeks at room temperature.

Essential Oil Extraction

The extraction of the essential oils was carried out by hydrodistillation for three hours using a Clevenger-type apparatus according to the European Pharmacopoeia, 1997 [START_REF]European Pharmacopoeia[END_REF].

GC Analyses

Analytical gas chromatography was performed on a Thermo Electron Corporation gas chromatograph fitted with a flame ionization detector (FID), a DB-5 MS capillary column (30 m × 0.25 mm) with 0.1 µm film thickness or a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm, film thickness 0.20 µm).

Helium was the carrier gas (0.7 ml/min). The column temperature was initially set to 35 ˚C before being gradually increased to 85 ˚C at 5 ˚C/min, held for 20 min at 85 ˚C, raised to 300 ˚C at 10 ˚C /min and finally held for 5 min at 300 ˚C. Diluted 1 µl samples (1/100, v/v) were injected at 250 ˚C manually and in the splitless mode. Flame ionisation detection (FID) was performed at 310 ˚C.

GC-MS Analyses

The GC/MS analyses were performed using an Agilent 6890 gas chromatograph coupled with 5975 Mass Detector. The 7683 B auto sampler injected 1 µL of each diluted oil sample (1/100, v/v). A fused silica capillary column DB-5 MS (30 m × 0.25 mm internal diameter, film thickener 0.1 µm) or a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm, film thickness 0.20 µm) was used. Helium was the carrier gas (0.7 ml/min). The oven temperature program was identical to that described above (cf. GC Analysis). The mass spectra were recorded at 70 eV with an ion source temperature of 310 ˚C and a transfer line heated to 320 ˚C.

The acquisition was recorded in full scan mode (50 -400 amu).

Qualitative and Quantitative Analyses

Most constituents were identified by gas chromatography by comparing their retention indices (RI) with those from the literature [START_REF] Jennings | Qualitative analysis of flavour and fragrance volatiles by glass capillary gas chromatography[END_REF] [16], or with those of authentic compounds obtained from Sigma-Aldrich (Lebanon and France). The retention indices were determined relatively to a homologous series of n-alkanes (C8 to C24) analysed under the same operating conditions. Concomitantly, their mass spectra on both columns were compared with those provided in the NIST and Wiley 275 libraries, our home-made library constructed with pure compounds and EOs of known composition or with mass spectra from the literature [15] [17].The relative concentration of each compound was calculated based on the GC-FID peak areas without using correction factors.

Statistical analysis

A principal components analysis based on Pearson correlation between essential oils chemical analyses was conducted with XLstat version 2014. In the biplot, samples (EOs) are displayed as vectors and EO components are displayed as points. The antimicrobial activity of the EOs was measured using a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines with minor modifications [19 -24]. The essential oils and its major components were diluted in DMSO and were tested at concentrations ranging from 512 to 16 μg/ml. The maximum DMSO concentration didn't exceed 5% (v/v). Sterility and positive controls in Müller-Hinton broth (MHB) or Roswell Park Memorial institute (RPMI) medium alone and with 5% of DMSO (v/v) were included. Positive control wells contained microorganisms without antibiotics or antifungals. The microplates were incubated at 37°C for 24 h for bacteria, and at 35°C for 48 h for yeasts and 5 days for dermatophytes. The minimal inhibitory concentrations (MIC) refer to the lowest concentrations preventing visible microbial growth (Table 2, Table 3). Oxacillin and gentamicin (16 -0.03 μg/ml) were used as reference antibiotics, while itraconazole (16 -0.03 μg/ml) and fluconazole (64 -0.125 μg/ml) were used as positive controls for the antifungal assays. The antimicrobial standards were purchased from Molekula -Dorset, UK and the pure terpenes and the culture media from Sigma-Aldrich, France. Bold numbers indicate the percentages higher than 3% to show main components a ) Retention index on a DB-5MS column.

Antimicrobial activity

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  Cones from Cedrus libani were collected from Bcharre, North Lebanon (34°10'40.91'' N 36°02'55.89'' E), at approximately 1916 m altitude in September 2011. -Leaves from Cupressus macrocarpa were collected from Achkout, Mount Lebanon (33°59'33.16" N 35°41'59.48" E), at approximately 1048 m altitude, in January 2013. -Leaves from Cupressus sempervirens were collected from Kartaba, Mount Lebanon (34° 5'42.71" N 35°50'51.18" E), at approximately 1052 m altitude in December 2012. Twigs from Juniperus excelsa were collected from Kartaba, Mount Lebanon, (34°05'55.35" N 35°50'27.77" E), at approximately 1400 m altitude in October 2011. Twigs from Juniperus oxycedrus were collected from Kartaba, Mount Lebanon, (34°05'55.35" N 35°50'27.77" E), at approximately 1400 m altitude in September 2011. The plants identification was based on 'Nouvelle flore du Liban et de la Syrie' by Paul

  Gram (-) bacterial strain Pseudomonas aeruginosa CIP 82118, Gram (+) bacterial strain Staphylococcus aureus ATCC 29213, yeast Candida albicans ATCC 10231 and the clinical isolates of dermatophyte Trichophyton rubrum SNB-TR1, Trichophyton mentagrophytes SNB-TM1, Trichophyton soudanense SNB-TS1, Trichophyton violaceum SNB-TV1 and Trichophyton tonsurans SNB-TT1 [18], were used in this study.

b)Figure 1 .

 1 Figure 1. Biplot from the principal component analysis of the six conifers EOs

Table 1 .

 1 Chemical composition of six conifers essential oils

	Compounds (%)

Table 2 .

 2 Antimicrobial activity (MIC in µg/ml) of the six conifers essential oils

Acknowledgments

This work benefitted from an "Investissement d'Avenir" grant managed by the Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01) and from the support of CNRS Lebanon.