Olivier Mentré

Abdelhakim Laachir

Salaheddine Guesmi

Mostafa El

Mohamed Ketatni

Lahcen El Saadi

Siham Ammari

Mohammed Esserti

Fouad Faize

Bentiss

Ketatni

Mohamed Saadi

Lahcen El Ammari

Siham Esserti

Mohammed Faize

Fouad Bentiss

magnetic properties and antimicrobial activities

Keywords: 3,5-Bis(2-pyridyl)-1,2,4-triazolate, Homobimetallic Co II complex, Crystal structure, Hirshfeld surface analysis, Magnetic property, Antimicrobial activity

    A new homobimetallic cobalt(II) complex based on the tetradentate 3,5-bis(2-pyridyl)-1H-1,2,4-triazole ligand: Synthesis, crystal structure, Hirshfeld analysis, spectroscopic characterization,

Introduction

There is interest in dimeric complexes of odd electron metals with close metal-metal contacts and appropriate bridging atoms for their magnetic properties [1][2][3][4][5][6][7][8][9][10][11]. These properties may be modified by the character of the bridging species and by the geometry of the metal atoms and organic ligands. Thus there is an interest in multidentate ligands which may serve to orient the d orbitals of the two metals to achieve maximum interaction. 1,2,4-Triazole and its derivatives are very interesting chelating ligands, which represent a hybrid of pyrazole and imidazole and are able to act as bridges between metal centers [2,3,12]. 1,2,4-Triazoles with opyridine substitution at the 3 and 5 positions may serve as appropriate matrix ligands in either neutral or anionic form (Scheme 1). When one of the three nitrogen atoms of the 1,2,4-triazole ring bears an hydrogen, the ligand is neutral, it's the 3,5-bis(pyridin-2-yl)-1H-1,2,4-triazole abbreviated as Hbpt. This laster can be often deprotonated to form an anionic 3,5-bis(2pyridyl)-1,2,4-triazolate (bpt) in the formation process of complexes. However, only few mono or dinuclear transition metal complexes as well as tetranuclear or even polymeric complexes containing anionic bpt ligand were characterized by X-ray diffraction [4,6,7,[12][13][14][15][16][17][18][19][20], which indicate that these coordination compounds have relatively poor crystallization behaviour.

Different coordination modes can be occurred with bpt ligand and transition metal (Scheme 2). Indeed, when the four nitrogen atoms N', N 1 , N 2 et N" of the btp ligand are on the same side, a double-bridged bimetallic complex in close proximity is obtained (cisoid conformation); with metal-to-metal distance varying approximately from 4 to 4.5 Å [7,12,16,19,20]. In contrast, when one pyridine ring rotates 180° to chelate a metal ion with the contribution of the nitrogen atom in position 4, a mono-briged bimetallic complex is obtained (transoid conformation) [15,21,22]. Generally, bpt complexes would prefer the cisoid conformation [16].

Moreover, bpt ligand has formed complexes with appropriate transition metals (M) displaying either antiferromagnetic or ferromagnetic behaviour [12,17]. Magnetic exchange involving the M-N=N-M bridge has been postulated to imply a  overlap superexchange pathway exploiting the electrons of the heterocyclic ligands [17]. Spin crossover from one magnetic behaviour to the other has been observed with iron(II) complexes based on 3,5-di(2pyridyl)-1,2,4-triazoles and 3,5-di(2-pyridyl)-1,2,4-triazolates ligands [7]. Structural parameters such as the coplanarity of metal and heteroatom containing ligand, and the magnitude of the M-N=N angles have been discussed as playing a role in the magnetic comportment [19]. In addition, the triazoles and their complexes possess a variety of interesting biological activities, such as anticonvulsant, antifungal, anticancer, anti-inflammatory and antibacterial properties [23][24][25][26]. In view of these reports and as part of our going researches on the synthesis, structural investigation and property of heterocyclic metal complexes, we have chosen Hbpt as a bridging ligand to construct novel homobimetallic cobalt complex. We report herein the synthesis, structural and spectroscopic characterizations, and Hirshfeld surface analysis, as well as magnetic property and antimicrobial activity of dinuclear cobalt(II) nitrate complex with 3,5bis(2-pyridyl)-1,2,4-triazolate (bpt), [Co2(bpt)2(H2O)4](NO3)2.2H2O.

Experimental

Materials

The ligand, namely 3,5-bis(2-pyridyl)-1H-1,2,4-triazole (Hbpt) was synthesized according to the literature procedure [24]. The molecular structure of Hbpt is shown in Scheme 1. All the solvents and reagents for synthesis were commercially available and used as received.

Synthesis of the title compound [Co2(bpt)2(H2O)4](NO3)2.2H2O

A solution of Hbpt (

Single crystal X-ray data collection

A suitable single crystal for X-ray diffraction analysis of the title compound was selected under a microscope (0.33 × 0.29 × 0.25 mm). The red single crystal for the X-ray diffraction (XRD) data collection was mounted on a Bruker APEX-II CCD diffractometer equipped with a CCD bi-dimensional detector and MoKα graphite-monochromatic radiation ( = 0.71073 Å). SAINT + 6.02 program was used for extraction and integration of diffraction intensities [28] and SADABS program was carried out for correction of absorption effect [29].

The coordination complex structure was solved by direct methods using SHELXTL-2014/4 [30] and refined (by weighted full matrix least-square on F 2 techniques) to convergence using the SHELXL-2016/6 program [31]. All non-hydrogen atoms were refined anisotropically.

Hydrogen atoms were located in a difference map and treated as riding with C-H = 0.96 Å for the aromatic CH, with Uiso(H) = 1.2 Ueq (aromatic). Crystal data, data collection and structure refinement details are summarized in Table 1. The bond distances and angles are given in Table 2. The plot of the title complex and the three-dimensional drawing of the crystal structure are obtained using the Ortep3 and Mercury programs, respectively [32,33].

Hirshfeld surface analysis

The Hirshfeld surfaces calculated for the title complex provide additional information on the distinctive contributions made to the molecular packing. Thus, a Hirshfeld surface analysis [34] and the associated two-dimensional fingerprint plots [35] were performed using CrystalExplorer17.5 [36] to figure out the normalized contact distance (dnorm), which depends on contact distances to the closest atoms outside (de) and inside (di) the surface. The 3D dnorm surfaces are mapped over a fixed color scale of -0.8273 to 1.0993 a.u. for Co complex. The electrostatic potential was generated using the STO-3G basis set at the Hartree-Fock level of theory, energy in the range -0.1099 to +0.1470 a.u for water molecule, -0.0543 to +0.1035 a.u for NO3 -anion and -0.1830 to +0.2303 a.u. for the investigated complex.

Physical measurements

UV-Visible absorption spectra in dimethyl sulfoxide (DMSO; 10 -4 M) solvent were recorded in the range 200-900 nm using a SHIMADZU 2450 spectrophotometer. FTIR spectra were recorded on a SHIMADZU FT-IR 8400S spectrometer with samples dispersed in KBr pellets in the range 500-3000 cm -1 . Element analyses (C, H and N) were performed on a VARIO-ELEMENTAR analyzer.

Magnetic measurements (thermal dependence of the susceptibility and field dependence of the magnetization) have been collected using the VSM module of a Physical Property Measurement System (PPMS) Dynacool, maximum field = 9T.

Antifungal and antibacterial activity

The antifungal activity of the title complex was examined against three strains of the fungal plant pathogen V. dahliae (Strains SJ, SH and SE) and one strain of the fungal plant pathogen F. oxysporum fsp. melonis (FOM). Strains of V. dahliae were isolated from olive trees in different regions of Morocco while FOM was isolated from a melon field in Doukkala region, Morocco. They were sub-cultured on potato dextrose agar (PDA) plates and incubated at 27 °C in the dark. The cobalt complex was dissolved in 0.2% DMSO and added at concentrations of 5, 50 and 100 g mL -1 to the PDA medium before being autoclaved and cooled. The amended media was poured into Petri dishes and inoculated using 5 mm plugs of agar and mycelium taken from actively growing cultures of fungus on PDA. Petri plates were incubated in the dark at 27°C. The mycelial growth was assessed by measuring 2 orthogonal diameters of each colony after 7 days of incubation. Mycelial growth was compared with growth on PDA amended with 0.2% DMSO (control) and the percentage of inhibition growth was expressed [37] relative to the control. The experiment was repeated 3 times.

The in vitro antibacterial activity of the investigated complex was performed against the following phytopathogenic bacteria: Agrobacterium tumefaciens (Ach5, A281 and C58 strains), Pseudomonas syringae pv. tabaci (strain CFBP2106) and Pseudomonas syringae pv.

syringae (strain CFBP692). A. tumefaciens strains were sub-cultivated on LB solid medium while P. syrnigae pv. syringae and P. syringae pv. tabaci were cultured on King B (KB) medium at 28°C. Antibacterial activities of the dimeric complex was carried out using the agar disc diffusion method as described in [38]. Bacteria that were grown overnight were diluted to a concentration of 3 × 10 8 CFU. mL -1 . Sterile cellulose discs of 6 mm diameter were impregnated with the products (200 µg mL -1 in 0.2% DMSO) and discs were placed on either LB or KB solid medium previously amended with 3 × 10 8 CFU mL -1 of bacteria. DMSO (0.2%) was used as a negative control. The inoculated plates were incubated at 28°C for 48 h. The antimicrobial activity was evaluated by measuring the clearance zone around the discs which indicates a positive antibacterial activity of the respective products. Each experiment was repeated 3 times.

All data of antimicrobial activities were expressed as the mean and confidence intervals.

They were subjected to ANOVA, and means were compared using Tukey's test and p values of 0.05 were considered to be significantly different.

Results and discussion

Crystal structure determination and description

The dinuclear cobalt(II) complex of 3 Symmetry codes: (i) 1+x, y, z; (ii) -x, 1-y, 1-z; (iii) -x, -y, 1-z; (iv) 1-x, -y, -z; (v) 1-x, -y, 1-z.

Hirshfeld surface analysis study

The As illustrated in Fig. 6, the corresponding fingerprint plots for the title compound are shown with characteristic pseudo-symmetric wings in the de and di diagonal axes and those 4, providing additional stability in 3D architecture. The Hirshfeld surfaces in the coordination geometry formed by the N4O2 about the Co(II) centre in the complex molecule that has been mapped to a normal surface are shown in

delineated into H•••H, H•••O/O•••H, C•••H/H•••C, N•••H/H•••N and C•••C.
•••H, (c) O•••H/H•••O, (d) C•••H/H•••C, (e) N•••H/H•••N and (f) C•••C contacts.
b) H•••H 31.6 (c) O•••H/H ••• O 22.1 O•••H 2.2 H ••• O 19.9 H•••C 6.7 C•••H 11.4 (d) C•••H/H•••C 18.1 (e) N•••H/H•••N 12.9 N•••H 8.1 H•••N 4.8 (f) C•••C 4.6 (a) Overall a b c

Spectroscopy studies

Ultraviolet-visible spectroscopy

The electronic spectra of Hbpt and title complex are shown in Fig. 9. The absorption maxima with the corresponding extinction coefficients are given in Table 5. The UV-Vis spectrum of [Co2(bpt)2(H2O)4](NO3)2.2H2O in DMSO reveals the presence of two absorption bands in the ultra-violet range at 236 and 260 nm. By analogy with the Hbpt proligand spectrum, these bands were attributed to 𝜋→𝜋* ligand transitions, characteristic of their 𝜋-electronic system [40,43]. The significant red shift of the transitions attributed to the ligand in the complex (Table 5) can be due to the stabilization of the 𝜋* orbitals and to a better electronic delocalization along the conjugated system [44]. The d-d transitions of the Co complex appear in the visible domain as three low-intensity bands at 479 (λ1), 509 (λ2) and 785 nm (λ3). These maxima correspond respectively to λ1: 4 T1g (F) → 4 T1g(P), λ2: 4 T1g(F) → 4 A2g(F) and λ3: 4 T1g(F) → 4 T2g(F) transitions for a Co(II) ion in an octahedral geometry [44][45][46][47].

Table 5

Assignments of the main UV bands of the Hbpt ligand and of its Co complex in DMSO (10 -4 M).

FTIR spectroscopy

The FTIR spectrum of [Co2(bpt)2(H2O)4](NO3)2.2H2O is very similar to that of the Hbpt ligand (Fig. 10), except for the presence of a low intensity band around 3400 cm -1 and another thinner at 1387 cm -1 attributable to water molecules [48,49] and nitrate ions [48,50,51] respectively. The presence of nitrate ions reveals the cationic nature of the product synthesized and the analogy between the two spectra indicates the presence of the ligand in the complex characterized by its main absorption bands, attributable to the (C-H), (C=N) and (C=C) bonds as shown in Table 6.

Magnetic properties

The temperature dependence of the inversemolar magnetic susceptibility collected at 0.1 T is given in Fig. 11a using one Co 2+ per formula unit. (T) values exhibit a broad maximum at T = 5 K typical of the low-dimensional Co 2+ dimers units [START_REF] Carlin | Magnetochemistry[END_REF]. It also shows ZFC/FC divergence below 7.5 K which signs a minor ferromagnetic impurity responsible for the constant low temperature magnetization of the zero-field cooled (ZFC) branch. This later represents a major drawback for the accurate (T) fit (see inset Fig. 11a), signing a weak but real net magnetic moment. The exchange coupling within the dimers was first estimated from the high-temperature Curie-Weiss law fitted between 25 and 400 K (Fig. 11a). We found eff = This "mean field" approximated solution is expected to give good results in such a "weak J" context, compared to other analytical Heisenberg or more accurate Ising solutions and prevails in our study [START_REF] Spasojevic | [END_REF]. One must emphasize that the maximal distances between next dimers being > 8.1 Å (Fig. 2), inter-dimer exchanges were neglected. Fig. 10b shows the best fit of the lowtemperature (T) part using a Ising dimer model after powder averaging of // and  ⊥ [START_REF] Spasojevic | [END_REF]54]. For reasons given above there are strong incertitudes on g// and g⊥, both scaling the peak height finally fixed. In contrast the temperature of the  maximum mainly depends on the J values giving J = -6.61(8) K very close to the Curie-Weiss deductions, revealing weak antiferromagnetic coupling between the two cobalt centers. This value falls in the range usually found for binuclear triazole-and triaolate-N 1 ,N 2 -bridged cobalt complexes [2]. At low temperature, assuming the ferromagnetic extrinsic contribution constant, the Fisher heat capacity given by d(.T)/dT shows a sharp maximum, suggesting magnetic ordering below TN = 3.11 K (Fig. 11c). Taking into account spatially well isolated dimers, the anisotropy of the Ising dimer exchanges account for such ordering. Finally, the M(H) plots are rather informative (Fig. 11d), their evolution is typical of a paramagnetic regime above 7K. Then at 2K, the magnetization turns weaker due to AFM exchanges in the dimers, but abruptly increases above 6.5 T, suggesting a metamagnetic transitions, here the alignment of Co 2+ in the field is possible until the value 2 B/Co at 9 T.

Antimicrobial activities

The effect of [Co2(bpt)2(H2O)4](NO3)2.2H2O was assessed through the mycelial growth inhibition of three strains of V. dahliae and one strain of F. oxysporum fsp. melonis. V. dahliae and F. oxysporum are the causal agent of wilt that affects several crops of economic interest [55,56]. Results showed that the investigated dimeric complex exhibited a great antifungal activity against the strains SH and SJ of V. dahliae and against F. oxysporum fsp. melonis. Indeed, at least 70% of inhibition of radial growth was achieved at 50 µg mL -1 (Fig. 12a).

However, these inhibitions were relatively lowers with the strain SE of V. dahliae since only 46 and 66% of inhibition was recorded at 50 and 100 µg mL -1 , respectively. Such a differential sensitivity of these fungal strains to the cobalt complex could be explained by their different genetic background. The elevated activity of this complex may rely on its high lipophilicity allowing its penetration through the lipid bilayer membrane and consequently inhibition of fungal growth. An illustration of the antifungal activity of the dimeric complex against the tested fungal strains was shown in Fig. 12b. In this study we also evaluated the effect of the Co II complex against several phytopathogenic bacteria of economic interest including A. tumefaciens, the causal agent of crown gall disease [57]. The cobalt complex revealed to effective in the inhibition of the strains A281, C58 and Ach5 (Fig. 13). However, no significant inhibition was recorded for P. syringae pv. tabaci and P. syringae pv. syringae, the causal agent of wild fire disease of tobacco and various diseases in several crop [58]. These bacteria are epiphytic and exhibit higher ability to produce form a biofilm allowing them to survive epiphytically. This may explain different sensitivities between tested bacteria. Indeed, the exopolysaccharides (EPS) polymers, which form a capsule involved in biopolymer formation, may act as an effective permeability barrier against the dimeric complex [59].

Control Complex

3 , 5 -Scheme 2 .

 352 Scheme 1. 3,5-Bis(2-pyridyl)-1H-1,2,4-triazole (Hbpt) and its anionic form (bpt).

  ,5-bis(2-pyridyl)-1,2,4-triazolate (bpt), dinitrate dihydrate [Co2(bpt)2(H2O)4](NO3)2.2H2O, crystallizes in triclinic crystal system with P1 ̅ space group and results from the association of the asymmetric unit and its inversion symmetric as illustrated in Fig. 1. The crystallographic study showed that there are two [Co2(bpt(H2O)4] 2+ cation moieties, two NO3 -anions and two solvated water molecules in the unit cell. This result indicated that the central triazole ring of Hbpt (ligand) was deprotonated to produce the anionic bpt ligand (Scheme 1). The Co II centres are bridged by the two triazolate moieties and the bpt ligand adopts the cis-(N',N 1 ,N 2 ,N'')2 double bridging coordination mode (Scheme 2).

Fig. 1 .

 1 Fig. 1. Plot of the crystal structure of the binuclear cobalt complex, [Co2(bpt)2(H2O)4](NO3)2.2H2O, showing hydration water molecule and nitrate group linked to the complex by hydrogen bonds (dashed lines).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Projection of the structure of [Co2(bpt)2(H2O)4](NO3)2.2H2O showing the intra and interdimer Cu⋯Cu separations.

Fig. 4 .

 4 Fig. 4. π-π stacking interactions and O•••π intermolecular interactions (between the O-atom of NO3 - conteranion and pyridyl ring) in crystal structure of [Co2(bpt)2(H2O)4](NO3)2.2H2O.

  three dimensional dnorm surface of [Co2(bpt)2(H2O)4](NO3)2.2H2O using a standard surface resolution is shown in Figs. 5a-e. The intense red spots on the surface are due to the H2O•••H-O-H, H-O-H•••O3Nitrate and H-O-H•••Ntriazolate hydrogen bonds resulting from the interactions between the coordinated water and water molecule of crystallization and nitrate group and between the water and N atom of the triazolate ring. On the Hirshfeld surface mapped over the electrostatic potential (Fig. 5f) shows blue regions around the metal coordinating water molecules indicating the positive electrostatic potential, while bright-red spots indicate atoms with negative electrostatic potential. Figs. 5g-h also illustrated the positive and negative electrostatic potential for uncoordinated water molecule (free) and nitrate conterion, respectively.

  The relative contributions of all the contacts to the Hirshfeld surface are summarized in Table 4. The presence of water molecules and nitrate ion in the crystal of Co-complex increases the percentage contribution from O•••H/H•••O contacts to the Hirshfeld surface of the asymmetric unit compared with the complex molecule alone. The molecular interactions in terms of H•••H contacts are reflected in the distribution of the scattered points in the fingerprint plots, which extend to de = di = 1.15 Å in the Co complex, which comprise 31.6% of the total Hirshfeld surface area (Fig. 6b). The Hirshfeld surface does not show a similar proportion of O•••H/H•••O with highest contributions of 22.1%, of the total Hirshfeld surface area. In complex, the H•••O (19.9% interactions make a larger contribution than the O•••H (2.2 %) interactions, corresponding to O-H•••O interactions, is represented by the spikes in the bottom right and left region, de + di  1.75 Å (Fig. 6c). In the absence of weak C-H••• interactions in the crystal, the pair of characteristic wings resulting in the fingerprint plot delineated into H•••C/C•••H contacts with a 18.1% contribution to the Hirshfeld Surface have a symmetrical distribution of points, Fig. 6d, with the tips at de + di  3.2 Å, corresponding to C-H•••O (nitrate) interactions. The N•••H contacts (8.5%), are represented by two signals with thick edges in the region de + di  1.75 Å (Fig. 6e), which indicates which refer to formation of O-H•••Ntriazolate interactions. The distribution of points in the de = di  1.7 Å range in the fingerprint plot delineated into C•••C contacts, indicate the existence of weak - stacking interactions between the triazolate rings (N2-N4/C6-C7) and pyridine rings (N1/C1-C5) (Figs. 6f and 7a-b). The presence of the lp••• (lone-pair•••) contact in the structure, involving nitrate-O6 and pyridine rings (N5/C8-C12) (Figs. 7a and 7c), is also evident from the contribution of C•••O/O•••C contacts to the Hirshfeld surface as summarized in Table

Fig. 5 .Fig. 6 .

 56 Fig. 5. Viewed Hirshfeld surfaces are shown mapped with dnorm of (a) and (b) [Co(bpt)(H2O)2], (c) [Co2(bpt)2(H2O)4], (d) Uncoordinated water molecule and (e) NO3 -conterion. Different views of the Hirshfeld surfaces for the coordinated and the uncoordinated the Co parts mapped over the electrostatic potential: (f) [Co2(bpt)2(H2O)4] (g) uncoordinated water molecule and (h) NO3 -conterion.

Fig. 7 .

 7 Fig. 7. (a) - and lp••• interactions in crystal structure of Co complex and views of the Hirshfeld surface mapped with the shape-index property for the complex molecule in (I) from -1.0 to +1.0 a.u. highlighting (b) - interaction and (c) the N6-O6••• interaction through black and purple dashed lines, respectively.

(

Fig. 8a .

 8a Fig. 8a. The large red and rectangular area on the dnorm surface for Co show the contact between

Fig. 8 .

 8 Fig. 8. Atomic Hirshfeld surface of Co in the tittle complex with the descriptor (a) dnorm, (b) shape-index mapped onto the surfaces and (c) corresponding fingerprint plot for cobalt.

Fig. 9 .

 9 Fig. 9. Electronic spectra of Hbpt ligand and its Co complex.

Fig. 10 .

 10 Fig. 10. FTIR spectra of the Hbpt ligand and its Co complex.

  B/Co, above the spin-only value of 3.87 B/Co but very close to the commonly reported value ca. 4.8 B/Co due to orbital contribution. The J exchange between two Co 2+ ions intradimer is J/kB = -5.6 K using the Curie-Weiss temperature  = -14.1 K, and J/kB = 3 / [2S (S+1)].

Fig. 11 .

 11 Fig. 11. Temperature dependence of magnetic susceptibility for [Co2(bpt)2(H2O)4](NO3)2.2H2O measured at 0.1 T in (a) m -1 and (b) m forms. The inset in part (a) shows ZFC and FC magnetization curves. Solid lines correspond to the best fit of the experimental data using the Curie-Weiss law in the 25 -400 K temperature region. (c) Temperature dependence of m.T for the cobalt complex. (d) Isothermal magnetization of cobalt complex at different temperatures.

Fig. 12 .

 12 Fig. 12. (a) In vitro inhibition of mycelial growth of the strains SH, SE and SJ of V. dahlia and of F. oxysporum fsp melonis (FOM) using the cobalt complex at 50 and 100 µg mL -1 . Data are means and confidence intervals from three replicates ( = 5%). Percentage of inhibition growth was determined at 7 days after incubation. Letters indicate significant differences according to Tukey HSD test (P < 0.55). (b) Photos showing antifungal activity of the title complex against SE, SJ, SH and FOM.

Fig. 13 .

 13 Fig. 13. Antibacterial activity against the strains Ach5, A281 and C58 of Agrobacterium tumefaciens, Pseudomonas syringaepv. Syringae (PSS) and Pseudomonas syringaepv. Tabaci (PST) using the studied cobalt complex. Data are means and confidence intervals from three replicates ( = 5%). Inhibition diameter was recorded 48 h after incubation at 28 °C with sterile cellulose discs impregnated with 200 µg mL -1 of the two products. Letters indicate significant differences according to Tukey HSD test (P < 0.55).

Table 1

 1 Crystal data and summary of the measurement parameters for [Co2(bpt)2(H2O)4](NO3)2.2H2O.

	Chemical formula	C12H14CoN6O6
	Mr (g mol -1 )	397.22
	Temperature (K)	296
	Wavelength	0.71073 Å
	Crystal system, space group	Triclinic, P1 ̅
	a, b, c (Å)	9.242(3), 9.327(3), 11.149(4)
	, ,  (°)	81.067(12), 69.692(12), 60.699(11)
	Volume (A 3 )	785.8(4)
	Z	2
	Calculated density (g/cm 3 )	1.679
	 (mm -1 )	1.14
	Crystal shape and color	Block, Red
	Crystal size (mm)	0.33×0.29×0.25
	Data collection
	Tmin, Tma.	0.6621; 0.7464
	Rint	0.044
	Refinement
	Refinement method	Full-matrix least-squares on F 2
	Data / restraints / parameters	3996/0/226
	R[F 2 > 2(F 2 )], wR(F 2 ), S	0.031; 0.079, 1.03
	H-atom treatment	H atoms parameters constrained
	Δρmax, Δρmin (e Å -3 )	0.51, -0.31

Diffractometer 'Bruker APEX-II CCD' Absorption correction Multi-scan (SADABS) Theta range for data collection (°) 2.505 -30.508 Index ranges -13  h 13, -13  k 13, -15  l 15

Table 2

 2 Selected bond distances (Å) and angles (°) for [Co2(bpt)2(H2O)4](NO3)2.2H2O.

	N1-Co1	2.2415 (14)	C6-N4	1.3515 (19)
	N2-Co1	2.0506 (13)	C7-N3	1.3279 (18)
	O1-Co1	2.0877 (13)	C7-N4	1.3481 (19)
	O2-Co1	2.0771 (14)	C8-N5	1.353 (2)
	N3-Co1 i	2.0505 (13)	C12-N5	1.339 (2)
	N5-Co1 i	2.2388 (14)	C1-C2	1.385 (2)
	C1-N1	1.3386 (19)	C2-C3	1.368 (3)
	N2-N3	1.3557 (16)	C3-C4	1.386 (2)
	N6-O5	1.231 (2)	C4-C5	1.379 (2)
	N6-O6	1.238 (2)	C7-C8	1.475 (2)
	N6-O4	1.258 (2)	C8-C9	1.376 (2)
	C5-N1	1.3524 (19)	C9-C10	1.391 (2)
	C5-C6	1.470 (2)	C10-C11	1.368 (3)
	C6-N2	1.3247 (19)
	C1-N1-Co1	128.17 (11)	N3 i -Co1-N2	93.35 (5)
	C5-N1-Co1	114.45 (9)	N3 i -Co1-O2	98.80 (6)
	C6-N2-Co1	120.52 (10)	N2-Co1-O2	97.05 (6)
	N3-N2-Co1	133.34 (10)	N3 i -Co1-O1	90.55 (5)
	C7-N3-Co1 i	120.58 (10)	N2-Co1-O1	95.16 (6)
	N2-N3-Co1 i	133.31 (9)	O2-Co1-O1	164.13 (5)
	C12-N5-Co1 i	127.54 (11)	N3 i -Co1-N5 i	74.62 (5)
	C8-N5-Co1 i	114.84 (9)	N2-Co1-N5 i	167.88 (5)
	N1-C1-C2	122.98 (16)	O2-Co1-N5 i	83.67 (6)
	C3-C2-C1	119.15 (15)	O1-Co1-N5 i	86.52 (5)
	C2-C3-C4	118.87 (16)	N3 i -Co1-N1	166.86 (5)
	C5-C4-C3	118.95 (16)	N2-Co1-N1	74.69 (5)
	N1-C5-C4	122.70 (14)	O2-Co1-N1	88.25 (6)
	N1-C5-C6	113.65 (13)	O1-Co1-N1	85.23 (5)
	C4-C5-C6	123.62 (14)	C6-N2-N3	106.00 (11)
	N2-C6-N4	113.41 (12)	C7-N3-N2	105.97 (12)
	N2-C6-C5	116.68 (13)	C7-N4-C6	101.22 (12)
	N4-C6-C5	129.83 (13)	C12-N5-C8	117.62 (14)
	N3-C7-N4	113.39 (12)	O5-N6-O6	120.02 (17)
	N3-C7-C8	116.54 (13)	O5-N6-O4	118.25 (17)
	N4-C7-C8	130.06 (13)	O6-N6-O4	121.72 (17)
	N5-C8-C9	122.66 (14)	O5-N6-O6	120.02 (17)
	N5-C8-C7	113.32 (12)	C11-C10-C9	119.25 (16)
	C9-C8-C7	124.02 (14)	C10-C11-C12	118.90 (16)
	C8-C9-C10	118.61 (16)	N5-C12-C11	122.95 (16)
	N1-C5-C4	122.70 (14)	C1-N1-C5	117.36 (13)
	N1-C5-C6	113.65 (13)	C4-C5-C6	123.62 (14)

Symmetry code: (i) -x+1, -y, -z+1.

Table 3

 3 Hydrogen-bond geometry (Å, º) for [Co2(bpt)2(H2O)4](NO3)2.2H2O.

	D-H⋯A	D-H (Å)	H⋯A (Å)	D•••A (Å)	D-H⋯A (°)
	C1-H1⋯O6 i	0.9300	2.5800	3.450(3)	156.200
	C9-H9⋯O4 ii	0.9300	2.5300	3.324(3)	143.200
	C12-H12⋯O6 iii	0.9300	2.5600	3.405(3)	151.500
	O2-H2A⋯O6 iv	0.7700	2.1500	2.891(2)	163.000
	O2-H2B⋯O4	0.8400	1.9700	2.801(2)	169.400
	O2-H2B⋯O5	0.8400	2.5300	3.133(2)	129.800
	O1-H1A⋯O3 v	0.8000	2.0000	2.7874(18)	169.500
	O1-H1B⋯O3 i	0.7900	1.9300	2.7117(17)	173.500
	O3-H3A⋯O4	0.7800	1.9700	2.740(2)	167.800
	O3-H3B⋯N4 ii	0.8000	2.0300	2.8331(18)	174.900

Table 4

 4 Percentage contributions of interatomic contacts to the Hirshfeld surface for the Co(bpt)(H2O)2 and

	[Co2(bpt)2(H2O)4](NO3)2.2H2O.
	Contact		Percentage contribution (%)
		Co(bpt)(H2O)2	[Co2(bpt)2(H2O)4](NO3)2.2H2O
	H⋯H	31.6	26.7
	O⋯H/H⋯O	22.1	41.0
	C⋯H/H⋯C	18.1	17.9
	H⋯N/N⋯H	12.9	5.4
	C⋯C	4.6	4.4
	Co⋯N/N⋯Co	5.5	⎯
	C⋯N/N⋯C	1.8	1.9
	N⋯O/O⋯N	1.3	1.1
	N⋯N	1.1	0.3
	C⋯O/O⋯C	0.9	0.7
	H⋯Co/Co⋯H	0.2	⎯

Table 6

 6 FTIR data (cm -1 ) of the Hbpt ligand and its complex.

	Compound	ν (NO3 -)	ν (C=C)	ν (C=N)	ν (C-H)	ν (H2O)
	Hbpt	⎯	1565 -1589	1604	3050	⎯
	Complex	1387	1584 -1600	1636	3066	3400

Acknowledgements

CUR CA2D of Chouaib Doukkali University is acknowledged for its technical support and UATRS-CNRST for the use of the Bruker X8 diffractometer. The Fonds Européen de Développement Régional (FEDER) and contrat plan état région (CPER), CNRS, Région Hautsde-France and Chevreul Institute (FR 2638) are also acknowledged for funding of the Physical Property Measurement System (PPMS). We thank Laurence Claire Minaud for her magnetic experimental contribution.

Conclusions

The reaction of Co II nitrate with Hbpt generates a novel homobimetallic cobalt(II) complex, [Co2(bpt)2(H2O)4](NO3)2.2H2O, and its structure has been characterized by singlecrystal X-ray diffraction as well as by UV-Visible and FTIR spectroscopic techniques. The crystal structure determination of Co II complex showed that the dinuclear Co2N8 unit is almost planer in which each Co II ion is coordinated by four nitrogen atoms from the anionic ligand (bpt) equatorially and two water molecules axially in a distorted octahedral geometry. The structural stability of the investigated complex results from the establishment of different intermolecular hydrogen bonds, π-π stacking and intermolecular lone-pair•••π interactions. The 3-D Hirshfeld surface analysis revealed that the cohesion and stabilization of the crystal network is for the most part ensured by means of O⋯H/H⋯O (41.0%) intermolecular hydrogen interactions. Magnetic susceptibility study revealed the presence of weak antiferromagnetic exchange coupling between two Co II centers mediated by two central triazolate bridges of bpt ligand. This homobimetallic complex showed also elevated antifungal activity against various strains of phytopathogenic fungus suggesting its possible use as a fungicide in crop protection.

Supplementary material

CCDC 2006865 contains the supplementary crystallographic data for the [Co2(bpt)2(H2O)4](NO3)2.2H2O complex. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam. ac.uk, or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.


Rose Ndemah

Bruno Pierre Le Ru

Claire Capdevielle-Dulac

Fritz Schulthess

Laure Kaiser

Rachid Hanna

Alexandre Depoilly

Julius Obonyo

Paul-André Calatayud

Albert Abang

Stéphane Dupas
email: stephane.dupas@egce.cnrs-gif.fr

Managing and monitoring genetic isolation and local adaptation of endemic and introduced Cotesia sesamiae for the biological control of the cereal stemborer Busseola fusca in Cameroon

Keywords: Cotesia sesamiae, biological control, Polydnavirus, Wolbachia, genetic

The success of biological bontrol (BC) introductions can be enhanced by considering theory and knowledge of biological systems. The gregarious braconid parasitoid Cotesia sesamiae (Cameroon) is one of the best studied biological control agent from the perspective of molecular ecology. Its evolutionary adaptation to the target host involves symbiotic partners. Polydnaviruses are responsible for immune and developmental adaptations whereas Wolbachia bacteria may reinforce this local adaptation though genetic isolation mechanisms. The noctuid Busseola fusca is a major stemborer pest of maize in sub-Saharan Africa. In contrast to eastern Africa, C. sesamiae is rarely found on B. fusca in western Africa. It is however often obtained from other stemborer species feeding on wild grasses. A biological control project was launched in 2006-2007 by introducing to Cameroon seven crosses of Kenyan populations of C. sesamiae collected in different ecozones. They included populations adapted to B. fusca that develop on maize as well as populations adapted to other hosts feeding on wild plants to allow carryover between cropping seasons. Wolbachia strains responsible for cytoplasmic reproductive incompatibilities with endemic strains were included in the crosses to limit genetic exchanges between introduced and endemic C. sesamiae and preserve genetic adaptation to B. fusca of the introduced populations, while at the same time preserving their ability to survive on wild plants. Six post release surveys were carried out on maize from 2007 to 2013, and on wild grasses in 2013. A total of 393 C. sesamiae individuals, each from one cocoon mass, were genotyped for 11 microsatellite loci. Multidimensional scaling analysis, STRUCTURE and GENECLASS analyses assigned almost all the parasitoids recovered from maize to those introduced from Kenya. The introduced strains were also recovered from wild host plants with little genetic exchanges with endemics. Each population remained strongly associated with its original Wolbachia component, suggesting that Wolbachia may contribute to genetic isolation between endemics and introduced populations in wild host plants when maize is absent, thereby conciliating biological control success and safety.

Introduction

Classical Biological control (BC) operations can be optimized using evolutionary ecology knowledge, which should also minimize the risks toward non target organisms (Szücs et al. 2019). In contrast to chemicals or genetically modified organisms, biological control agents can theoretically evolve to overcome the evolution of resistance in the host [START_REF] Holt | When is biological control evolutionarily stable (or is it)?[END_REF]. There is empirical evidence of evolution in BC which can affect its control success (Szücs et al. 2019). In addition to adaptation to target species, BC agents can evolve adaptation to non targets which can potentially endanger endemic biodiversity [START_REF] Simberloff | Risks of biological control for conservation purposes[END_REF][START_REF] Le Hesran | Next generation biological control -an introduction[END_REF]. Research aiming at understanding evolutionary ecology of host specialization has been considered as a particularly essential aspect of the efficiency and safety of BC [START_REF] Brodeur | Host specificity in biological control: Insights from opportunistic pathogens[END_REF].

In Cameroon, indigenous parasitoids do not exert sufficient control over the predominant stemboring maize pest Busseola fusca (Fuller) (Lepidoptera, Noctuidae) [START_REF] Ndemah | Natural enemies of lepidopterous borers on maize and elephant grass in the forest zone of Cameroon[END_REF][START_REF] Ndemah | Factors Affecting Infestations of the Stalk Borer Busseola fusca (Lepidoptera : Noctuidae ) on Maize in the Forest Zone of Cameroon with Special Reference to Scelionid Egg Parasitoids[END_REF][START_REF] Ndemah | Lepidopteran cereal stemborers and associated natural enemies on maize and wild grass hosts in Cameroon[END_REF]. A comparison between regions of Africa showed that certain species and populations of parasitoids are lacking in this country and in western Africa in general [START_REF] Schulthess | Exchanging natural enemies of lepidopteran cereal stemborers between african regions[END_REF]. The gregarious braconid Cotesia sesamiae (Cameron) is one of the most studied biological control agents in terms of its ecology and genetics [START_REF] Kaiser | Ongoing ecological speciation in Cotesia sesamiae, a biological control agent of cereal stem borers[END_REF][START_REF] Kaiser | The Cotesia sesamiae story: insight into host-range evolution in a Hymenoptera parasitoid and implication for its use in biological control programs[END_REF]. It is commonly found on B. fusca in East and Southern Africa. By contrast, in Cameroon it was found only on noctuid stemborer species feeding in wild grasses but never on B. fusca on maize [START_REF] Ndemah | Natural enemies of lepidopterous borers on maize and elephant grass in the forest zone of Cameroon[END_REF]2007). Adaptation to B. fusca in Kenya involves immune suppression mediated by symbiotic polydnaviruses (PDV) found in the calyx fluid of the parasitoid ovary, which is injected into the insect host together with the eggs during oviposition [START_REF] Ngi-Song | Suitability of Busseola fusca and Sesamia calamistis (Lepidoptera: Noctuidae) for the development of two populations of Cotesia sesamiae (Hymenoptera: Braconidae) in Kenya[END_REF][START_REF] Gitau | An association between host acceptance and virulence status of different populations of Cotesia sesamiae, a braconid larval parasitoid of lepidopteran cereal stemborers in Kenya[END_REF]. PDVs are directly related to parasitism success, enabling the parasitoid to overcome the immune system of its host [START_REF] Gitau | An association between host acceptance and virulence status of different populations of Cotesia sesamiae, a braconid larval parasitoid of lepidopteran cereal stemborers in Kenya[END_REF][START_REF] Herniou | When parasitic wasps hijacked viruses: Genomic and functional evolution of polydnaviruses[END_REF]. They disable and prevent host haemocytes from encapsulating parasitoid eggs [START_REF] Gitau | An association between host acceptance and virulence status of different populations of Cotesia sesamiae, a braconid larval parasitoid of lepidopteran cereal stemborers in Kenya[END_REF]. The difference in virulence of C. sesamiae populations on B. fusca is correlated with differences in expression of the nucleotide sequence CrV1 of the parasitoid PDV gene [START_REF] Gitau | Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron)[END_REF]. [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF] reported polymorphism in PDV haplotypes across Africa. In western Kenyan C. sesamiae, the PDVs are virulent against B. fusca, while in Cameroon the PDVs are not virulent against B. fusca. Further DNA analysis on a larger numbers of host species and on a wider geographic range confirmed the association between the hosts in which the parasitoid had developed, and the genotype of the PDV CrV1 gene [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF].

Cotesia. sesamiae populations are also infected by different strains of Wolbachia spp. responsible for cytoplasmic incompatibilities when inter-crossed, and potentially reinforcing specialization and local adaptation to different hosts [START_REF] Branca | Maintenance of adaptive differentiation by Wolbachia induced bidirectional cytoplasmic incompatibility: the importance of sib-mating and genetic systems[END_REF]).

Incompatibilities can be unidirectional (when infected males mate with a non-infected females) or bidirectional (through matings between insects harboring different strains of the bacterium). Four Wolbachia variants have been identified from C. sesamiae collected in Kenya on maize [START_REF] Mochiah | Wolbachia infection in Cotesia sesamiae (Hymenoptera: Braconidae) causes cytoplasmic incomptatibility: implication for biological control[END_REF][START_REF] Gounou | Reproductive compatibility of several East and West African Cotesia sesamiae (Hymenoptera: Braconidae) populations and their crosses and backcrosses using Sesamia calamistis (Lepidoptera: Noctuidae) as the host[END_REF][START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. In western Kenya, the parasitoid developing on B. fusca is infected by the Bwest variant, while in the eastern coastal region the parasitoid developing on the noctuid cereal stemborer Sesamia calamistis Hampson, it is infected by the A, Beast and the co-infection

A+Beast(co-infection).

In hybridization experiments, the number of females in the offspring is reduced in both directions of mating. Incompatibility is total for ♀east x ♂west and weak for ♀west x ♂east [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF] supplementary data) estimated an infection rate of 92% across Africa, which somewhat reduces incompatibility.

Because of the differences in the ecology and economic importance of C. sesamiae on B. fusca in Cameroon and eastern Africa, a BC project to redistribute Kenyan C. sesamiae populations on B. fusca in Cameroon was proposed by [START_REF] Schulthess | Exchanging natural enemies of lepidopteran cereal stemborers between african regions[END_REF].

Non-target effects were assessed through detailed studies on other borer species occurring in wild habitats as well as on associated Wolbachia and PDVs [START_REF] Ndemah | Lepidopteran cereal stemborers and associated natural enemies on maize and wild grass hosts in Cameroon[END_REF][START_REF] Gitau | Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron)[END_REF][START_REF] Dupas | Evolution of a polydnavirus gene in relation to parasitoid-host species immune resistance[END_REF][START_REF] Branca | Maintenance of adaptive differentiation by Wolbachia induced bidirectional cytoplasmic incompatibility: the importance of sib-mating and genetic systems[END_REF][START_REF] Gitau | An association between host acceptance and virulence status of different populations of Cotesia sesamiae, a braconid larval parasitoid of lepidopteran cereal stemborers in Kenya[END_REF][START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]. Busseola fusca is the target species but wild stemborers enable the parasitoid to over-season when green maize stalks are not available and B. fusca is diapausing in dry maize stalks. There was a trade-off between the necessity to maintain introduced parasitoid populations on neighboring wild habitats and the need to prevent non target impact on endemic parasitoid populations. In Cameroon, endemic C. sesamiae in wild habitats are infected by the A or A+Beast Wolbachia strains [START_REF] Branca | Maintenance of adaptive differentiation by Wolbachia induced bidirectional cytoplasmic incompatibility: the importance of sib-mating and genetic systems[END_REF].

Interestingly the incompatibility of the Cameroonian strains is stronger than that of the Kenyan strains [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]; therefore, differences in Wolbachia strains could be used to protect endemic Cameroonian populations from hybridization with introduced Kenyan strains.

Using knowledge on polydnavirus and Wolbachia molecular ecology, emphasis was given in this project to introducing strains that (1) maximize impact on crops, by harboring genes adapted to B. fusca and maize, ( 2) can perennate the off-season on other stemborers attacking wild plants, by harboring some genes adapted to wild host, but (3) do not interbreed with endemic C. sesamiae present on these plants, by harboring Wolbachia strains different from the endemics to avoid non-target impact.

The specific objectives of the project were: (1) to select and cross strains expected to maximize BC success while minimizing ecological side effects to Cameroonian C. sesamiae and wild borers; and (2) to survey the genetic material of endemic and introduced strains in wild and cultivated habitats and their relationship with Wolbachia strain status to test the predictions of establishment of Kenyan populations on B. fusca on maize and survival on wild host plants without gene flow from introduced to endemic strains of C. sesamiae.

Materials and methods

Constitution of Kenyan Cotesia sesamiae populations for introduction in Cameroon: Role of Wolbachia

The parental populations of C. sesamiae were collected in Kenyan maize fields in Kitale (Ki), Meru (Me), and Kakamega (Ka) from B. fusca, in Mount Kenya 1 (MK1) and Mombasa (Mb) from S. calamistis , and in Tana River (Ta) from Sesamia firmata Moyal infesting the Napier grass Pennisetum purpureum Schumach (Fig. 1, suppl. Table 1). Maize or P. purpureum plants that exhibited signs of stem borer feeding were collected and dissected.

Borer larvae and cocoons of C. sesamiae were placed individually in glass vials (7.5 cm x 2.5 cm). The larvae were provided with a piece of maize stem or artificial diet [START_REF] Onyango | Continuous rearing of the maize stem borer Busseola fusca on an artificial diet[END_REF]. A total of 395 cocoon masses were collected. The number of cocoon masses collected per locality is given in suppl. Table 1. The larvae and cocoons were kept in the laboratory at icipe (African Insect Science for Food and Health, Nairobi, Kenya) and observed for cocoon formation and parasitoids emergence. Adult Cotesia spp.

that emerged from cocoons were identified using the shape of the male genitalia or female propodia (Kimani Njogu & Overholt 1995) at icipe`s biosystematics unit. Cotesia sesamiae progeny were allowed to mate in a vial for at least 2 hours. under a strong source of light Because of lack of a rearing protocol, C. sesamiae from Tana River were reared on S. calamistis instead of their original host, S. firmata.

Parasitoid rearing

Adults were fed a 20% honey solution. After exposing the larvae to the parasitoids using the hand stinging method by [START_REF] Overholt | Rearing and Field Release Methods for Cotesia flavipes Cameron (Hymenoptera: Braconidae), a Parasitoid of Tropical Gramineous Stem Borers[END_REF], the parasitized larvae were placed on artificial diet at 26 ± 1 ºC until cocoon formation and parasitoids emergence (Onyago and Ochieng'Odero 1994). Time to cocoon formation took 12 to 14 days. The progeny that emerged were used to start the F1 colony for mass production in the laboratory. Fifteen, 14, 2, 2, 10 and 13 cocoon masses were used to initiate the Cs-Ki, Cs-Me, Cs-Ka, Cs-MK1, Cs-Mb and Cs-Ta colonies, respectively.

Wolbachia curing

The aim was to produce Kenyan strains for release that were either not infected or infected by a Bwest Wolbachia strain, not present in Cameroon [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. The variant of Wolbachia present in each strain was identified with the wsp gene AFLP test [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]) using two individuals per population, before and after mass production in the laboratory. The Ki, Me and Ka populations that were infected with the Bwest variant of the bacterium were not cured of Wolbachia. The populations infected by both the A and Beast were cured from Wolbachia using a rifampicin (Sanofi-Aventis, France) treatment for one generation [START_REF] Mochiah | Wolbachia infection in Cotesia sesamiae (Hymenoptera: Braconidae) causes cytoplasmic incomptatibility: implication for biological control[END_REF]. The absence of Wolbachia after treatment was confirmed by PCR test on DNA extraction.

Crosses for constructing hybrid release strains

Hybrid populations were produced by crossing populations infected by the same Wolbachia Bwest strain (i.e., Me, Ki and Ka) or cured from the A, Beast and A/Beast strains present in Cameroon (i.e., MK1, Ta and Mb) (Table 1). Each individual cocoon was placed in a vial. Freshly emerged males and females from the different populations were selected and observed to cross-mate. Mated females were offered B. fusca larvae for parasitism using the hand stinging method.

The first hybrid population (named Hy) was initiated from reciprocal crosses between Cs-Ki and Cs-Me, after 11 and 9 generations of rearing in the laboratory, respectively (Table 1, 20 males and 20 females for each cross). The progenies of the reciprocal hybrids resulting from each cross were then mixed to obtain the Hy population, and then reared for 1 generation before being sent to Cameroon.

The second hybrid population named Ke1 resulted from crosses between Ki, Me and Ka (Table 1). The progenies of the hybrids resulting from each cross were then mixed to obtain the Ke1 population and then reared for 1 generation before being separated for cured and uncured procedures (see below).

The third hybrid named Ke2 resulted from the cross between all six populations (MK1, Ta, Mb, Ka, Me and Ki) (Table 1). All reproductively compatible crosses (males not infected by Wolbachia) were realized. The progenies were then mixed to obtain the Ke2 population and reared for 1 generation before being separated for cured and uncured procedures (see below). Ke1 and Ke2 hybrid population were cured of Wolbachia using the rifampicin protocol described above [START_REF] Mochiah | Wolbachia infection in Cotesia sesamiae (Hymenoptera: Braconidae) causes cytoplasmic incomptatibility: implication for biological control[END_REF] to obtain the Ke1 W-and Ke2 W-populations.

Considering the cross proportions described above and in Table 1, the genetic material introduced in the Hy, Ke1 and Ke2 populations originated at 100%, 100% and 89%, respectively, from individuals collected on B. fusca larvae the rest being collected on Sesamia calamistis and Sesamia firmata, and at 100%, 100% and 93% from individuals collected on maize, the rest being collected on Pennisetum purpureum. The aim was to introduce populations harboring genes permitting to counteract the immune reaction of B. fusca [START_REF] Dupas | Evolution of a polydnavirus gene in relation to parasitoid-host species immune resistance[END_REF]) and genes permitting to switch host plant and host species.

In total, two parental (Ki, Me) and five hybrid populations (Hy, Ke1 and Ke2 infected by B west Wolbachia, and Ke1 W-and Ke2 W-, cured from Wolbachia) were released in Cameroon.

Releases of parasitoids in Cameroon

The releases were conducted in 193 sites from 44 localities between 2006 and 2008. At each site a specific strain was released (Fig. 2, suppl. material for details). First releases were conducted in inland valleys (wetlands) maize fields of the forest zone, during the off-season. Cocoon masses were packed into boxes with cotton wool and sent under ambient environmental conditions via express air mail from Nairobi to the International Institute of Tropical Agriculture (IITA) in Yaoundé, where they were kept at room temperature (26° C ± 2) in vials with cotton wools.

In 2006-2007, the five (Hy, Ke1U, Ke2U, Ke1C, Ke2C) hybrids as well as the two parental (Ki, Me) populations of C. sesamiae were released as mated adult females. Infested larvae, received from Kenya in a 1 liter plastic jar covered with a fine insect proof screen mesh, were inspected daily for adult emergence. Adults were fed with a 20% honey solution soaked in cotton wool. They were allowed to mate for three hours under artificial light in the laboratory. Adults emerging from a single cocoon mass were released in each of 147 fields in 11 villages in the low altitude forest zone and in high altitudes zone with the agreement of the farmers (Fig. 2a).

In 2007-2008 the same strains (Ki, Me, Hy, Ke1U, Ke2U Ke1C, and Ke2C crosses) were released as parasitized B. fusca larvae in tunnels drilled into maize stem, in 119 maize fields in 42 localities, in the forest zone and high altitudes zone. The longitude, latitude and altitude of the release localities (Fig. 2a) as well as the number of parasitized larvae released were recorded (suppl. material).

Surveys in maize fields

Post release surveys were conducted in 2007, 2008, 2009, 2010, 2011 and 2013 in the release villages. In the high altitudes, there is one long maize season from March to October while in the forest zone there are two to three maize seasons, two during the two rainy seasons from March to June and from end of August to early December, respectively, and a last one during the dry season (January to April). The fields in the forest zone were surveyed mostly during the second rainy season in October/November, when pest densities are expected to be high, while in the high altitude most of the fields were surveyed in June/July when B. fusca is in the right stage for parasitization by C. sesamiae. Surveys were also conducted in Yaoundé where the parasitoid might have escaped from the lab and invaded farmers' fields.

Each field was divided into four quadrants, and six maize plants per quadrant were randomly sampled. In addition, four plants per quadrant that showed borer damage were sampled. During 2013, only three infested plants were sampled per quadrant. In each field, the maize plants sampled were dissected and checked for stem borer larvae and pupae, and C. sesamiae cocoon masses. The larvae found were reared on stem and cob pieces of maize in the lab until emergence of adult moths or parasitoid cocoon formation.

Each cocoon mass was placed in a separate transparent plastic container. Once the adults had emerged and died, the number of cocoons per mass, the total number of emerged adults as well as the number of females and males were counted. The adult parasitoids were preserved in 95% ethanol and sent to the Institut de Recherche pour le Développement (IRD) lab at Gif Sur Yvette in France for molecular analyses. The postrelease sites surveyed are shown in Fig. 2b. 2).

Surveys on wild host plants

Surveys for C. sesamiae on wild noctuids and B. fusca were conducted in roadside patches of wild grasses in the forest zone and highlands of Cameroon between 2001 and 2003 [START_REF] Ndemah | Lepidopteran cereal stemborers and associated natural enemies on maize and wild grass hosts in Cameroon[END_REF][START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]). After release, in 2013, wild noctuids were collected from grasses at the edges of maize fields in the sites as shown in Fig. 2c and prepared for molecular analysis.

Analysis of Cotesia sesamiae specimens for microsatellite markers and

Wolbachia infection

All the insects emerging from larvae collected during the surveys were preserved in alcohol. From each cocoon mass emerging, one individual was analyzed for 11 microsatellite loci [START_REF] Jensen | Characterization of microsatellite loci in the parasitoid, Cotesia congregata (Say) (Hymenoptera Braconidae)[END_REF]Abercrombie et al., 2009[START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF] and Wolbachia wsp loci [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]. DNA of was extracted individually using the NucleoSpin Tissue XS (Macherey-Nagel) following the manufacturer's protocol. Next, approximately 10ng of DNA was added to 5 µl of 2X Platinum Multiplex PCR Master Mix (Life Technologies), 1 µl of Primer mix (final concentration of 0,1 µM for each primer) and 3 µl of water in a PCR to amplify. The list of primers and amplification protocol are given in supp. Table 2 The PCR was carried out at a denaturation temperature of 95⁰C for five minutes, then 25 or 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C, followed by a final elongation of 5 minutes at 72°C. PCR products were then run on an ABI 3130 XL sequencer at the IRD lab at Gif-sur-Yvette in France, and the profiles analyzed with GeneMapper v3.1 (Life Technologies).

Wolbachia strain infection was detected by amplifying the wsp gene using the protocol developed by [START_REF] Zhou | Phylogeny and PCR-Based Classification of Wolbachia Strains Using WSP Gene Sequences[END_REF]and characterized by the AFLP PCR test developed by [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]. For 13 individuals sampled, Wolbachia was amplified but the strain could not be correctly identified.

Population genetic structure analysis

The genetic diversity of individuals (microsatellite data) was represented by multidimensional scaling according to their category or recovery site (MDS) (Fig. 3). In addition, the microsatellite data were analyzed using GENECLASS (INRA/CIRAD) and STRUCTURE [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF][START_REF] Hubisz | Inferring weak population structure with the assistance of sample group information[END_REF] softwares. The post release samples recovered from maize or wild host plants were assigned to either the endemic population identified before the release , or to the introduced parental Kenyan strains.

Results

Recovery of Cotesia sesamiae from maize and wild host plants and

GENECLASS assignation

The distribution of C. sesamiae on the different plant and stemborer hosts before and after release of Kenyan populations is presented in Table 2. Before its release, C. sesamiae was obtained only from noctuids stemborers feeding on wild host plants and never from B. fusca on maize, whereas after its release, of a total of 192 C. sesamiae cocoon masses 96 were collected on maize and 96 on wild host plants. On maize, the stemborer species composition collected from 2007 to 2013 and the percentage of infestation by C. sesamiae are shown in Table 3. Busseola fusca was by far the most abundant stemboring species on maize, in both highland and forest regions, followed by Eldana saccharina (Walker) and S. calamistis. Parasitism on maize stemborers varied between 0 to 1.0 % with an average of 0.15 % (Table 3). Among the 96 parasitoid samples collected on maize 45 were found in fields around the released sites and 51 in the vicinity of the parasitoid rearing unit in Yaoundé.

Genetic structure of introduced and endemic Cotesia sesamiae populations in wild and cultivated compartments

Individual females from 395 C. sesamiae cocoon masses were genotyped using microsatellite markers; 192 cocoon masses were from the seven introduced Kenyan strains released in Cameroon, 9 were from wild host plants before the releases of the Kenyan strains, 98 were collected from maize post-release, and 96 from wild host plants post-release. The representation of genetic distance by multi-dimensional scaling (MDS) is shown in Fig. 3. Eighty-six percent of the variation is explained by the two axes of the model presented in Fig. 3. We observed three genetic groups. A first "wild" group included the samples collected on wild host plants, namely all the 9 samples collected before release of the Kenyan strains, with 80 of the 96 genotyped insects collected on wild host plants post-release, and 3 of the 97 genotyped insects collected from maize post-release. A second "cultivated" group on the right of the first axis contained 185 of the 192 genotyped insects from the Kenyan released strains, 95 of the 98 genotyped insects collected from maize post-release, and 16 of the 96 insects collected on wild host plants post-release. Finally, a third group on the top of the second axis contained four insects from the released "Hy" Kenyan strain. The percentage of "migrants" i.e.

genetically from Kenya but observed on wild plants along with endemics (first group), or genetically from Cameroon but observed for the first time in cultivated fields (second group) was 16/98=16% for the first group and 3/96 = 3% for the second group.

The GENECLASS analysis provided similar percentages of "migrants". Of the 96 specimens collected post-release from wild habitats, 75 could be assigned to the endemic populations and 21 to the introduced populations (21% for the first group). Of the 96 specimens analyzed and recovered from maize post-release, 92 could be assigned to "migrants" and 5 to endemics (5% for the second group). According to GENECLASS, the number of "migrants" were 21 of 96 (22%) in wild habitats and 5 of 92 (5%) in cultivated habitats. If we consider only the individual with probability above 95% of being migrants, these values dropped to 19 (20%) and 4 (4%), respectively.

Structure analysis performed on all individuals, pre-release, introduced and post-release led to an optimal number of cluster based on Evanno's method of K = 2 (suppl. Fig. 1, [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study[END_REF]. It showed the same results as GENECLASS with 80.2% assignation to the same cluster of individuals collected post-release in wild habitats and individuals collected pre-release wild plant insects (19.8% of "migrants" in the first group) and 96.6% assignation to the same cluster of individuals collected post-release on maize and individuals from introduced strains (3.4 % of "migrants" in the second group) (Table 5).

Wolbachia infection was detected in 84% of the 194 post-release samples . Wolbachia strain status, endemic or introduced matched by more than 95% that of the microsatellite markers. Sixty-one individuals were infected by endemic Wolbachia, of which 58 could be assigned to endemic microsatellite populations (95.1%). Eighty-four individuals were infected by introduced Wolbachia, of which 81 could be assigned to the introduced microsatellite population (96.4%).

Discussion

Results of the surveys show that the three specific objectives of the BC project were met Almost all the populations recovered from maize after the biological control introductions could be assigned to the genetic cluster of the Kenyan strains released indicating that they established on maize in Cameroon. In contrast to the situation in high altitudes zones, the Kenyan populations successfully survived the dry season in the Inland Valley forest zone and were still recovered five years after the releases, probably because of the presence of continuous maize cropping [START_REF] Thenkabail | Capabilities of Landsat-5 Thematic Mapper (TM) data in regional mapping and characterization of inland valley agroecosystems in West Africa[END_REF]Nolte 1996, 1998;[START_REF] Ndemah | Towards an integrated crop management strategy for the African stalk borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in maize systems in Cameroon[END_REF]Almekinders and Hardon 2006) that have been shown to be reservoirs for nondiapausing B. fusca and S. calamistis and their natural enemies during the off-season [START_REF] Chabi-Olaye | Role of Inland Valleys in the Management of Stem Borers and Their Natural Enemies in Upland Maize Fields in the Humid Forest Zone of Cameroon[END_REF]). These fields along with wild grass habitats thus help perennating C. sesamiae during the off-season, when B. fusca in up-land fields are in diapause as larvae. Thus, [START_REF] Chabi-Olaye | Role of Inland Valleys in the Management of Stem Borers and Their Natural Enemies in Upland Maize Fields in the Humid Forest Zone of Cameroon[END_REF] showed that during the dry season inland valleys harbour the scelionid egg parasitoids Telenomus spp., which move to upland maize fields at the beginning of the succeeding rainy season. Similarly in East Africa, cultivated and wild sorghum harbour non diapausing B. fusca during the dry season as well as several Sesamia spp. that perennate the parasitoid during the off season [START_REF] Mailafiya | Factors affecting stem borer parasitoid species diversity and parasitism in cultivated and natural habitats[END_REF]. The presence of introduced C. sesamiae on wild host plant in the present study also underlines the role of this habitat during dry season.

In the high altitude, recoveries were made mostly during the same year as the releases in 2007 and 2008. Thereafter, only three cocoon masses were recovered in 2013. This scarcity of descendants from the introduced population may reflect the fact that the parasitoid may still be acclimatizing to its new environment, because it can be a long process as found for C. flavipes in East Africa [START_REF] Midingoyi | Assessing the long-term welfare effects of the biological control of cereal stemborer pests in East and Southern Africa: Evidence from Kenya, Mozambique and Zambia[END_REF]. Such a delay is common in biological control [START_REF] Fauvergue | The biology of small, introduced populations, with special reference to biological control[END_REF]. The reasons classically invoked are:

(1) time for genetic adaptation to new environment, (2) Allee effect due to small initial population size, or (3) environmental stochasticity [START_REF] Fauvergue | The biology of small, introduced populations, with special reference to biological control[END_REF]). In our case, adaptation might be related to ability to survive in diapausing host. In Potchefstroom, South Africa, which is characterized by a five-month cool season, the size of the first flight of B. fusca was largely dependent on the incidence of parasitism of diapausing larvae by C. sesamiae [START_REF] Rensburg Van | Ecology of maize stalk borer, Busseola fusca (Fuller) (Lepidoptera : Noctuidae)[END_REF]). Similarly, van Rensburg et al. (1987) observed high mortality of field-collected larvae caused by C. sesamiae in cold storage. Thus, it appears that C. sesamiae is able to survive during the non cropping season in diapausing B. fusca larvae. This was verified by B. Le Ru (pers. Observations), who obtained C. sesamiae from diapausing B. fusca larvae collected in Kitale, which is situated in the highland zone in Kenya. In the present study, the sample size might have been too small to allow detection of C. sesamiae in the highlands of Cameroon so soon after the first releases. Also, additional releases should be considered to help establishment and spread of the parasitoid in the highlands.

The results show that despite the co-occurrence of endemic and introduced populations on refuge host plants, the two populations did not interbreed at a significant rate. Five years post release there appeared to be a strong separation of the introduced genomes on one hand and that of the endemics on the other hand. Wolbachia is likely to contribute to this separation. Endemic Wolbachia were associated at more than 95% with endemic nuclear genes and introduced Wolbachia were associated at more than 95% with introduced nuclear genes. There is therefore some permeability of the Wolbachia compartments. This is consistent with the observation that cytoplasmic incompatibility is incomplete [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF] also observed that cytoplasmic incompatibility was stronger for A+Beast (endemic) Wolbachia variants than for Bwest (introduced) variants. There should therefore be less gene flow from introduced to endemics than the reverse. On maize, endemic Wolbachia were not recovered while on wild host plants introduced Wolbachia were present. The genetic exchanges are therefore likely to occur in wild habitats first. Most of the introduced individuals found on wild host plants were also infected by introduced Wolbachia strains but there were also three introduced individuals with endemic Wolbachia recovered and five individuals not infected. This asymmetric and incomplete cytoplasmic incompatibilityI can be due to an effect of parasitoid genome [START_REF] Bordenstein | Effects of A and B Wolbachia and host genotype on interspecies cytoplasmic incompatibility in Nasonia[END_REF]. A possible scenario is therefore introgression in wild habitats due to incomplete cytoplasmic incompatibility.

Models predict that linkage disequilibrium between Wolbachia and nuclear genes would break down quickly if other forces do not maintain isolation (Telshow et al. 2002). [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF] confirmed that host species contributed to genetic differentiation even more than Wolbachia on an African scale. Since C. sesamiae mostly reproduce immediately after emergence on host larvae, the role of host in genetic differentiation may be effective, even for populations present in the same habitat [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]. The reduction of genetic exchanges between the introduced insects and the endemic compartments is therefore likely to be reinforced by the difference in insect host species associated to these compartments.

The key for conciliating biological control efficiency and safety in our case was the use of scientific knowledge on population biology of the biological control agents and their wild relatives and molecular genetics tools to allow refuge habitat to contribute to population dynamics without negative interaction between introduced and endemic populations. It was shown that introduced populations need wild habitats but must not interbreed with endemics. It is necessary to conduct long term follow-up studies in both host plant compartments to confirm this hypothesis of Wolbachia driven genetic protection of endemic populations from introduced conspecific biological control agents. The confirmation of such hypothesis, namely that reproductive isolation by Wolbachia would play a role in keeping local adaptations of endemic insects toward their natural hosts and may preserve their native ecological functions, would be a further success of the project. This would require a genetic marker of adaptation. The CrV1 gene would be a good candidate because it has an allele that is almost endemic to Cameroon and associated with the wild hosts Poeonoma serrata (Hmps.) (Lep.: Noctuidae) [START_REF] Branca | Intraspecific specialization of the generalist parasitoid Cotesia sesamiae revealed by polyDNAvirus polymorphism and associated with different Wolbachia infection[END_REF]). The CrV1 virulence gene could also be used as a diagnostic marker of adaptation to B. fusca by harbouring alleles linked to parasitoid success on this host [START_REF] Dupas | Evolution of a polydnavirus gene in relation to parasitoid-host species immune resistance[END_REF]). The gene evolves through natural selection and is genetically linked to factors of suppression of local host resistance [START_REF] Dupas | Evolution of a polydnavirus gene in relation to parasitoid-host species immune resistance[END_REF][START_REF] Jancek | Adaptive selection on bracovirus genomes drives the specialization of Cotesia parasitoid wasps[END_REF]). Yet recent genomic studies suggest CrV1 is not the causal factor but just a marker due to the co-segregation of the virus in the parasitoid genome [START_REF] Gauthier | Chromosomal resolution reveals symbiotic virus colonization of parasitic wasp genomes[END_REF]. Diagnostic PCR tests will have to be developed in order to follow up populations as well as to bring in new introductions by confirming the cohesion between Wolbachia, microsatellites and polydnavirus markers. Table 1. Constitution of the Hy | Cs-Kenya 1 | Cs-Kenya 2 release strains. The column name is the strain of the father (F.) and the line name, the strain of the mother (M.) .

Legends of figures

The number of parental pairs for each introduced populations are given in the following order: Hy | Cs-Kenya 1 | Cs-Kenya 2 (Hy|K1|K2) . Parental strains are : Ki : Kitale, Me: Meru, Ka: Kakamega, MK1: Mount Kenya 1, Ta: Tana River, Mb: Mombasa. Total G : total genetic contributions of each parent in the cross. Wb : Wolbachia variants : WE : Wolbachia variants found endemically in cameroon (A and Beast strains, [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. WI: Wolbachia variants not found endemically in cameroon (Bwest strain, [START_REF] Branca | Relative influence of host, Wolbachia, geography and climate on the genetic structure of the Sub-Saharan parasitic wasp Cotesia sesamiae[END_REF]. *: the strains harboring WE were cured from Wolbachia before the crosses so that the resulting Cs-Kenya 1 and Cs-Kenya 2 strains were WI+WE-(infected by WI and cured from WE). Insect : species of insect host from which the strian was collected in Kenya : B. fusca: Busseola fusca, S. calam.: Sesamia calamistis, S. firm.: Sesamia firmata. Plant: plant species from which the insect was collected in Kenya : P. purp.: Pennisetum purpureum. Z. mays: Zea mays. Superscripts Bf : rate of genetic contribution of Table 4. Geneclass Assignation of Cotesia sesamiae individuals collected post release to endemic or introduced pouplations based on Wolbachia and nuclear microsatellite (SSR) compartments. Nuclear microsatellite asignement (in lines) was performed with the Geneclass software assignment (P>0.5). Wolbachia strain asignment (in columns) was based on bacterial sequence. F|H: number of insects collected in Forest zone|Highlands (see Figure 2). Underlined are the number of insects exhibiting SSR and Wolbachia of distinct origin.

Wolbachia Total

Endemic

  Before releasing the Kenyan populations of C. sesamiae in Cameroon, pre-release surveys were conducted on four wild host plants, namely Pennisetum purpureum (Schumach), Setaria megaphylla (Steud.) Dur. & Schinz, Setaria spp. and Typha domingensis (Table

:

  (1) the introduced C. sesamiae strains genetically designed to attack the major Cameroonian crop pest B. fusca were successfully recovered from B. fusca on crops; (2): the introduced insects were also recovered from wild hosts plants where 16% (MDS) to 21% (GENECLASS) of the insects collected were assigned to introduced C. sesamiae ; (3):although endemic and introduced insects co-occur on wild host plants, the introduced Wolbachia remained associated at more than 95% with the introduced nuclear genome and the endemic Wolbachia remained associated at more than 95% with the endemic nuclear genome.

Figure 1 .

 1 Figure 1. Populations collected in Kenya and their Wolbachia strain infection status.

Figure 2 .

 2 Figure 2. Release and recoveries of C. sesamiae from maize and wild host plants in highlands (northern points) and forest Zone (southern points) of Cameroon. A: Release sites on maize. B: post release sampling on maize. C: Post release sampling on wild host plants. Me: Meru, Ki: Kitale, Hy: Me ✕ Ki, Ke1: Meru ✕ Kitale ✕ Kakamega, Ke2: Meru ✕ Kitale ✕ Kakamega ✕ Muranga ✕ Mombasa ✕ Tana River. C: Cured from Wolbachia. U: Uncured from Wolbachia. ✕: crossing.

Figure 3 .

 3 Figure 3. Multidimensional scaling representation of two main axes of microsatellite genetic variation among wild pre-release (collected on wild host plants before releases of Kenyan C. sesamiae), introduced (Kitale, Meru, Hybrid : Kitale ✕ Meru, Ke1: Meru ✕ Kitale ✕ Kakamega, Ke2: Meru ✕ Kitale ✕ Kakamega ✕ Muranga ✕ Mombasa ✕ Tana River. C: Cured from Wolbachia. U: Uncured from Wolbachia. ✕: crossing), Wild Post-Release (collected on Wild host plants after Kenyan strains releases), Wild Post-Release (collected on cultivated host plants after releases).

Table 5 .

 5 STRUCTURE assignation of the different populations: Prerelease (Wild plants), introduced (Me: Meru, Ki : Kitale, Hy: Hybrid Me x Ki, Ke1: Kenya 1 strain, Ke2: Kenya 2 strain, U : Uncured from Wolbachia, C: cured from Wolbachia), and recovered in wild (Post-release (Wild plants)) and cultivated (Post-release (Cultivated plants)) compartments after introduction. #Ind. : number of individuals. The model was admixed. Best number of cluster was 2 (S1 and S2 cluster)

		S1	S2	#
				Ind.
	Pre-release (wild plants)	0.02	0.97	9
		3	7
	Me	0.99	0.00	10
		8	2
	Ki	0.98	0.01	17
		9	1
	Hy	0.99	0.00	15
		8	2
	Ke1U	0.99	0.00	43
		8	2
	Ke1C	0.78	0.21	47
		7	3
	Ke2U	0.97	0.02	30
		4	6
	Ke2C	0.98	0.01	30
		7	3
	Post-release (Wild plants)	0.19	0.80	96
		8	2
	Post-release (Cultivated	0.96	0.03	98
	plants)	6	4

Funding: This work was funded by IRD DEEIT, IRD UMR 247, ANR Biodiversité BioInv4i (2006-2010), and Biodiversité ABC-Papogen (2012-2016), and by the German Federal Ministry for Economic Cooperation (BMZ). Partial funding for parasitoid collections in Cameroon were provided by core funds from the International Institute of Tropical Agriculture.

Data statement

The molecular data is given as supplementary data (SupplementaryData.xlsx).

Aknowledgement


Marie Colmont

Anastasiya Zadoya

Sylvie Daviero-Minaud

Nora Djelal

Olivier Mentré

Original Oxocentered frameworks in Bi 3 (VO 4 )O 3 and Bi 3.5 (AsO 4 )(OH) 0.5 O 3.5 by Supercritical Steam

ABSTRACT: Two new bismuth oxo-vanadate Bi3(VO4)O3 and oxo-arsenate Bi3.5(AsO4)(OH)0.5O3.5 were prepared using supercritical hydrothermal pressure. Dealing with the anionic sublattice, both crystal structures are built on anti oxo-OBi4/OBi3, or OBi4/OBi5 units connected together in infinite corrugated 2D layers surrounded by isolated XO4 (X= V or As) tetrahedra. These edifices complete a series initiated by the recent Bi3(PO4)O3 prepared in similar conditions. The latter being assigned to the "simplest" Bioxophosphate in terms of structural complexity, this aspect was investigated among the other compounds in their chemical ternaries. These phases are suggested to be high-pressure polymorphs not possible to tackle working at ambient pressure and temperature conditions.

A plethora of inorganic crystal structures have been described using the antiphase approach in which, unlike the standard model, the crystallographic units used for description are centered on the anions. [START_REF] Krivovichev | Anion-Centered Tetrahedra in Inorganic Compounds[END_REF] While anti-perovskites are clearly best suited for such structural descriptions, fluorites such as the 3D -Bi2O3, clearly show the interest to analyze the oxo-centered frameworks, in several low dimensionality Bi-based compounds. It leads to the emergence of motifs made of oxo-centered O(Bi,M)x groups, best adapted for mega-series descriptions. [START_REF] Abraham | Crystal Structure Approach of the Disordered New Compounds Bi∼1.2M∼1.2PO5.5 (M=Mn, Co, Zn): The Role of Oxygen-Centered Tetrahedra Linkage in the Structure of Bismuth-Transition Metal Oxy-phosphates[END_REF][START_REF] Aliev | Labile Degree of Disorder in Bismuth-Oxophosphate Compounds: Illustration through Three New Structural Types[END_REF][START_REF] Colmont | Bi2O3-CuO-P2O5 system: two novel compounds built from the intergrowths oxocentered polycationic 1D-ribbons[END_REF] In the last decades, many related structures based on OBi4 building units were isolated and fully characterized. The anion-centered topologies also allow to predict new architectures of original oxo-centered based frameworks, surrounded by isolated spacers such as phosphates, vanadates, arsenates, molybdates, tungstates, or sulfates… [START_REF] Colmont | Bi∼3.785Cd∼3.575Cu∼1.5(PO4)3.5O5.5, a new arrangement of double and triple [M4Bi2n-2O2n] x+ polycationic ribbons in the bismuth-transition metal oxy-phosphate series[END_REF][START_REF] Debreuille-Gresse | The crystal structure, phase transition, and dielectric properties of K2Bi3(PO4)3O, a new oxyphosphate[END_REF][START_REF] Mentré | Structural features of the modulated BiCu2(P(1x)V(x))O6 solid solution; 4-D treatment of x = 0.87 compound and magnetic spin-gap to gapless transition in new Cu 2+ two-leg ladder systems[END_REF][START_REF] Deacon | Cu3Bi4V2O14, a new compound[END_REF][START_REF] Lü | Revised Bi/M Layered Oxo-Sulfate (M = Co, Cu): A Structural and Magnetic Study[END_REF][START_REF] Lü | Investigation of New Alkali Bismuth and Oxophosphates with Original Topologies of Oxo-Centered Units[END_REF][START_REF] Radosavljevic | Synthesis and structure of bismuth copper arsenate, BiCu2AsO6[END_REF][START_REF] Vannier | Bi26Mo10Oδ Solid Solution Type in the Bi2O3-MoO3-V2O5 Ternary Diagram[END_REF] Recently, Bi3(PO4)O3 [START_REF] Aksenov | Bi3(PO4)O3, the Simplest Bismuth(III) Oxophosphate: Synthesis, IR Spectroscopy, Crystal Structure, and Structural Complexity[END_REF] announced as "the simplest bismuth(III) oxophosphate ever synthesized" was prepared under supercritical hydrothermal pressure, around 50 MPa at 700K. Being aware of the size effects of PO4 for AsO4 or VO4 substitution, the replacement of phosphate by bigger tetrahedral groups may give rise to innovative architectures and properties. For example, in BiCu2O2(PO4) and BiCu2O2(AsO4), [START_REF] Aksenov | Bi3(PO4)O3, the Simplest Bismuth(III) Oxophosphate: Synthesis, IR Spectroscopy, Crystal Structure, and Structural Complexity[END_REF] different arrangements between same polycationic units exist and pressure-induced transformations from one form to another one, [START_REF] Colmont | Compressibility of BiCu2PO6 : Polymorphism against S = 1 /2 Magnetic Spin Ladders[END_REF] with important outcome concerning the associated low-D magnetism. In Bi6ZnO7(XO4)2 (X= P, As), [START_REF] Olchowka | Original oxo-centered bismuth oxoarsenates; critical effect of PO4 for AsO4 substitution[END_REF] the modification induced by P/As substitution breaks the spatial inversion (centrosymmetric/CS-PO4: CS vs. NCS-AsO4) which is of great interest for nonlinear optical applications. Combining the HPwater method used by Aksenov et al. [START_REF] Aksenov | Bi3(PO4)O3, the Simplest Bismuth(III) Oxophosphate: Synthesis, IR Spectroscopy, Crystal Structure, and Structural Complexity[END_REF] and chemical substitution is the leitmotif of this work. Supercritical water pressure conditions in autoclave yields two new crystal structures, isolated and structurally studied: (1) Bi3(VO4)O3 and (2) Bi3.5(AsO4)(OH)0.5O3.5. Their topologies are analyzed and compared to their oxo-phosphate variant Bi3(PO4)O3 ranking their structural complexity.

Single crystals of (1) and (2) were obtained from the starting materials (BiO)2CO3, NaVO3, K2CO3 (ratio 3:1:1) and (BiO)2CO3, KH2AsO4 (ratio 3:1) respectively. In both cases, the potassium act as a mineralizer. [START_REF] Feger | Hydrothermal Synthesis and Characterization of M2Te3O8 (M=Mn, Co, Ni, Cu, Zn): A Series of Compounds with the Spiroffite Structure[END_REF] 1 mg of the reactants mixture was sealed in a gold capsule half-filled of water, and placed during 3 days under counter supercritical water pressure in a Novaswiss autoclave, working at 450°C -150 bars for (1) and 700°C -200 bars for (2). At the end, the furnace was cut off and the autoclave left open at room temperature (RT) after cooling. The products were extracted from the tubes and washed several times with distilled water, filtered and finally dried at RT. Both samples contain colorless polycrystalline material and needle-shaped single crystals corresponding to the novel vanadate (1) (Figure S1) and arsenate (2) (Figure S2) respectively. The sample containing vanadium is inhomogeneous whereas the one containing arsenic is pure (Figure S3). Crystal structures of (1) and (2) were refined by the single crystal X-ray diffraction method. The experimental data collection and refinement results are listed in tables S1 (for (1)) and S2 (for (2)). Crystal structure of (1) was refined in P-1 space group with unit cell parameters a= 5.682 These two new compounds are pertinent examples of well-adapted antiphase description. [START_REF] Krivovichev | Anion-Centered Tetrahedra in Inorganic Compounds[END_REF] Here, OBi4 tetrahedra are associated to OBi3 triangles and/or OBi5 units leading to polycationic bidimensional (2D) oxo-centered units. In the vanadate (1), six independent oxygen are involved in OBi4 tetrahedra (Td): O1, O2, O3, O5, O6 and O9. As in the homologous systems, the OBi4 tetrahedra are strongly distorted with Bi-O distances varying from 2.128(11) Å for Bi1-O1 to 2.733(11) Å for Bi5-O6. They share edges to build a 4-Td-wide ribbon, decorated on both sides by OBi5 square pyramids (SP) centered on O5, sharing their square-face with the ribbons. These polycationic ribbons are connected together through two edges connecting OBi3 triangles (Tr) centered on O3, forming all together [Bi3O3] 3+ corrugated layers parallel to (010). For a better understanding, the layer is shown along different orientations figure S4. These layers sandwiched slabs of isolated VO4 tetrahedra (figure 1a)), according to the refined formula of (1): Bi3(VO4)O3. An EDX study confirmed the presence of Bi and V in ratio 2.92/0.98 (figure S5). It involves very complex oxo-centered layers compared to the [BiO] + fluorite ones. Due to the steric effect of VO4 groups, this new phase is different from the referenced Bi3(PO4)O3 (figure 1b). In the latest, the structure shows also corrugated layers but built on double-Td-wide ribbons connected through association of two OBi3 Tr. All crystallographic information are gathered tables 3-6. The arsenate compound (2) displays the same kind of quadruple ribbons as found in (1), built on the edge sharing of four OBi4 Td centered on O1, O4, O8 and O10. There are also additional OBi5 SP units (centered on O3) decorating the ribbons in their middle. The main difference with (1) lies in the layers (presented along different directions figure S6) where the ribbons are connected through triangles OBi3 Tr (centered on O5, O15 and O16) by corner instead of edge as in (1). Their associations form four member rings units hosting a local Bi-disorder (i.e. half-occupied Bi6a/Bi6b and Bi7a/Bi7b pairs, figure 2a andb) forming in-layer channels along b direction. However, on the Fourier map some residual electronic density still present close to the O15 atom suggesting the presence of hydroxyl groups pointing to the vacant space of the channels. The bond valence sum calculations (table S9) validate the assignment of the hydroxyl groups hold by O15, H1 and H2 atoms, and their presence is confirmed by infra-red spectroscopy (figure 2c). The wide and intense peaks observed in the 300-900 cm -1 region can be attributed to: a) the stretching vibration modes of arsenate groups AsO4, see PbCu(Fe 3+ ,Cu)(AsO4)2(OH,H2O)2 [START_REF] Brooker | Infrared and Raman spectra and X-ray diffraction studies of solid lead(II) carbonates[END_REF] , and b) to Bi-O-Bi vibrations, see -Bi2O3 (figure 2c). [START_REF] Díaz-Guerra | Formation of β-Bi2O3 and δ-Bi2O3 during laser irradiation of Bi films studied in-situ by spatially resolved Raman spectroscopy[END_REF] The broad weak bands around 1300 cm -1 are attributed to O-H vibration in Bi-OH units, Bi(OH)3. [START_REF] Hobosyan | A novel nano-energetic system based on bismuth hydroxide[END_REF] The broadness of this latter can plausibly be due to the hydroxides local disorders. Indeed, two 50% occupied OH bonds exist on O15. This disorder is linked to the local disorder on Bi6 and Bi7 mentioned above and allows local ordering such as sketched on figure 2. It significantly modifies Bi-O distances i.e. Bi7a/b-O16 = 2.395(14)/2.045(13) Å and Bi6a/b-O15= 2.091(12)/2.236(12) Å depending on the proton presence or not. Finally, as in (1) the structure can be described by corrugated layers sandwiching isolated AsO4 tetrahedra, showing similarities with structures detailed in ref 25. An EDX study confirmed the presence of Bi and As in expected ratio (figure S7). After structural refinement, the final formula is Bi3.5(AsO4)(OH)0.5O3.5 and the corresponding crystal structure shown figure 2a). All main crystallographic information are detailed in tables S7-10. To date, it was not possible to synthesize the Bi3VO7 phase at ambient pressure using solid-state synthesis from mixtures of Bi2O3, V2O5. The samples were systematically composed of known phases (BiVO4, 26 Bi8V2O17 27 and Bi3.5V1.2O8.25 28 ) (figure S8). Also using solid state synthesis with Bi2O3, Bi(OH)3 and As2O5 did not allow to obtain Bi3.5(AsO4)(OH)0.5O3.5. It gave mixture of only known phases (α-Bi2O3, 29 Bi12As2O23, [START_REF] Watanabe | Bismuth Multiple Oxide and Its Production[END_REF] and Bi12.38As0.62O20.12 31 ) (figure S9). Up to now, we tackled these two new phases by using only the supercritical water route. In addition, only (2) was prepared as a single material under conditions given above. (figure S3) As it is usually the case for such synthesis, the mineralizer and at the end of the reaction, is evacuated in the filtrate. Bi3(PO4)O3 was analyzed on the basis of its structural complexity [START_REF] Aksenov | Bi3(PO4)O3, the Simplest Bismuth(III) Oxophosphate: Synthesis, IR Spectroscopy, Crystal Structure, and Structural Complexity[END_REF] using Shannon information content per unit cell (IG,total). [START_REF] Krivovichev | Which Inorganic Structures are the Most Complex?[END_REF] In this prior work, it was described laconically as the simplest term in the oxo (hydroxo)-phosphate series. Indeed IG,total value varies from 76.104 to 2592.966 bits/unit cell for Bi3(PO4)O3, with 2D corrugated layers, to Bi14O15(PO4)4 with 2D-thick blocks respectively. It denotes a versatile crystal-chemistry of oxo-centered polyhedra while associated with small phosphate groups (rP5+=0.17Å). One could also plausibly suggest the effect of steam pressure for Bi3(PO4)O3 leading to a more symmetrical edifice. A similar analysis on series of oxo (hydroxo)-vanadates and arsenates concerned here is summarized table 1.

The structural complexity per unit cell (IG,total) were also calculated for (1) and (2) using details of calculation summarized in [START_REF] Aksenov | Bi3(PO4)O3, the Simplest Bismuth(III) Oxophosphate: Synthesis, IR Spectroscopy, Crystal Structure, and Structural Complexity[END_REF] and [START_REF] Krivovichev | Which Inorganic Structures are the Most Complex?[END_REF] . The structural information amounts of (1) and (2) are respectively equal to 196.22 and 488.85 bits/unit cell.

The comparison with other vanadate and arsenate phases clearly reveals for bigger XO4 groups (rAs5+-V5+ = 0.33-0.35 Å), a more homogeneous distribution of complexities (IGtotal varying from 24.054 to 1016.355 bits unit cell for vanadates and from 64.426 to 488.85 bits/unit cell for arsenates). One must note that the bigger XO4 groups drive less complex edifices, which pictures well a less labile matching between the XO4 groups and the BiOx oxo-centered framework. The most complex phase is a vanadate: Bi14V4O30 (1016.35 bits/unit cell). [START_REF] Labidi | Crystal structures and conductivity properties of MBi6V2O15 family type compounds (M=Pb, Sr, Ca, Cd, Na0.5Bi0[END_REF] In comparison, the number of phases referenced in the literature for oxo (hydroxo)-arsenates is very limited: only three to date and two of them hosting hydroxides.

Clearly (2) is the most complex (hydro)oxo-arsenate of bismuth, it is mainly due to the structural disorder and IG,total drops to 513.53 bits/unit cell considering local ordering only. (1) stands in the lowest range in terms of complexities within its chemical series. Dealing within absence of O/OH tandem, similarly to the Bi3(PO4)O3 case, the high-pressure conditions may be responsible for its relatively simple (i.e. symmetrical) structure. It is of course far below the complexity calculated for some Bi-based minerals such as the Bouazzerite Bi6(Mg,Co)11Fe14(AsO4)18O12(OH)4(H2O)86 [START_REF] Britvin | Polyoxometalate chemistry at volcanoes: discovery of a novel class of polyoxocuprate nanoclusters in fumarolic minerals[END_REF] (IG,total = 3035.201 bits/cell) in which natural conditions brewed a diversity of cationic species. Taking into account that our experimental conditions using super-critical water steam vapor may simulate some mineralogical conditions, it emphasizes the broad perspectives offered by working in more rich and more diversified chemical systems. Conclusion. Considering the title compounds and the phosphate equivalent of (1), Bi3(PO4)O3, the XO4 size effect drastically impact the crystal structure substituting P 5+ by V 5+ or As 5+ . We observed a true preference of the oxo-centered framework for fluoritetype 2D slabs separated by XO4 entities, which topology and complexity are versatile. [START_REF] Olchowka | Original oxo-centered bismuth oxoarsenates; critical effect of PO4 for AsO4 substitution[END_REF] In these rich chemical systems, the supercritical water-pressure parameter is barely used now, while it is

shown here to open wide prospective possibilities. It seems very promising to further stabilize stable or metastable compounds into more complex Bi-based chemical systems. Especially, the possibility to isolate new bismuth vanadate is promising considering the broad impact of BiVO4 in the current development of new catalytic, luminescent and photocatalytic materials. [START_REF] Reddy | Constructing ordered paths to improve the charge separation and light harvesting capacity towards efficient solar water oxidation performance[END_REF][START_REF] Yu | Luminescence Amplification at BiVO4 Photoanodes by Photoinduced Electrochemiluminescence[END_REF]

  (1) Å, b= 8.479(2) Å, c= 14.243(3) Å, α= 81.39(1)°, β= 86.91(1)° and γ= 88.53(1)° (Robs= 0.045, Rall= 0.051, GOF= 2.38). (2) was refined in P21/n space group with unit-cell parameters a= 11.7264(14) Å, b= 5.4063(7) Å, c= 24.213(3) Å, β= 98.66(1)° (Robs= 0.039, Rall= 0.088, GOF= 1.09).

Figure 1 :

 1 Figure 1: Projection of a) Bi3(VO4)O3 and b) Bi3(PO4)O3 along a axis. The anion-centered units are drawn in yellow for OBi4 (Td), green for OBi3 (Tr) and grey for OBi5 (SP). XO4 tetrahedra (X= P and V) are shown in red. The inset shows the crystal shape of (1).

Figure 2 :

 2 Figure 2: a) General overview of the new Bi3.5(AsO4)(OH)0.5O3.5 along the b axis. The anion-centered units are drawn in yellow for OBi4 (Td), green for OBi3 (Tr) and grey for OBi5 (SP) whereas AsO4 groups are shown in red. The inset shows the crystal shape of (2). b) Possible ordering between two consecutive ribbons, c) Infra-Red spectrum of crushed crystals of Bi3.5(AsO4)(OH)0.5O3.5. The right inset shows the weak bands due to Bi-OH vibration.

TABLE 1 . Complexity of each Bi/V/O +H and Bi/As/O +H members of existing phases Containing Oxo-Centered Tetrahedrons.

 1

	Compound/Mineral	S.G.	a (Å),	b (Å),	c(Å),	V [Å 3 ]	v (atoms)	IG,total	Synthesis route
		Z	α (°)	β (°)	γ (°)			(bits/u.c.)
	Bi4(VO3.5)2O4 35	C2/m	5.6120(2)	15.2829(4)	16.60139(50	1423.85	11	24.054	Solid state synth at
		6		89.756(1)	)				850°C
	Bi6 (VO3.33)3O6 36	Pnma	5.4721(1)	17.2542(3)	14.9174(2)	1408.45	100	384.386	Sealed tube under
		4							vaccum at 650°C
	Bi4(VO3.5)2O4 37	I4/mmm	3.99172(5)	3.99172(5)	15.4309(3)	245.87	11	24.054	Solid state synth at
		1							800°C
	Bi4(VO3.5)2O4 37	Amam	11.2331(3)	5.6491(2)	15.3469(3)	973.87	36	102.117	Solid state synth at
		4							800°C + slow cool-
									ing at 300°C.
	Bi46(VO4)8O57 38	C2/m	20.02938(5)	11.59621(3)	21.12860(6)	4571.07	150	834.323	Solid state synth at
		2		111.3366(2)					825°C
	Bi14(VO4)4O14 39	C2/c	9.9345(3)	11.5464(2)	27.9515(4)	6011.09	184	1016.355	Solid state synth at
		8		110.8813(9)					800°C
	Bi23(VO4)4O28 40	Immm	12.1371(4)	11.6935(4)	16.0191(6)	2273.51	84	342.955	Solid state synth at
									800°C
	Bi2(VO4)O(OH) 41	P21/c	6.971(1)	7.535(1)	10.881(1)	546.57	36	114.117	Mineral
		4
	Bi3(VO4)O3 [this work]	P-1	5.6823(12)	8.4789(19)	14.2428(26)	677.39	44	196.22	This work
		2	81.39(1)	86.91(1)	88.5287(1)
	Bi24(AsO4)1.7O33.2 42	I23	10.2094(5)	10.2094	10.2094	1064.14	33	64.426	Precipitation
		1
	Bi3(AsO4)2(OH)O 43	P-1	9.993(3)	7.404(3)	6.937(3)	430.07	30	117.207	Mineral
		2	87.82(2)	115.01(2)	111.07(2)
	Bi2(AsO4)(OH)O 44	P21/c	7.000(2)	107.08(2)	10.831(2)	538.48	36	114.117	Mineral
		4
	Bi3.5(AsO4)(OH)0.5O3.5	P21/n	11.7264(14)	5.4063(7)	24.2131(30)	1517.51	104	488.85	This work
	[this work]			98.66(1)

ACKNOWLEDGMENT

The Fonds Européen de Développement Régional (FEDER), CNRS, Région Nord Pas-de-Calais, and Ministère de l'Education Nationale de l'Enseignement Supérieur et de la Recherche are acknowledged for funding the X-ray diffractometers.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: XX.XXXX/acs.inorgchem.xxxxx. SEM images, EDS analysis, powder XRD data, and results of crystal structure refinements.

AUTHOR INFORMATION

Corresponding Author * E-mail: marie.colmont@centralelille.fr. ORCID Marie Colmont : 0000-0002-6120-0618 Anastasiya Zadoya: 0000-0002-7761-7979 Sylvie Daviero : 0000-0003-3887-5585 Olivier Mentré: 0000-0002-1822-6003

Author Contributions

The manuscript was written through contributions of all authors and all of them have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interests. Two new bismuth oxo-vanadate Bi3(VO4)O3 and oxo-arsenate Bi3.5(AsO4)(OH)0.5O3.5 were prepared using supercritical hydrothermal pressure. Their crystal structure and complexity are compared together and with the previously reported phosphate analogue Bi3(PO4)O3.

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