Although a significant degree of frustration is expected, we find that Sr2MgOsO6 orders in a type I antiferromagnetic structure at the remarkably high temperature of 108 K. The measurements presented allow for the first accurate quantification of the size of the magnetic moment in a 5d2 system of 0.60(2) μB –a significantly reduced moment from the expected value for such a system. Furthermore, significant anisotropy is identified via a spin excitation gap, and we confirm by first principles calculations that SOC not only provides the magnetocrystalline anisotropy, but also plays a crucial role in determining both the ground state magnetic order and the size of the local moment in this compound. Through comparison to Sr2ScOsO6, it is demonstrated that SOC-induced anisotropy has the ability to relieve frustration in 5d2 systems relative to their 5d3 counterparts, providing an explanation of the high TN found in Sr2MgOsO6.The influence of spin-orbit coupling (SOC) on the physical properties of the 5 A2BB′O6, where A and B are non-magnetic cations, and B′ is a magnetic 4d or 5d transition metal cationB′ cations forms a quasi-face-centered cubic (fcc) type lattice where antiferromagnetic interactions are highly frustrated as each B′ cation has twelve nearest neighbor B′ cationsd and 5d transition metals. It’s imperative to understand the role SOC plays in such materials due to its role in lifting the orbital degeneracy and enhancing multipolar interactions, leading to rich phase diagrams456There is a great deal of interest in materials with inherently frustrated magnetic exchange interactions and the resulting unusual magnetic ground states1d3 electronic configuration, the relatively large S = 3/2 spins are expected to have a quenched orbital contribution resulting in classical behaviord3 configuration for 4d or 5d cations, the reduction in the magnetic moment due to SOC is small relative to the effects of covalency, which is large for cations with high oxidation states8d3 or 5d3 ions tend to exhibit frustration indexes (|Θ|/TN) ranging from 4 to 14 and adopt a type I antiferromagnetic structure upon ordering810111214In the case of double perovskites containing a single magnetic cation with a d1 configuration, SOC is expected to play a significant role and antiferromagnetic, ferromagnetic, and quadrupolar order have all been predicted as a result52LiOsO6 and ferromagnetic order in Ba2NaOsO6172MgReO62YMoO620d4 configuration has also begun, such as in A2BIrO6 , where questions have arisen concerning the strength of SOC and the magnetism of the resulting ground state222324In the case of the much smaller S = 1/2 d2 S = 1 configuration, by studying the double perovskite Sr2MgOsO62627d2 S = 1 scenario, however there has been difficulty in sorting known materials in this context. Ba2YReO6, Sr2YReO6, and Ca2MgOsO6 appear to be spin glasses, which have not been predicted28292LiReO6 and Sr2InReO6 host non-predicted spin singlet states292CaOsO6 orders antiferromagnetically, though with moments too small for detection in the reported neutron scattering experiment2MgOsO6, we are able to provide insight into the nature of frustration and influence of SOC in this material.In this work, we investigate the influence of SOC on the magnetic state for the intermediate 52MgOsO6 orders at 108 K with a type I antiferromagnetic structure, shown in 6+ moments of 0.60(2) μB are significantly reduced from the 2 μB spin only value expected for an S = 1 ion. Density functional theory (DFT) confirms that this substantial reduction in moment occurs through a combination of both covalency and SOC, and furthermore predicts that SOC-induced anisotropy is essential in the selection of the magnetic ground state. The presence of this anisotropy is experimentally confirmed by the observation of a spin gap in the magnetic excitation spectrum via inelastic neutron scattering. Sr2MgOsO6 is therefore a rare example of a compound where the Néel order, rather than just the anisotropy, is set by the spin-orbit interaction. Furthermore, we find that SOC-induced anisotropy is responsible for the reduced magnetic frustration in Sr2MgOsO6 relative to d3 double perovskites, therefore explaining the enhanced TN in Sr2MgOsO6.Sr2MgOsO6 were synthesized by grinding stoichiometric amounts of SrO2, MgO, Os, and OsO2 together using a mortar and pestle according to the following chemical equation:Powder samples of Sr2MgWO6 samples were synthesized in air following the procedure outlined in the literatureGround mixtures of up to 3 g were contained in high-density alumina tubes and sealed in evacuated silica ampoules  for heatings of 48 hours at 1000 °C in a box furnace located within a fumehood. This was followed by regrinding and identical reheating for an additional two cycles. Larger sample sizes were produced by synthesizing multiple aliquots which would be ground together in the intermittent grindings and redistributed for the subsequent heatings. Powdered Sr2MgOsO6 powders was measured using a Quantum Design MPMS SQUID magnetometer. Data were collected over the temperature range 2.5 to 400 K under zero-field-cooled (ZFC) and field-cooled conditions (FC) in an applied field of 10 kOe. Powders were contained in gel capsules and mounted in straws for insertion into the device for measurement. An analogous data set was collected using an empty sample mount and subtracted from the temperature dependent magnetization data of Sr2MgOsO6 in order to remove the background response.The temperature dependence of the magnetization of Sr2MgOsO6 and Sr2MgWO6 were cold pressed and sintered at their synthesis temperatures overnight (in an evacuated ampoule for Sr2MgOsO6) to prepare polycrystalline pellets. The specific heat measurements were conducted on the pellets mounted with Apiezon grease using a Quantum Design PPMS instrument using a relaxation technique.Powders of Sr2MgOsO6 at Oak Ridge National Laboratory’s (ORNL) Spallation Neutron Source (SNS) on the POWGEN beamline346+ from ref. Laboratory x-ray powder diffraction measurements were conducted at room temperature on a Bruker D8 Advance equipped with a Ge (111) monochromator and a Cu radiation source. Time of flight neutron powder diffraction (NPD) measurements were conducted on Sr2MgOsO6, and on a 16.5 g sample of Sr2ScOsO6 that was previously examined in ref. mf.u./ms, where mf.u. is the formula unit mass and ms is the sample mass for each of Sr2MgOsO6 and Sr2ScOsO6. The presented magnetic scattering intensity is therefore per Os ion.Inelastic neutron scattering experiments were performed on an 11 g sample of SrFirst principles calculations were performed using the generalized gradient approximation (GGA) of Perdew, Burke and Ernzerhof (PBE)2MgOsO6 crystallizes in the tetragonal I4/m space group as previously reported272BOsO6 compositions42434445I4/m space group is associated with the a0a0c− Glazer tilt system, where out of phase tilting occurs about the c-axis2+ and Os6+ Sr2MgOsO6 from POWGEN are given in 6+ in the double perovskite structure42466 and OsO6 octahedra are slightly elongated, with two slightly longer and four slightly shorter M−O bonds. For the d2 cation Os6+, this is the expected Jahn-Teller distortion. The unit cell is tetragonally distorted as compared to the cubic cell, with a c-axis which is greater than √2 of the a-axis. The tetragonal distortion is enhanced at lower temperatures, with a c/√2a ratio of 1.0068 at 300 K and 1.0206 at 10 K. The distortion manifests as a combination of enhanced tetragonal elongation of the octahedra and octahedral tilting as evidenced by a reduced Mg−O−Os bond angle. No structural phase transition or change in symmetry occurs within the temperature range studied. The short Os−O bond lengths compared to the d3 osmates reflect the contraction and increased covalency that can be anticipated as the oxidation state is increased.The results of the refinement of neutron powder diffraction data on Sr2MgOsO6 is given in 27χT)/dT, shown in TN from this approach is typical of double perovskites27B, in close agreement with reported values, and a Weiss constant of Θ = −269 K, in approximate agreement with reported values27B for S = 1, indicating that the influence of spin-orbit coupling is significant for Os6+. The ratio between the Weiss constant and ordering temperature TN yields a relatively low frustration index, (|Θ|/TN), of 2.5.The temperature dependence of the magnetization of Sr2MgOsO6 is shown in 2MgWO6, was measured to approximate the nonmagnetic lattice contributions to the specific heat. The solid line, also shown in 2MgWO6 scaled by mass. The difference of these two data sets, plotted as Cmag/T and shown in 2MgOsO6. Clearly, there is a large peak at the antiferromagnetic transition, but there is also a significant tail up to temperatures much higher than TN, indicating persistent magnetic fluctuations. The magnetic specific heat Cmag/T is integrated to obtain the magnetic entropy, plotted against the right axis in Smag ~ 11.2 J/mol K, which is in between the theoretical values for a simple L-S scheme for a d2 cation with an expected total spin J = 2 (Smag = 13.38 J/mol K) and a spin-only scenario with S = 1 (Smag = 9.134 J/mol K). These results clearly contrast from a similar analysis recently conducted on 3d8 S = 1 Sr2NiWO62MgOsO6 is significantly impacted by the orbital contribution to the magnetic moment in this compound.The specific heat of SrQ region of the neutron powder diffraction data collected on the POWGEN instrument, shown as the inset of d2 Ba2CaOsO6, where NPD data from the C2 diffractometer at the Canadian Neutron Beam Centre at Chalk River National Laboratories did not yield any observable magnetic reflections despite evidence of long range magnetic order from muon spin relaxation experimentsB per Os6+.In order to obtain a microscopic insight into the ordered magnetic structure, we examined the low TN, two magnetic reflections previously anticipated due to type I antiferromagnetic order were observed, see 6+ moments of 0.60(2) μB, which are aligned within the a-b plane. The resulting value is just below the proposed detection limit from the case of Ba2CaOsO6−1 peak is shown in TN, and confirms the TN = 108(2) K transition temperature associated with this magnetic peak. This is a remarkably high Néel temperature for a double perovskite with B = Mg, since the Os6+ ions are on a quasi-fcc lattice and are separated by more than 5.5 Å.In order to search for the presence of weak magnetic reflections, additional neutron powder diffraction data was collected on the HB-1A beamline using an 11 g sample, nearly 10 times the mass measured on POWGEN, and with the sample contained in an aluminum can to minimize incoherent scattering. HB-1A was utilized for this particular investigation because of its excellent signal-to-noise ratio, arising from the combined use of a double-bounce monochromator and an analyzer. Below a-b plane of the tetragonal cell. We find that PBE-GGA calculations without SOC predict an incorrect magnetic order, specifically a ferromagnetic ground state. Only when spin-orbit coupling is included do we obtain the correct type I order as the lowest energy state. This conclusion is robust, as when spin orbit coupling is included we obtain type I order independent of the moment direction and for both PBE and PBE + U calculations. It follows that Sr2MgOsO6 is a rare example of a material where the Néel order itself, rather than just the anisotropy is set by the spin-orbit interaction–a reflection of the strong SOC.For our DFT calculations, we considered different magnetic orders including ferromagnetic, the observed type I order, and checkerboard antiferromagnetic order in the basal I4/m cell. The anisotropy is sizable and increases with U. For the PBE calculations we find that the <110> direction is disfavored by 1 meV/Os, while the <001> direction is disfavored by 5 meV/Os. The easy axis and plane do not depend on U. This anisotropy and the symmetry breaking due to the tetragonal lattice no doubt partly explain the relatively high ordering temperature on a dilute fcc-like lattice. The other needed ingredient in obtaining the ordering is the intersite exchange interaction. The value of this coupling depends on the choice of U, and as such we cannot directly predict the precise magnitude of this coupling. However, as seen from the energy differences, regardless of the choice of U the correct ground state is predicted, i.e. there is sufficient intersite exchange coupling. We find that the ferromagnetic ordered state is 31 meV above the ground state in the PBE + U calculation and 168 meV above the ground state without U.Also for both PBE and PBE + U calculations with SOC we find the lowest energy spin direction to be along the tetragonal <100> direction in the tetragonal B consisting of a spin moment of 0.77 μB and an orbital moment of −0.29 μB. In the probably more realistic PBE + U calculation we obtain 0.57 μB, from a spin moment of 1.07 μB and an orbital moment of −0.50 μB, in close agreement with the experimental result of 0.60(2) μB from NPD. We also find sizable moments on the O ions, which is a result of strong covalency. This can be seen in the density of states, shown for the ground state with PBE + U in eg σ bonding states, and one can see very strong Os character in this region reflecting the covalency. The Os t2g states, which are the active orbitals here, extend from −0.5 eV to + 1.8 eV and are split by U to give a gap of 0.22 eV. The t2g band width is similar in the PBE calculation. The Os eg states extend from 4.3 eV to 6.3 eV. The large crystal field splitting is another reflection of very strong covalency.Turning to the moment size, based on integration within the LAPW spheres (2.0 bohr for Os) we obtain moments that are strongly reduced from the nominal values due both to covalency and SOC. The total Os moment in the PBE calculation is 0.48 μ6+d orbitals can be regarded as largely inside a 2 bohr LAPW sphere, this is not the case for O2−p orbitals with a 1.55 bohr sphere. In order to estimate the O contribution, we turn to the ferromagnetic case. In the PBE + U calculation the total spin moment in the unit cell is 1.93 μB per formula unit, while the Os spin moment is only 1.14 μB. Thus ~40% of the spin moment is distributed over the six neighboring O ions. This moment will be active in specific heat and susceptibility data, but not in refinements of neutron diffraction data as the moment is spread across neighboring oxide ions and bonds. The O moments provide an explanation for the sizable intersite exchange. Although the double perovskite lattice can be regarded from a Zintl perspective as touching (OsO6)6− anions held together by interstitial Mg2+ and Sr2+ cations, the fact that the O atoms on the exterior of these contacting polyanions carry sizable moments provides a mechanism for the intersite exchange.A consequence of this covalency is the presence of sizable moments on the O sites. In the double perovskite structure each O has only one Os nearest neighbor. The O moments are parallel to those of the neighboring Os regardless of the treatment (PBE or PBE + U), the magnetic order or the inclusion of SOC. While Os2MgOsO6. Confirmation of this can be found by examining the inelastic neutron scattering spectra of both Sr2MgOsO6 and Sr2ScOsO6 shown in 2MgOsO6 to Sr2ScOsO6 because Sr2ScOsO6 also shows high-TN type I AFM order with TN = 92 K, but has Os5+ 5d3 ions which are expected to show significantly reduced SOC due to a S = 3/2 state. In both materials we observe scattering emanating from the type I antiferromagnetic wavevector at Q ≈ 0.8 Å−1, 2MgOsO6 and 2ScOsO6. The Sr2MgOsO6 spectrum at 6 K is remarkably similar to that of Sr2ScOsO6, in which the gap has been extensively characterizedHaving shown that SOC has a significant influence on the moment size observed by neutron scattering, we anticipate that SOC may have a major effect on the magnetic dynamics in Sr2MgOsO6 and Sr2ScOsO6 does, however, support a picture of SOC having stronger influence in Sr2MgOsO6. The microscopic mechanism by which SOC typically produces the spin gap is associated with either exchange anisotropy or single-ion anisotropy (or a combination)1015382MgOsO6 has a smaller magnetic moment, the similarity of observed gap to that in Sr2ScOsO6 demonstrates that SOC is stronger in Sr2MgOsO6 resulting in a comparable gap.The similar size of the gaps in SrTN the intensity of the observed scattering is very different between the two compounds, see Q < 1 Å−1 and 3 < E < 12 meV. For both materials the integrated intensity in this region increases with temperature, because of both the modification of the scattering due to the closing of the gap and the Bose thermal population factor. The integrated intensity per Os ion for each of the samples is similar at low temperatures, with a slightly higher value for Sr2ScOsO6, as expected for the larger spin system. However, as the magnetic transition temperatures are approached the integrated intensities diverge dramatically. This difference in fluctuation intensity above TN is indicative of the level of frustration in each system - a strong signal implies strong correlations despite the absence of long range magnetic order.Despite the similarity in the spectra at 6 K, above Q < 1 Å−1 and 3 < E < 5 meV following the method described in ref. In d2 cation6+ 5d2 cation in Sr2MgOsO6. Despite this, we have shown that the ground state remains in the “AFM100” region of the phase diagram predicted in ref. d3 and 5d3 double perovskites8101112d2 state would inherently infer anisotropic interactions on the Os6+ ions, with the strong Os−O hybridization in Sr2MgOsO6 ensuring that the anisotropy has significant influence on the collective properties. We have confirmed anisotropy is present in Sr2MgOsO6 via observation of the spin gap in the magnetic excitation spectrum, A phase diagram depicting seven potential magnetic ground states, including three potential antiferromagnetic configurations, has been proposed for double perovskites with a single magnetic 5B is considerably reduced (70%) from the expected high-field spin-only value of 2 μB. Covalency has been shown to play a significant role in the reduction of the moment in the case of 4d3 and 5d3 transition metal oxides, resulting in 37–47% reductions of the 5d3 Os5+ 3 μB moment to 1.6 to 1.9 μB891011126+ 5d2 case. Here, however, the magnetization and specific heat analyses strongly suggests that an orbital contribution is also important, consistent with the recent x-ray magnetic circular dichroism (XMCD) study of Os6+ in the related material Ca2CoOsO62CaOsO6 on a similar instrument in order to conclusively place the magnetic ground state among those known and predicted.The moment observed by neutron diffraction of 0.60(2) μd2 double perovskites the type I AFM structure was anticipated via theory including strong SOC in ref. d2 state would inherently infer anisotropy, with the strong Os−O hybridization in Sr2MgOsO6 ensuring that the anisotropy has significant influence on the collective properties. Via inelastic neutron scattering we have indeed observed anisotropy in Sr2MgOsO6 via the spin gap.For 5d2 and 5d3 systems. Sr2MgOsO6 orders at a higher temperature than other double perovskites with a single magnetic iond3 and 5d3 double perovskites like Sr2ScOsO6 have larger magnetic moments, which should yield stronger interactions and higher ordering temperatures, they also have significantly larger frustration indices ranging from 4 to 1481011122MgOsO6. The similarity of the QE-space dependence and the intensity of the excitation spectra of Sr2MgOsO6 and Sr2ScOsO6 at 6 K, shown in 2ScOsO6 above TN is far greater than the intensity in Sr2MgOsO6 above TN. This implies strong correlations persist in Sr2ScOsO6 in the absence of long range magnetic order–a hallmark of frustration. While the tetragonal symmetry of Sr2MgOsO6 may play some role in reducing the geometric frustration, it is less distorted than monoclinic Sr2ScOsO6, which has angles that deviate more substantially from 180° It is interesting to investigate the comparison between 52MgOsO6 compared to Sr2ScOsO6? The strength of hybridization plays a major role in determining the strength of the interactions, but does not directly explain relief of frustration. Similarly, the unit cell volume and B/B′ site disorder affect interaction strengths, and all these mechanisms likely contribute to differences in TNs amongst the AFM d2 DPs Sr2MgOsO6 (|Θ|/TN = 2.5) [this work], Ba2CaOsO6 (|Θ|/TN = 3.1)2CaOsO6 (|Θ|/TN = 3.0)d3 DP Sr2ScOsO6. Our DFT results reveal that the SOC induced anisotropy leads to selection of the magnetic ground state, as also found experimentally for Sr2ScOsO62MgOsO6 and the multiple sources of evidence we have presented for stronger SOC in this d2 material, we conclude that SOC induced anisotropy is the dominant factor in the relieved frustration in Sr2MgOsO6.The question, therefore, is why is the frustration relieved in SrTN should vary with the strength of anisotropy in such systems, due to reduced competition between possible ground states2MgOsO6 and Sr2ScOsO6, 2MgOsO6, we conclude that the anisotropy is indeed much larger in Sr2MgOsO6. Therefore, the results presented demonstrate the relief of frustration via SOC induced anisotropy, and represent the experimental demonstration of the evolution of TN with SOC.This conclusion is supported by an earlier theoretical prediction that 2MgOsO6 has been synthesized and characterized by magnetometry, specific heat measurements, and elastic and inelastic neutron scattering. The combined results demonstrate that spin-orbit coupling is essential to describe the magnetic properties of the system. The Os6+ moments order antiferromagnetically at 108 K in a type I configuration on the Os fcc sublattice as theoretically predictedd2 material, refinements of neutron powder diffraction data yields a moment of 0.60(2) μB per Os cation which is significantly reduced due to the combined effects of SOC and covalency. Through comparison of inelastic neutron spectra, it is shown that SOC induced anisotropy has the ability to relieve frustration in d2 systems, relative to analogous d3 materials which have systematically higher frustration indices, promoting high magnetic transition temperatures.The double perovskite osmate SrHow to cite this article: Morrow, R. et al. Spin-orbit coupling control of anisotropy, ground state and frustration in 5d2 Sr2MgOsO6. Sci. Rep.6, 32462; doi: 10.1038/srep32462 (2016).


Our findings reveal that the sulphur-vacancy defects preferentially serve as active sites for both hydrogen chemisorption and physisorption. Unexpectedly the sulphur vacancy instantly dissociates the H2 molecules and strongly binds the split hydrogen at the exposed Mo atoms. Thereon the additional H2 molecule is adsorbed with enabling more hydrogen physisorption on the top sites around the sulphur vacancy. Furthermore, the increase of the sulphur vacancy on the MoS2 surface further activates the dissociative hydrogen chemisorption than the H2 physisorption.We provide a new insight that the sulphur-depleted MoS During the last few decades, numerous hydrogen storage materials were extensively investigated, including carbonaceous materials2, metal–organic frameworks3, and metal hydrides4. Among them, the molecular adsorption (physisorption) on the porous materials constitutes a main avenue of research due to the fast reversibility of adsorption and desorption5. However, high storage densities have been obtained only at temperature of 80 K or below, because of the weak van der Waals (vdW) interaction between hydrogen molecules and the surface of most porous materials6. Therefore, the capacity of the H2 physisorption at high or room temperatures is of the utmost importance.For realization of the hydrogen economy, a number of strategies have been developed to tackle key technical barriers such as hydrogen generation, storage and applications in fuel cells. Especially, the most critical problem among them is to search for suitable materials that can store hydrogen at ambient temperature and atmospheric pressure2 has been intensively studied as a promising and inexpensive alternative to platinum (Pt) for the prominent hydrogen evolution reaction (HER) catalysis nearing the efficiency of Pt7, together with its unique electronic and optical properties8. It has long been realized that the HER activity stems from the coordinatively unsaturated sites at the edges of the MoS2, i.e., the exposed Mo sites, while the basal plane of MoS2 is catalytically inactive11. Hence, designing MoS2 nanostructures with more edge sites has become a significant topic13. Very recently, the basal plane of monolayer 2H-MoS2 has been catalytically activated and optimized for the HER by applying the strain on the single sulphur vacancies (VS). It is understood that the dissociated hydrogen atoms or ions from the water splitting are bound at the significantly increased number of the exposed Mo atoms on the basal plane of the MoS214. More interestingly, the first-principles calculations have predicted that the H2 molecule dissociates at the VS15. Furthermore, it has been reported that the hydrogen molecules adsorb dissociatively on the 4-fold symmetric Mo exposed surface of substoichiometric MoSx phase at room temperature16. These results need to reveal how the VS on the MoS2 surface intrinsically interacts with the hydrogen.On the other hand, the layered 2H-MoSS defect is occasionally obtained in the MoS2 samples prepared by mechanical exfoliation17, we have investigated the hydrogen gas interaction on the various single-crystalline MoS2 surfaces in the UHV chamber using angle-resolved photoemission spectroscopy (ARPES) supported by density functional theory (DFT) calculations. Here we unexpectedly find that the VS defects serve as active sites for both hydrogen chemisorption and physisorption at room temperature. The H2 molecules instantly and preferentially dissociate at the VS defect on the MoS2 surface. Additionally more H2 molecules are adsorbed on the top site of the dissociative hydrogen chemisorption at the VS defect with expanding the other top sites of Mo (TMo), S (TS), and hollow, i.e., the center of a hexagon (TH) around the VS. This new insight makes attractive for onboard storage applications with providing ways to modify very small specific surface area of the MoS2 surface18 to increase both the adsorption potential and the number of available sorption sites.Since the predominant V2 layers  between the center of mass of the H2 molecule and the top Mo-layer of the MoS2 sheet is 4.65 Å. The length of bonds in the hydrogen molecule is 0.76 Å, which is a little larger than the limiting value of 0.74 Å in the ref. 2 molecules on the defect-free MoS2 surfaces has a weak contribution at −4.3 eV of total density of states (DOSs) as shown in Fig. 2 molecules favor the TH site21.In the defect-free MoSers Fig. , the H2 S defect is introduced, the preferential adsorption position of H2 molecules changes to be the on-top site of a remained bottom S atom [T(VS)] with the h[T(Vs)] ≈ 5.20 Å [h(TMo) ≈ 3.64 Å] as shown in Fig. S have slightly lower adsorption energies than that of the first one (TMo) as shown in Fig. S) and S introduces a defect state in the bandgap as indicated by (red) arrow in Fig. S. Nevertheless, the adsorption of H2 molecules on the defective MoS2 surface also has a small influence on the DOSs  for monolayer (bilayer) system, compared to the physisorption of H2 molecule at the T(VS) site  arrow in Fig. 2 molecule is located on the T(VS) site  = 5.31 Å [h(TMo) = 3.80 Å] .On the other hand, it has been found that the Hite Fig. . When th7 Å Fig. , each H gap Fig.  are weakgap Fig.  due to tite Fig.  with theion Fig.  is rever2 surface decreased rather quickly when exposed to the hydrogen gas in the UHV chamber at room temperature. In Fig. −6 Torr•s) and further reduced to 34% in 1 h (3600 L). The entire surface of the cleaved MoS2 is supposed to be fully and physically covered with hydrogen gas.Figure 2 exposure leads to slightly shift the valence band maximum (VBM) of the cleaved surface (0.45 eV) toward high binding energy side (0.51 eV) and then the further H2 exposure reverses the shift of VBM toward EF (0.43 eV). This subtle change is more elucidated in the S 2p core-level spectra  and the surface  contributions, respectively22. As the cleaved MoS2 surface is exposed to the hydrogen gas, the intensity of the surface state significantly decreased from 43 to 32%  corresponds to the low-valence-state sulphur (S¯) due to the presence of the VS defect23. It is away from the bulk state of C1 by −0.75 eV, which is comparable to the energy difference between the VBM and defect state in Fig. 3  appeared at the higher binding energy side (162.41 eV) with the 2 molecules (5 L). The relative intensity further increased from 6 to 14% at the H2 exposure of 3600 L. The energy difference between the C1 and C4 components (0.7~1.0 eV) is quite comparable to that (0.6 eV) between the Mo-H bonds-induced peak (−6.6 eV) and the edge of main DOSs  at the coordinatively unsaturated Mo edge sites24. On the other hand, it is notable that the hydrogenation of the MoS2 single crystal by the S-H bonds induces the weak ferromagnetism and formation of the atomic stripes on the MoS2 surface26. The further hydrogenation at higher temperature (above 500 °C) resulted in more severe decomposition of MoS2 with H2S desorption27. Thus, instead of the annealing, in order to effectively desorb the physically adsorbed H2 molecules, the alternative method such as the application of the compressive strain is considered, because it returns to the poor activity of the basal plane28.More importantly, a new component of COSs Fig. . Thus, tles Fig.  togetherles Fig. . On the 2 exposed  around 2 physisorption. Additionally, the tail of the band structures at the S as shown in the DOSs of Fig. 4 feature in Fig. On the other hand, the comparison of the ARPES data between the cleaved Fig.  and H2 esed Fig.  MoS2 sur2 surface. By the Bi deposition, the VBM (0.43 eV) of the 3600 L H2 exposed MoS2 surface was shifted away from EF by 0.51 eV with partially occupied Bi related-bands at EF . First of all, the Bi induced electron doping into the MoS2 surface elucidates that the H2 molecules are adsorbed physically on the MoS2 surface. And we notice that the reduced intensities are quite similar to the case of the Bi deposition on the cleaved MoS2 surface, i.e., without the hydrogen exposure at the same condition of the Bi thickness (not shown here). This ensures that the desorbed H2 molecules are no more than the monolayer, because the probing depth at the current photon energy is comparable to the thickness of 1L-MoS2 (~6.5 Å) at least30. Based on these results, we could materialize the H2 molecules covered MoS2 surface based on the dissociative chemisorption of H atoms around the VS defects in Fig. 2 molecules are 3.57 (TH), 7.31 (TMo), and 10.34 Å (TS), respectively, with respect to the Mo plane. They have the tilt axes to the basal plane due to the interaction among the H2 molecules. The estimated coverage of the H2 physisorption on the 3 × 3 monolayer MoS2, i.e., a gravimetric storage density is of 3.6 wt.%, which is much higher than the capacity of the MoS2 nanotubes31.Finally, in order to confirm the hydrogen physisorption and estimate the coverage, we performed the subsequent bismuth (Bi) deposition on the hydrogen covered MoS EF Fig. , and the0 s Fig. . Accordi0 s Fig. . Notablytra Fig. . In addi2 surface from interacting with the hydrogen  were cleaved in the UHV chamber at a base pressure better than 2 × 10−10 Torr and then exposed to the H2 gas by filling the chamber with a pressure of 1 × 10−6 Torr. The cleanliness and structural order were verified by the quality of the valence band dispersion and low energy electron diffraction (LEED) pattern.ARPES and photoemission experiments were performed at the 4A2 and 10D beamlines of the Pohang Accelerator Laboratory (PAL), respectively. All photoemission data were collected at room temperature. The energy and angle resolutions of the ARPES apparatus (4A2) for the ARPES data obtained at the photon energy of 100 eV were better than 130 meV and 0.4°. Natural, single crystalline MoSnd light order at the photon energy of 100 eV. For comparison, they were shifted by + 101.3 eV after the energy calibration. In fitting of S 2p core-level spectra23, the natural (Lorentzian) line width, representing the core-hole lifetime, was determined to be ~0.07 eV while the Gaussian width was fixed at the instrumental resolution of 380 meV. The values of the spin-orbit coupling (3/2)/I(2p1/2)] were 1.18 eV and 0.5, respectively.The S 2p core-level photoemission spectra were obtained by the 2S defects fundamentally alters the catalytic property of the basal plane of MoS2, we calculated the adsorption energies of the H2 molecules on mono- and bilayer MoS2 samples with and without VS defects on each surface. The DFT calculations were performed by adopting the generalized gradient approximation (GGA) of the PBEsol33 functional for the exchange correlation potential and the projector augmented wave (PAW) method34 as implemented in the Vienna Ab initio Simulation Package (VASP)35. The electron wave function was expanded in a plane wave basis set with an energy cutoff of 400 eV. A vacuum region is thicker than 10 Å in order to avoid the coupling of the interlayer. Integration over the Brillouin zone was carried out by using 15 × 15 × 1 Monkhorst-Pack k-point mesh for all systems considered. All atomic positions for 3 × 3 cell of mono- (a = 3.141 Å) and bilayer (a = 3.142 Å) MoS2 were fully optimized. For comparison of the calculations with and without including the vdW interaction, the optB86b-vdW36 functional was also calculated with the lattices of mono- (a = 3.163 Å) and bilayer (a = 3.165 Å) MoS2 (Supplementary Fig. In order to understand how the presence of VSupplementary Information


By increasing the size of the residue R from methyl to tert-butyl in several steps, we find that the dynamics drastically change from backward to dominant forward scattering of the leaving ion relative to the reactant RY velocity. This characteristic fingerprint is also confirmed by direct dynamics simulations for ethyl as residue and attributed to the dynamics of elimination reactions. This work opens the door to a detailed atomistic understanding of transformation reactions in even larger systems.The competition between bimolecular nucleophilic substitution and base-induced elimination is of fundamental importance for the synthesis of pure samples in organic chemistry. Many factors that influence this competition have been identified over the years, but the underlying atomistic dynamics have remained difficult to observe. We present product velocity distributions for a series of reactive collisions of the type X The competition between chemical reactions critically affects our natural environment and the synthesis of new materials. Here, the authors present an approach to directly image distinct fingerprints of essential organic reactions and monitor their competition as a function of steric substitution. N2) are probably the two most fundamental exchange reactions in organic chemistry1. Both processes are stereo-specific, occur in a concerted way and always appear in competition with each other. Due to the ubiquity and importance of this competition, immense experimental and theoretical effort has historically been devoted to investigate both the intrinsic reactivity and atomistic dynamics of these reactions6. In the past years, new experimental and theoretical developments have provided detailed insight into bimolecular nucleophilic substitution reactions between monatomic anions and methyl halides (X− + CH3Y)14. While early studies already revealed non-statistical reaction kinetics and dynamics and thereby showed that such systems cannot be described using common statistical models such as Rice–Ramsperger–Kassel–Marcus (RRKM) or phase space theory16, very recent investigations have theoretically postulated quantum state specificity in SN2 reactions18. Furthermore, new mechanisms beyond the classical collinear attack have been revealed and characterized19. All these advances reflect that the understanding of the underlying mechanistic features of nucleophilic substitution is a complex and ongoing subject of research.Base-induced elimination (E2) and bimolecular nucleophilic substitution  affects both the dynamics and reactivity of such systems. The addition of bulky substituents is supposed to hinder the well-known Walden inversion and promote the E2 reaction. Such an elimination process is assumed to occur via an anionic attack on a β-carbon-bonded hydrogen atom in periplanar orientation with respect to the α-carbon-Y bond 35 techniques  we have obtained energy- and angle differential cross-sections for a series of reactions of types (1)–(4), as indicated in Fig. N2 pathways. For each of these combinations reactive scattering experiments have been performed on reactions with four increasingly methylated neutral species  that represent the transition from primary to tertiary alkyl halides. The corresponding reaction enthalpies are listed in Table Using an ion-molecule reactive scattering apparatusof types –(4), as of types –(4), as 35Cl product ions from reactions between F− and increasingly substituted alkyl chlorides at a collision energy of 1.9 eV. For F− + CH3Cl dominant backward scattering of the Cl− product ion is observed with respect to the incoming CH3Cl velocity, while scattering into the forward hemisphere and isotropic scattering barely contribute to the velocity distribution. This backward scattering feature corresponds to the commonly assumed collinear substitution mechanism under Walden inversion and has already been reported in previous studies on this and other similar systems36. Both backward and forward scattering are present in the reaction F− + C2H5Cl, whereby backward scattering events appear at velocities considerably lower than in F− + CH3Cl, indicative of a higher degree of energy partitioning among the larger number of C2H5Cl rovibrational modes. Ions produced from F− + iC3H7Cl and F− + tC4H9Cl are predominantly forward scattered and no backward scattering is observed, as can be extracted from the scattering angle distributions on the upper right panel of Fig. Figure − ion images from reactions between F− and stepwise substituted alkyl iodides. As discussed in a previous work, the reaction between F− and CH3I features dominant indirect as well as direct stripping dynamics37. The reaction F− + C2H5I presents a combination of forward, backward and sideways scattered events, whereas both F− + iC3H7I and F− + tC4H9I show dominant forward scattering of the I− product. There are clear differences between the velocity distributions of F− + CH3Cl and F− + CH3I, which was recently shown to arise from a substantially different relative orientation of the respective reactants13. In contrast, we observe forward scattering of the leaving Cl− or I− ion in reactions with tC4H9Y. As such a scattering pattern has never been observed for nucleophilic substitution reactions, we assign this feature to the atomic-level mechanism of base-induced elimination. This is in line with more indirect previous findings that tC4H9Y reactions occur via E2 due to the strong steric hindrance at the α-carbon31.Fig. − and different alkyl iodides have been performed. The resulting centre-of-mass product velocity distributions are presented in Fig. − + CH3I toward a forward scattering mechanism in reactions with tC4H9I, as clearly illustrated in the lower right scattering angle distribution of Fig. − + C2H5I a high fraction of backward scattered events show velocities above the kinematic limit of the E2 reaction, unambiguously indicating nucleophilic substitution events. In contrast to the nucleophile-leaving group combinations discussed before, backward scattered events are still observed both in reactions with C2H5I and iC3H7I. This fact can be explained by the lower proton affinity of Cl− and consequently by the smaller tendency of undergoing E2 reactions at increasing CH3-substitution. The observed mechanistic transition as a function of methylation is very similar to the reactions with F− ions and reinforces the indication that scattering into the forward hemisphere is a mechanistic fingerprint of E2 reactions.In order to confirm the inherent character of this mechanism irrespective of the attacking anion, additional experiments between Cl− + C2H5I reaction were investigated by quasi-classical trajectory direct chemical dynamics simulations38 at a relative collision energy of 1.9 eV . The resulting potential energy profiles for the different SN2 and E2 pathways are shown in Fig. 39. Both SN2 and E2 exhibit strongly bound pre- and post-reaction complexes separated by central transition states (TSs). Three of the TSs are lower in energy than the reactants, while a significant potential energy barrier of 0.49 eV is obtained for the front-side SN2 pathway. However, this barrier is much less than the collision energy of 1.9 eV. The back-side SN2 and anti-E2 TSs are very similar in energy and lie substantially below the syn-E2 TS.To complement the experimental findings, the main atomic-level mechanisms involved in the Fanti-E2 trajectories proceed via a direct pathway. Representative snapshots of such a trajectory are presented in Fig. anti-periplanar TS geometry followed by simultaneous formation of HF and I−. As shown in the cartoon, the anion usually approaches at large impact parameters and abstracts a β-hydrogen without modifying its original direction. Such an atomic-level mechanism is very similar to the stripping dynamics observed in several X− + CH3Y reactions12. All E2 reactive trajectories were found to depend on the initial relative orientation of the anion and the β-hydrogen, as no rotation of the CH3 group was observed. This is expected given a CH3 rotational barrier of 0.15 eV, which cannot be surmounted under the internal temperatures of the neutral reactant (see Methods section).Fig. 2H4] + I− or [CH3CH2I–F−]. The roundabout pathway8 also participates in the indirect E2 mechanism. In addition, both dihalide formation and proton transfer reactions were observed after overcoming the syn-E2 and anti-E2 TSs, respectively. The formation of these products has been recently reported to be efficient in F− + CH3I40 and is also observed to a minor extent in the current experiments. As also found for a variety of X− + CH3Y reactions12, the SN2 pathway occurs via direct rebound, direct stripping and indirect mechanisms. Average direct anti-E2 reactions finish in about 500 fs . The concentration and backing pressure of the neutral reactant are optimized in order to avoid possible clustering. The pulsed valve is heated to avoid condensation of the nozzle. The velocity and translational temperature of the neutral molecules are obtained by electron impact ionization of the supersonic beam and imaging of the resulting non-fragmented ions. We obtain translational temperatures of 160–170 K, which are considered an upper limit due to the momentum transfer of the impinging high-energy electrons. The rotational temperature is assumed to lie in the same range, whereas a temperature between 165 and 345 K  is assumed for the vibrations, which are predicted to cool less efficiently during supersonic expansion.− and C2H5I has been characterized using the M06 density functional method using an effective core potential (ECP)/d basis set42. This method has been shown to give the best agreement with the CCSD(T)/pseudo-potential (PP)/t benchmark, as compared to other density functionals39. For the ECP/d, Dunning and Woons aug-cc-pVDZ basis set is used for the H, C and F atoms. For iodine, the Wadt and Hay ECP is used for the core electrons and a 3s,3p basis set for the valence electrons, which is augmented by a d-polarization function with a 0.262 exponent, and s, p and d diffuse functions with exponents of 0.034, 0.039 and 0.0873, respectively. For the PP/t basis set, the Peterson aug-cc-pVTZ basis with a PP is used for iodine and the aug-cc-pVTZ basis for all other atoms.The potential energy profile of the reaction between F43 and interfaced44 to the NWChem electronic structure computer program45. Including zero-point energy (ZPE) corrections, this theory gives reaction exothermicities of −46.5 and −32.4 kcal mol−1 for the SN2 product C2H5F + I− and the E2 product CH2=CH2 + HF + I−, respectively, in good agreement with the experimental values of −48.7 and −37.6 kcal mol−146. To compare with the experiments, the simulations were performed at a collision energy of 1.9 eV and C2H5I rotational and vibrational temperatures of 165 and 250 K, respectively. Quasi-classical sampling, which includes ZPE, was used to determine initial conditions for the trajectories, as described previously for the Cl− + CH3I simulations47. The impact parameter b was randomly selected between 0 Ã… and the maximum impact parameter, bmax, which was determined by incrementing b until no reaction occurred out of 200 trajectories, which happened at bmax = 6.0 Å. A total of 1500 trajectories were calculated in the simulations and their atomic-level mechanisms were determined by animating these trajectories. A representative snapshot is given in Fig. Chemical dynamics simulations were performed by ab initio direct dynamics at the M06/ECP/d level of theory. For this purpose, the VENUS general chemical dynamics computer program was usedThe experimental data sets generated during the current study are available from the corresponding author upon request.


Bacillus thuringiensis (Bt). Previous work showed that resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton, can be conferred by mutations disrupting a cadherin protein that binds this Bt toxin in the larval midgut. However, the potential for epistatic interactions between the cadherin gene and other genes has received little attention. Here, we report evidence of epistasis conferring resistance to Cry1Ac in the cotton bollworm, Helicoverpa armigera, one of the world's most devastating crop pests. Resistance to Cry1Ac in strain LF256 originated from a field‐captured male and was autosomal, recessive, and 220‐fold relative to susceptible strain SCD. We conducted complementation tests for allelism by crossing LF256 with a strain in which resistance to Cry1Ac is conferred by a recessive allele at the cadherin locus HaCad. The resulting F1 offspring were resistant, suggesting that resistance to Cry1Ac in LF256 is also conferred by resistance alleles at this locus. However, the HaCad amino acid sequence in LF256 lacked insertions and deletions, and did not differ consistently between LF256 and a susceptible strain. In addition, most of the cadherin alleles in LF256 were not derived from the field‐captured male. Moreover, Cry1Ac resistance was not genetically linked with the HaCad locus in LF256. Furthermore, LF256 and the susceptible strain were similar in levels of HaCad transcript, cadherin protein, and binding of Cry1Ac to cadherin. Overall, the results imply that epistasis between HaCad and an unknown second locus in LF256 yielded the observed resistance in the F1 progeny from the complementation test. The observed epistasis has important implications for interpreting results of the F1 screen used widely to monitor and analyze resistance, as well as the potential to accelerate evolution of resistance.Evolution of resistance by insect pests reduces the benefits of extensively cultivated transgenic crops that produce insecticidal proteins from  First, a field‐captured male from Langfang, China, was mated to a female from the resistant SCD‐r1 strain that is homozygous for the recessive resistance allele r1 at the cadherin locus (HaCad)  of the opposite sex from the SCD‐r1 strain. From each of 128 resulting single‐pair families, 48 of the F1 offspring were tested in bioassays (see below) at a diagnostic concentration of activated toxin (1 μg Cry1Ac per cm2 diet) that previously killed 100% of larvae from susceptible strains and F1 larvae from SCD‐r1 X SCD, but only 4%–8% of SCD‐r1 . Cry2Ab protoxin was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), China. Cry1A toxins and Cry2Ab protoxin were produced according to Monnerat et al.  and Wei 2.4H. armigera  for SCD and the resistant strain LF256. For each concentration, we tested 48 larvae from each strain against each toxin.We used a series of five to seven concentrations of each toxin (including a control with no toxin) to estimate the concentration of Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Ab that kills 50% of the larvae  were tested with 1 μg/cm2 Cry1Ac. To quantify the results of the complementation tests, we calculated the index of commonality (C), which measures the extent to which resistance alleles in two resistant strains are expected to share a common locus , a susceptible strain (SCD) and progeny from reciprocal crosses between each resistant strain and the susceptible strain SCD or the resistant strain SCD‐r1. For each mass cross, 30 males and 30 females were placed in one cage. At the same time, we set up single‐pair reciprocal crossed between LF256 and SCD‐r1 according to the method described by Jin et al. . Ninety‐2.7Premix Taq™  were used to amplify four overlapping gene fragments  according to the manufacturer's instructions and reverse transcribed with the Moloney murine leukemia virus reverse transcriptase (Promega). Specific primers and TaKaRa ts Table , and one2.8®Premix Ex Taq™ , and reactions were carried out using the 7500 RT‐PCR detection system . qRT‐PCR included an initial incubation of 30 s at 95°C followed by 40 cycles of amplification at 95°C for 5 s, 60°C for 34 s. Relative transcript levels of target genes in the LF256 larvae compared with the control SCD strain were calculated using the 2−ΔΔCT method. The reference gene EF‐1α, validated by and Yang, Chen, Wu, Yue, & Wu. . 3.5 ml of 24 mM MgCl2 was added and incubated on ice for 15 min. The homogenate was then centrifuged at 2,500 g for 10 min; the supernatant was transferred into a new tube and centrifuged at 30,000 g for 30 min; and the pellets were collected and dissolved in 800 μl of 10 mM Hepes buffer . Prepared BBMVs were kept at −80°C until used. The protein concentration was determined by Bradford method with BSA as a standard.BBMV were prepared from insect midguts by the differential magnesium precipitation method  were separated on SDS–PAGE (10%) and transferred to polyvinylidene difluoride (PVDF) membrane . The membrane was blocked with 2.5% (w/v) BSA in TBST  for 1.5 hr and then washed three times with TBST. After blocking, all filter incubations and washes were performed in TBST. Proteins were detected with polyclonal anticadherin antibody  and a goat anti‐rabbit secondary antibody coupled with horseradish peroxidase  , followed by Super Signal chemiluminescence substrate  as indicated by the manufacturer.For ligand blot analysis, BBMV proteins (10 μg) were also separated on SDS–PAGE (10%) and transferred to polyvinylidene difluoride (PVDF) membrane (Bio‐Rad). The membrane was blocked with 2.5% (w/v) BSA in TBST for 3 hr. The blocked membrane was incubated with 1 nM Cry1Ac toxin overnight at 4°C. After washing three times with 0.1% (w/v) BSA in TBST, proteins with bound Cry1Ac were detected with polyclonal anti‐Cry1Ac antibody  and a goat anti‐rabbit secondary antibody coupled with horseradish peroxidase  , followed by Super Signal chemiluminescence substrate  as indicated by the manufacturer.Both the anti‐Cry1Ac and anticadherin antibodies were produced in New Zealand white rabbits that were immunized subcutaneously by Abgent . A synthetic HaCad peptide (HGMFEFEVEATDSRR) found in the 11th cadherin repeat (CR11) was synthesized, conjugated to keyhole limpet hemocyanin (KLH) carrier protein, and used as the cadherin antigen by Abgent. In the first week, 200 μg of the purified polypeptides was homogenized with 1 ml of complete Freund's adjuvant and then injected hypodermically into the back of the rabbit. The second injection was performed 3 weeks later, 200 μg of polypeptide was injected into the muscle of the rabbit's back. After 1 week, 100 μg of polypeptide with incomplete Freund′s adjuvant was injected in the same location for intensification. The specificity and titer of the antiserums were examined by Western blot.2.111 progeny of LF256 × SCD to identify concentrations of Cry1Ac that enabled identification of live resistant and susceptible larvae. Using the bioassay method described above, second instars were exposed for 5 days to diet with either 0.5 or 5 μg Cry1Ac per cm2 diet. We tested 96 larvae from LF256 and F1 at each concentration . At the higher concentration, the percentage of larvae that survived and weighed >5 mg was 56% for LF256 and 0% for F1, indicating that survivors weighing >5 mg at the higher concentration could be characterized as resistant. At the lower concentration, the percentage of larvae surviving and weighing <5 mg was 0% for LF256 and 90% for F1, indicating that larvae surviving and weighing <5 mg after exposure to the lower concentration could be characterized as susceptible.In preparation for genetic linkage analysis, we used bioassays with LF256 and the Fxrx) and a female from the SCD strain (ss). One male from the resulting F1 progeny (putative rxs) was backcrossed with a female from LF256 (putative rxrx), which is expected to yield backcross progeny with rxrx and rxs in a 1:1 ratio. Using the bioassay method described above, second instars from the backcross progeny were exposed for 5 days to diet with either 0.5 or 5 μg Cry1Ac per cm2 diet. Based on responses in the preliminary bioassays summarized above, survivors at the higher concentration were scored as resistant and larvae that survived but weighed <5 mg at the lower concentration were scored as susceptible. The larvae scored as either resistant or susceptible were transferred to untreated artificial diet and reared for DNA extraction until they became fourth or fifth instars.To test for genetic linkage in LF256 between resistance to Cry1Ac and the cadherin locus, we made a single‐pair cross between a male from the LF256 strain (putative cadherin genotype rxs would weigh <5 mg at the lower concentration (susceptible) and only rxrx would survive at the higher concentration (resistant). To test these predictions, we identified the cadherin alleles in the parents , in the F1 male and the female he mated with  and in 30 larvae that survived and weighed >5 mg from the higher concentration . From each of the 64 individuals listed above, we isolated genomic DNA with phenol‐chloroform extraction, amplified PCR fragments using specific primers  and the L256 female used to make the backcross were both homozygous for thymine (T), whereas the SCD female in the parental cross was homozygous for adenine (A). As expected, the F1 male that mated with the LF256 female to generate backcross progeny was heterozygous (AT) at this site  in intron 8 of s Figure . At thise Figure .33.11 screen, we paired a field‐captured male (#256) from Langfang with a female from the resistant strain SCD‐r1, which was homozygous for the cadherin resistance allele  was 85.4% (n = 48), suggesting the male had two resistance alleles at the cadherin locus or that he was homozygous for dominant resistance at another locus. To evaluate these hypotheses, we conducted a series of experiments, including isolation of resistant strain LF256. Sequencing of the cDNA in the F1 survivors revealed one new cadherin allele from the field‐captured male with a 12‐bp deletion at 2,403 bp and insertions of 4 and 7 bp at 4,247 and 4,257 bp of the cDNA sequence, respectively, yielding a premature stop codon in the region coding for the eleventh cadherin repeat (CR11) of the ectodomain  and has 98.4% amino acid identity with a wild‐type cadherin allele from SCD (GenBank accession no. AFB74168.1). Nonetheless, to represent its hypothesized role in resistance, we tentatively named it rx.As part of an Fn Figure . Here, w1 survivors and the susceptible SCD strain. Using PCR with primers specific for r18 (Table 18 and discarded their offspring. We retained the offspring (F2 progeny) from the eight other families, which were produced by single‐pair crosses between r1rx and ss. Next, we made 100 single‐pair crosses among the F2 progeny. Using PCR with primers specific for r1 (Table 1 and discarded their offspring. We pooled the offspring (F3 progeny) from the other 10 families, which were produced by crosses between rxs and rxs. In the subsequent generations, we allowed mass mating, selected larvae at increasing concentrations of Cry1Ac (1–5 μg Cry1Ac per cm2 diet), and reared the survivors to continue the strain. After six successive generations of selection, LF256 had 220‐fold resistance to Cry1Ac relative to the susceptible SCD strain . These results demonstrate that effects of maternal inheritance and sex linkage were not evident, indicating autosomal inheritance of resistance.For the Fh) calculated from the pooled survival of the F1 progeny at the diagnostic concentration . The similar survival of the F1 progeny compared to the resistant parent strains indicates lack of complementation and yields a value of 0.91 for the commonality index (C), which suggests resistance in LF256 and SCD‐r1 is conferred by alleles at the same locus, that is, the cadherin locus.Survival at the diagnostic concentration was 75% 72/96) for LF256, 85% (82/96) for SCD‐r1, and 75% (144/192) for the F/96 for L3.5x allele inherited from male #256 . The observed frequency of 0.16 does not differ significantly from the expected frequency of 0.5 if resistance was not genetically linked with the cadherin locus .Based on sequencing of cadherin cDNA, no indels occurred in LF256, the predicted cadherin amino acid sequence varied within the LF256 and SCD strains, and no consistent differences occurred between strains Figure . The abs6 Figure . However3.61 male (from LF256 X SCD) with an LF256 female confirmed that resistance to Cry1Ac was not genetically linked with cadherin in LF256. For 30 of the backcross larvae that survived when exposed to diet treated with 5 μg Cry1Ac per cm2 diet and thus were scored as resistant, the observed ratio of cadherin genotypes was 17 rxrx to 13 rxs. This observed outcome differs significantly from the outcome of 100% rxrx survivors predicted under the hypothesis of tight linkage between cadherin and resistance . For 30 of the backcross larvae that survived but weighed <5 mg after exposure to diet with 0.5 μg Cry1Ac per cm2 diet and thus were scored as susceptible, the observed ratio of cadherin genotypes was 13 rxrx to 17 rxs. This observed outcome also differs significantly from the outcome of 100% of susceptible individuals with the genotype rxs predicted under the hypothesis of tight linkage between cadherin and resistance . In addition, the observed ratio of rxrx:rxs did not differ significantly from the 1:1 ratio expected under random segregation  for either group of 30 larvae . Moreover, despite the major difference in resistance phenotype between the two groups of 30 larvae, the observed ratio of cadherin genotypes did not differ significantly between them , which supports the conclusion that resistance in LF256 was not linked with the cadherin locus.Analysis of 60 larvae from a backcross family produced by crossing an F3.7SE = 0.08) and SCD .Analysis of cadherin RNA by qRT‐PCR revealed no significant difference between LF256  of H. armigera and a female from the SCD‐r1 strain homozygous for a recessive cadherin resistance allele yielded 85.4% resistant offspring. The standard interpretation of the results from this complementation test is that the male carried two resistance alleles at the cadherin locus (HaCad). However, sequencing of cDNA revealed that while one of the cadherin alleles (r18) from this male harbors a premature stop codon expected to confer resistance, the second allele (rx) lacks deletions, insertions, or stop codons and encodes an amino acid sequence 98.4% identical to that of the susceptible strain SCD  from male #256 confers resistance to Cry1Ac. Nonetheless, the results from DNA sequencing and analyses of genetic linkage, as well as levels of transcript, protein, and binding  using single‐pair crosses with susceptible strain SCD followed by family screening with PCR and selection with Cry1Ac . Sequencing the putative cadherin resistance alleles in F1 survivors from complementation tests in three studies yielded eight distinct alleles (r2 to r9) other than r1 with disruptive mutations, but also 10 individuals with no major deletions, insertions, or premature stop codons in their putative cadherin resistance alleles . Table Sequence data for two alleles of  Click here for additional data file.


Here we apply this new method to studying the triple oxygen  and hydrogen (2H/1H) isotope ratios of gypsum hydration water (GHW), which can provide information about the conditions under which the mineral formed and subsequent post‐depositional interaction with other fluids.The recent development of cavity ring‐down laser spectroscopy (CRDS) instruments capable of measuring in vacuo and cryogenically trapping the evolved water. The isotopic composition  of the GHW is subsequently measured by CRDS. The extraction apparatus allows the dehydration of five samples and one standard simultaneously, thereby increasing the long‐term precision and sample throughput compared with previous methods. The apparatus is also useful for distilling brines prior to isotopic analysis. A direct comparison is made between results of 17O‐excess in GHW obtained by CRDS and fluorination followed by isotope ratio mass spectrometry (IRMS) of O2.We developed a semi‐automated procedure for extracting GHW by slowly heating the sample to 400°C 17O values, ±0.13‰ for δ18O values and ±0.49‰ for δ2H values , and ±1.1‰ and ±8 per meg for the deuterium‐excess and 17O‐excess, respectively. Accurate measurement of the 17O‐excess values of GHW, of both synthetic and natural samples, requires the use of a micro‐combustion module (MCM). This accessory removes contaminants  from the water vapour stream that interfere with the wavelengths used for spectroscopic measurement of water isotopologues. CRDS/MCM and IRMS methods yield similar isotopic results for the analysis of both synthetic and natural gypsum samples within analytical error of the two methods.The long‐term analytical precision of our method of extraction and isotopic analysis of GHW by CRDS is ±0.07‰ for δ17O, δ18O and δ2H values, and the derived deuterium‐excess and 17O‐excess, can be obtained from GHW and brines using a new extraction apparatus and subsequent measurement by CRDS. This method provides new opportunities for the application of water isotope tracers in hydrologic and paleoclimatologic research. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.We demonstrate that precise and simultaneous isotopic measurements of δ This derived parameter is defined as:2H and δ18O denote 2H/1H and 18O/16O in water standardized with respect to V‐SMOW.The isotopic composition of crystallization water of gypsum CaSO4·H2O has b17O‐ δ18O pair (known as the 17O‐excess) in GHW has not been applied yet in hydrology and paleoclimatology. The excess (or depletion) of 17O in water is defined as:17O and δ18O denote 17O/16O and 18O/16O in water standardized with respect to V‐SMOW.Conversely, the relationship between the δ17O‐excess to the isotopic measurements of GHW can provide additional information on the environmental conditions under which the gypsum formed, as well as its post‐depositional interactions with other fluids. For example, the 17O‐excess has been shown to be less sensitive to temperature effects than the d‐excess during evaporation.17O‐excess and d‐excess recorded by GHW may provide information about the relative effects of humidity and temperature change at the time of gypsum formation.The addition of the The most common method for extracting GHW involves step heating a sample in a vacuum line and cryogenically trapping the water.et al.16O, 17O and 18O) and hydrogen (1H and 2H) isotope analysis using a new generation CRDS analyzer (Picarro L2140‐i).17O‐excess values obtained by CRDS, a synthetic gypsum standard and a variety of natural gypsum samples were measured for their δ17O and δ18O values by fluorination followed by IRMS of O2.Here we describe a modification of the extraction procedure introduced by Hodell in vacuo and cryogenically trapping the evolved water. The Water Analyzer Sample Preparation (WASP) system comprises an adapted, programmable gas chromatography (GC) oven and six individual extraction lines attached to a common backing line, coupled to a two‐stage rotary pump   Fig. . A liquiEach line is ~1 m in length and composed of ¼‐inch O.D. stainless steel tubing with an I.D. of ~4 mm  is fitted to each of the six lines for monitoring pressure via an Edwards TIC 6 head instrument controller. The pressure in each line is continuously monitored during the extraction using a computer interface to the WASP system. The change in pressure as a function of time allows identification of any abnormalities (e.g. leaks) during the GHW extraction procedure Fig. .Gypsum samples  were loaded into 12‐mm OD Pyrex tubes and topped with 3 cm of quartz wool to prevent loss of gypsum powder during dehydration. The experiments utilized analytical‐grade powdered gypsum  (denoted as NEWGYP standard in this work). In addition, seven natural samples of gypsum were ground to a homogenous powder in an agate mortar and the GHW was extracted using the WASP for subsequent analysis by CRDS and IRMS.2S and CH4 trapped in the mineral lattice, may produce spectral interferences in the CRDS measurements when GHW is analyzed, as has also been reported for the analysis of water extracted from the xylem of plants.The presence of organic matter and other contaminants in gypsum, including gases derived from microbial activity such as H−3 mbar for at least 3 h at room temperature to remove adsorbed water. This low‐vacuum pumping (10−2–10−3 mbar) is effective at removing adsorbed water with no detectable loss of hydration water.The samples were dried at 45°C for 24 h, and then placed under vacuum to a pressure of ~5 × 10−3 mbar), each branch was isolated from the vacuum and the glass 6‐mm break‐seal tubes at the end of each of the lines were immersed in LN2 for water trapping. GHW was released from the samples by slowly heating the sample to 400°C  and enriched  waters, following the procedure of Conley and Bundy.−3 mbar for 4 days before the first experiments to remove potential adsorbed water remaining from previous samples. The system was evacuated for 30 min between the two consecutive runs.The system was tested for memory effects by extracting the hydration water of isotopically depleted and enriched gypsum in two consecutive runs of the apparatus. These gypsum standards (DPL‐gyp and ENR‐gyp) were produced by hydrating anhydrous CaSODuring GHW extraction, the pressure changes with time can be utilized to identify anomalies caused by vacuum leaks Fig. , water aRoutine isotopic analysis of brines can produce significant contamination problems in CRDS analyzers due to the accumulation of salts in the valves and pipework and problems with the needle used to inject water into the vaporizer. To solve this issue, we utilized the WASP for the distillation of saline waters prior to CRDS analysis.in situ using a hydrometer, ranged from 1.13 g cm−3 (DEPO‐03) to 1.09 g cm−3 (DEPO‐06). Volume of 200 μL of water were loaded into 12‐mm OD tubes and then topped with 3 cm of quartz wool in order to prevent salts from moving through the vacuum line. The samples were loaded into the WASP and a LN2 trap was placed on each sample tube to freeze the water sample and avoid evaporation during vacuum pumping. Subsequently, the assembly was evacuated for 2 min (to a pressure of ~5 × 10−3 mbar) and the lines again isolated by closing the Swagelok valves. The LN2 traps were then removed from the sample tubes and placed on the 6‐mm break‐seal tubes. The heating stage was set to a temperature ramp of 5°C/min, with a final temperature of 150°C (maximum pressure ~8 mbar), and held isothermally for 40 min to facilitate complete sample recovery in the traps . The traps were raised 5 cm during this final stage. Finally, the lines were evacuated for 30 s (pressure ~5 × 10−3 mbar) prior to flame sealing the 6‐mm tubes. In addition to the brines, four internal water standards  were distilled using the same methodology in order to assess any isotopic fractionation during the extraction procedure. The brines were run in duplicate and analyzed by CRDS, along with the water standards.Evaporated marine waters from two different brine pools from a natural salt factory  were utilized for the experiments. The water density measured Isotope analyses were conducted at the Godwin Laboratory for Palaeoclimate Research (Department of Earth Sciences) at the University of Cambridge .i Picarro water isotope analyzer for triple oxygen isotope analysis,®) was used in the majority of the experiments. The MCM comprises an 8‐cm‐long cylindrical cartridge filled with metallic wool coated with a proprietary catalyst, and it was placed in‐line between the vaporizer and the water isotope analyzer. The MCM has been shown to remove combustible compounds from water samples.2) as the carrier gas.The water oxygen and hydrogen isotopes were measured simultaneously by CRDS using a L2140‐17O values, ±0.04‰ for δ18O values, and ±0.19‰ for δ2H values, as observed from repeated analysis of an in‐house water standard  over the period of study.After water extraction, the 6‐mm OD tubes containing 40–60 μL of hydration water were stored until ready for analysis. Prior to analysis, water was frozen into the base of the break‐seal tube by immersion in LN2. Subsequently, the tube was scored with a diamond cutter, broken to a fixed height of 25 mm, and then quickly inserted into the 2‐mL septum‐capped vials used by an A0325 autosampler (Picarro). Each sample was injected ten times into the vaporizer, which was heated to 110°C. Memory effects from previous samples were avoided by rejecting the first three analyses. The values for the final seven injections were averaged with a typical in‐sample precision (±1SD) of ±0.02‰ for δ17O values of 0.0‰ and −29.6986‰, respectively, and δ18O values of 0.0‰ and −55.5‰, respectively.17O‐excess values are given in per meg units (0.001‰).The results were normalized against V‐SMOW by analyzing internal standards before and after each set of 10 to 12 samples. To this end, four internal water standards  were calibrated against V‐SMOW and SLAP, using δ17O‐excess value of 25±12 per meg during the course of the study. This value is in good agreement with the results reported by Schoenemann et al.17O‐excess and d‐excess were calculated for each injection using the corrected δ17O, δ18O and δ2H values. The 17O‐excess and d‐excess from the seven repeated analyses were then averaged. Typical in‐sample 17O‐excess and d‐excess precisions (±1SD) in water standards  were 13 per meg and 0.2‰, respectively. This agrees with the guaranteed precision given by the manufacturer (15 per meg for 17O‐excess). No 17O‐excess or d‐excess values were rejected.The GISP standard water was analyzed during the calibration as an unknown, yielding an average 3 reactor held at 375°C which converts H2O into O2 and HF, following the method developed by Barkan and Luz.2 is then trapped using 5Ǻ molecular sieve held at LN2 temperature. Helium is pumped away after disconnecting the oxygen trap from the He stream and sample O2 is transferred to one tube in a 12‐fold sample manifold immersed in LN2. For this study a manifold typically contained four reference water extractions, leaving eight sample tubes for O2‐extractions from two or three unknown water samples. To minimize memory effects at least three sample injections were discarded when switching between waters of different isotopic values. The CoF3 line is LabView  controlled and runs fully automated overnight.Fluorination‐IRMS analyses were conducted at the Institute for Geology and Mineralogy at the University of Cologne . An autosampler injects water samples (2.8 μL) into a CoF2 was then analyzed on a MAT253 isotope ratio mass spectrometer (Thermo Scientific) in dual‐inlet mode at m/z 32, 33 and 34. Each measurement comprises 66 samples to reference comparisons divided into 3 blocks of 22 cycles each. Each block begins with a peak centre and adjustment of bellow pressure, such that the ion beam intensity at m/z 32 is 8.5 V. The idle time was set to 17 s and the integration time to 12 s. The baseline was measured for 240 s prior to the first sample. The δ17Osample and δ18Osample values were normalized to V‐SMOW via our internal lab standards that were measured with the samples. As for the CRDS data, SLAP of δ17O = −29.6986‰ and δ18O = −55.5‰ was used for SMOW‐SLAP scaling.17Omeasured‐SLAP and δ18Omeasured‐SLAP values, respectively. This slightly contracted scale relative to the IAEA‐scale appears to be typical for MAT253 instruments.The sample OOur extraction procedure for GHW has several advantages over previous methods, including higher sample throughput and greater precision. The ability to extract five unknowns and a gypsum standard in the same run improves the long‐term reproducibility of the method. In addition, our system allows continuous monitoring of the pressure to detect anomalies in the mineral water extraction procedure.−2 mbar at the end of the extraction process. These profiles for the lines with compromised O‐rings showed slightly greater pressures (up to 1.2 mbar) at 120°C  from that obtained from the properly functioning line   than those from the properly functioning line in the same run   to be detected. The percentage of water in the gypsum samples in our experiments was on average 20.7 ± 0.4  for analyses of NEWGYP  using CRDS  showed analytical errors (±1SD) of ±0.08‰, ±0.16‰ and ±0.24‰ for the δ17O, δ18O and δ2H values, respectively, and the errors for the isotopically depleted gypsum  were ±0.06‰, ±0.12‰ and ±0.53‰, respectively  gave an error (±1SD) of ±0.02‰ for δ17O values, ±0.03‰ for δ18O values, and ±0.16‰ for δ2H values  from the results obtained by IRMS . The 17O‐excess values measured by CRDS were considerably higher than those obtained by IRMS when the MCM was turned off. The difference in 17O‐excess values with MCM On and Off reflects the degree of spectral interference caused by contamination.For most natural samples, the δRMS Fig. . However17O‐excess values for the NEWGYP standard (15±8 per meg for CRDS and 8±4 per meg for IRMS) when the MCM accessory was used with the CRDS analyzer  when the MCM was configured in WARM mode , and the results from lake samples showed an even larger disagreement of up to ~200 per meg in GHW (e.g. PI 6C‐7H‐2 26 cm). For all samples, the 17O‐excess of hydration water was greater when the MCM was not powered on. By contrast, the 17O‐excess results from CRDS were similar to those achieved by IRMS when the MCM was used  during dehydration cause spectral interferences at the wavenumber used to determine 1H217O by CRDS (7193 cm−1),1H218O and 2H1H16O signals; thus, although the 17O‐excess is affected by these contaminants, the δ18O and δ2H values are not.In previous work on the isotopic composition of GHW in lake samples measured by CRDS, Hodell 17O‐excess determination. As stated above, the differences in 17O‐excess in natural samples in our MCM On/Off experiments are greater in the case of gypsum samples generated in organic‐ and microbe‐rich environments (e.g. lake sediments). This, as expected, points to higher concentration of contaminants in these types of material and suggests that the cause of the spectral interferences could be H2S or organic compounds released by natural gypsum samples during dehydration. Further evidence for the presence of impurities in GHW is given by a strong smell of H2S in water after extraction, especially in water from lacustrine gypsum samples.Use of the micro‐combustion module (MCM) removes impurities that affect the 17O‐excess in GHW by CRDS. In earlier work, this device was found to be necessary for isotopic analysis of water samples with high concentrations of organic compounds, such as water extracted from the xylem of plants that usually contains alcohols that spectroscopically interfere with the CRDS analysis.2S, VOCs and long‐chain compounds, and this is important for the analysis of GHW samples. Although this accessory removes most impurities, we suspect that organic‐rich gypsum samples (e.g. those from lake sediments) may require additional pre‐treatment to remove organic compounds . However, any pre‐treatment procedure will require testing to ensure that it does not alter the δ18O and δ2H values of the hydration water.Our results reveal that the use of the MCM accessory is crucial for accurate determination of the 17O‐excess determination by CRDS of the analytical‐grade gypsum standard (NEWGYP) was ±8 per meg (n = 37) .Using the MCM, the long‐term precision (±1SD) of our method for 7) Table . In addi meg n = 7  in different runs over the period of this study. No measurable memory effects were detected in our experiments with ENR‐gyp and DPL‐gyp for the 17O‐excess  of one natural gypsum sample Salina 1) by CRDS using the MCM produced an analytical error (±1SD) of ±7 per meg. This indicates that the long‐term  by CRDS 3 fluorination IRMSThe repeated extraction of our NEWGYP standard (n = 8) and subsequent analysis by IRMS produced a typical in‐sample precision of ±6 per meg  and an external precision of ±4 per meg, which is similar to the typical precision achieved for water standards using CoF2H/1H, along with 17O/16O and 18O/16O, on the same hydration water sample. This enables the calculation of both d‐excess and 17O‐excess in GHW. The long‐term d‐excess of the NEWGYP standard (n = 37) determined using CRDS was −53.4 ± 1.1‰  is known  of gypsum hydration water (GHW) using the newest generation of CRDS analyzer. Our procedure presents several analytical advantages over earlier methods, including better long‐term precision, higher sample throughput, reduced sample size and less memory effect between consecutive samples.The method described enables the determination of the isotopic composition (δ17O‐excess and d‐excess values can be obtained by this method in GHW and brines. This can provide additional information about the conditions under which the gypsum formed and subsequently interacted with other fluids after deposition. Although the present procedure has been initially tested for the analysis of gypsum, other hydrated minerals could also be extracted. This opens up a broad field of possible future applications using this technique.Simultaneous Supporting info itemClick here for additional data file.