The results show that the crystal structure, light absorption, and device efficiency do not degrade after storing for 100 d in air with 40% relative humidity, while the conventional solution‐processed perovskites are usually stable for less than 20 d in similar conditions. While in moderate moisture (40% RH), the t1/2 is only 26 d. The high sensitivity to moisture poses severe challenge in terms of the practical applications.Methylammonium lead iodide CHNH3PbI3 b2O3 as an interfacial coating layer to resist the moisture permeation; the stability can be improved by a few days.x could also enhance the stability by a few days.3NH3PbIx3‐Brx could noticeably improve the stability to 20 d.6H5(CH2)2NH3)2(CH3NH3)2[Pb3I10] was synthesized which was stable for 45 d in ambient condition; the device delivered a PCE of 4.7%.To improve the stability, one way is to protect the perovskite from water molecule attachment. In this regard, Niu et al. utilized AlNH3)2Pb3I was synt3NH3PbI3 film can be dramatically improved by tailoring the compositional purity and morphology of the perovskite film through a modified chemical vapor transport (mCVT) reaction approach  onto the TiO2‐compact‐layer‐coated fluorine‐doped tin oxide (FTO) and dried at 100 °C for 10 min. Scanning electron microscope (SEM) characterization shows that the film is composed of small crystallites hosting many randomly distributed pores .Here we demonstrate that the intrinsic stability of CHch Figure1a. In 3NH3PbI3 is responsible for diminishing pores in the original PbI2 film. Previously, the solid–gas reactions in either static gas atmosphere or two‐zone furnace generate pinholes or cracks in between the perovskite nanocrystals.3NH3I deposition on the surface of the as‐prepared film than the static gas atmosphere or the two‐zone apparatus.SEM image Figure c of the 3NH3PbI3 without impurity peaks  with the same stability to that synthesized with PbI2 and CH3NH3I as precursors.The phase purity of the as‐prepared film is characterized by X‐ray diffraction (XRD), displaying typical perovskite structure of CHks Figure2a. To 2 is first spin‐coated on the TiO2/FTO substrate; it is then dipped into the CH3NH3I solution. Afterward, the film is taken out and heated at 100 °C for 10 min. This is the conventionally applied “two‐step sequential deposition” method (denoted as sample 2).3NH3PbI3  with reduced annealing time. Trace amount of DMF is detected in the perovskite film with Fourier transform infrared spectroscopy at this condition . Lower temperature annealing could lead to more DMF retaining in the film and thus more available site for water attachment. The as‐prepared perovskite shows identical crystal structure to that annealed at 100 °C for 10 min, while the decomposition rate is much faster than that annealed at high temperatures , it decomposes nearly completely after 30 d. On the other hand, if we prolong the annealing time to detach the DMF molecules, the stability can be improved to certain extent depending on the annealing time.The difference in the synthesis of sample 1 and sample 2 is that the reaction for sample 2 is in DMF solution followed by annealing for 10 min at 100 °C. A fact is that DMF can coordinate with PbThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.2O molecule  intercalating between the perovskite crystals to generate more available areas of the perovskite films for H2O attachment and (2) accumulating H2O molecule through hydrogen bonding, on both the surface of perovskite film and gaps generated 4PbI6·2H2O as a result of water adsorption.Conventionally, the planar perovskite film is prepared by a casting‐and‐annealing processing approach; here we also prepare the film for a comprehensive study. A mixture solution of PbClFigure3NH3PbI3 is shown in Figure 3b. The initial PCE of sample 1 is 12.23%. The devices based on samples 2 and 3 generate PCE of 12.11% and 12.74%, respectively  performance was examined on the ground of planar heterojunction architectures as shown in Voc increasing substantially from 0.95 to 1.05 V, the Jsc slightly drops 5%, and there is no much variation in fill factor (FF) on average. These alternations finally render an increment on the overall PCE. Remarkably, after storing for 70 and 100 d, the devices show PCEs of 12.59% and 12.71%, respectively. The J–V curves are provided in Figure S6 (Supporting Information).To exclude the influence of the other components such as the HTM on the device stability and gain a clear conclusion regarding the stability of perovskite film, we test the photovoltaic performance of the aged perovskite films by using fresh HTM every time. The performance evolution of the mCVT‐synthesized perovskite is shown in Figure 2 even though with negligible concentration. The formation of PbI2 is favorable for a larger Voc, which has been confirmed by fabricating perovskite with residual unreacted PbI2 . The type‐I heterojunction between PbI2 and perovskite is the reason for the enlarged Voc.Notably, the device efficiency is increased in the first 30 d and the highest PCE of 15.15% is obtained . In all, this research provides a new, low‐cost, and adaptable fabrication method for the perovskite film synthesis with excellent stability. The mechanistic understandings regarding the intrinsic stability of the perovskite film would benefit further improvement on the life time of the devices for practical applications.In conclusion, for the first time we show that perovskite film is stable for 100 d in air with 40% RH. The synthesis by an mCVT reaction in an isothermal furnace is able to generate high‐quality perovskite film when compared with the two‐zone furnace or the solid–gas reaction in static atmosphere. The conventional solution processed perovskite film reduces its absorption intensity to a half of its initial value even in 0% RH after 76 d.3NH3PbI3) synthesis is conducted by a chemical vapor transport synthesis in an isothermal furnace  should be addressed to the authors.SupplementaryClick here for additional data file.


SAXS, 129Xe NMR and PALS were used to interrogate the relationship between the structure, free volume and physicochemical properties of ionic liquid mixtures. 4C1im]+ cation and different anions have been systematically explored using small angle X-ray scattering (SAXS), positron annihilation lifetime spectroscopy (PALS) and 129Xe NMR techniques. Anion size has the greatest effect on the spatial arrangement of the ILs and their mixtures in terms of the size of the non-polar domains and inter-ion distances. It was found that differences in coulombic attraction between oppositely charged ions arising from the distribution of charge density amongst the atoms of the anion also significantly influences these inter-ion distances. PALS and 129Xe NMR results pertaining to the free volume of these mixtures were found to strongly correlate with each other despite the vastly different timescales of these techniques. Furthermore, the excess free volumes calculated from each of these measurements were in excellent agreement with the excess volumes of mixing measured for the IL mixtures investigated. The correspondence of these techniques indicates that the static and dynamic free volume of these liquid mixtures are strongly linked. Consequently, fluxional processes such as hydrogen bonding do not significantly contribute to the free volumes of these liquids compared to the spatial arrangement of ions arising from their size, shape and coulombic attraction. Given the relationship between free volume and transport properties such as viscosity and conductivity, these results provide a link between the structures of IL mixtures, the thermodynamics of mixing and their physicochemical properties.The formation of ionic liquid (IL) mixtures has been proposed as an approach to rationally fine-tune the physicochemical properties of ILs for a variety of applications. However, the effects of forming such mixtures on the resultant properties of the liquids are only beginning to be understood. Towards a more complete understanding of both the thermodynamics of mixing ILs and the effect of mixing these liquids on their structures and physicochemical properties, the spatial arrangement and free volume of IL mixtures containing the common [C The toxicological profile of the resultant liquid would also be predictable as there would be no new ions present in the liquid. The successful use of this approach relies on the development of a thorough understanding of the effect of forming mixtures on the structure and physicochemical properties of ILs.Ionic liquids (ILs) are low melting salts which are of interest for numerous applications including: solvents for synthesis and catalysis,i.e. there is no preferential clustering of particular ions. It is important to emphasise that despite coulombic interactions being isotropic, the charge density of each atom will differ for polyatomic ions. This means that the electrostatic attraction between ions will be governed by the size, shape and charge density of the ions rather than simply their overall charge.A major finding across a variety of IL mixtures is that the structure of these mixtures is governed by coulombic interactions.Within the ionic superstructure there are subtle perturbations to the preferred conformation of each ion due to stabilisation provided by weaker interactions such as hydrogen bonding or π–π stacking.+–π+ interactions, have been hypothesised to account for the ability of these liquids to form ideal or non-ideal mixtures.The weaker interactions in ILs, such as hydrogen bonding and π4C1im][Me2PO4]x[NTf2]x1– imide) mixtures are non-ideal with small but significant positive excess molar volumes observed whereas [C4C1im][OTf]x[NTf2]x1– ([OTf]– = triflate) mixtures do not show significant deviations from ideality.2PO4]– anion which preferentially occupied in-plane locations with respect to the imidazolium ring to facilitate stronger hydrogen-bonding interactions. As a consequence, the [NTf2]– anion is displaced to a position above the plane of the imidazolium ring, increasing the distance between [C4C1im]+ cations.–1) despite the substantial cohesive energies of ILs .+–π+ interactions, it is important to note that experimental measures of hydrogen bond basicity also incorporate more general electrostatic interactions and hence perturbations to the coulombic attraction between ions caused by the different anion basicities may also be responsible for the non-ideal behaviour.From our earlier investigations it was found that [NTf2],4C1im]+ cation with a range of anions1C1pyrr][NTf2] and [C2C1pyrr][NTf2]  organic plastic crystals.o-Ps accessible free volume results have been successfully correlated with macroscopic properties including the ionic conductivity and viscosity of the ILs.PALS enables the measurement of the free volume of condensed phases. The technique involves introducing positrons into a sample where they either annihilate on contact with electrons or form metastable bound states, known as a positroniums (Ps).o-Ps , the inert Xe gas atom  is able to penetrate into cavities within liquids, provided they are large enough to accommodate a Xe atom. 129Xe NMR has been employed as a technique to measure the free volume within condensed phases including porous solids,δ represents the chemical shift (in ppm); δ0 is the reference chemical shift of an isolated Xe atom; δwall is the chemical shift effect arising from the interaction of Xe with the wall of the cavity it is solvated in; δXe–Xe represents the chemical shift arising from Xe–Xe interactions; ρ is the density of the gas phase; and δE accounts for the polarisation of Xe in response to an external field, in this case the IL or IL mixture.i.e. the free volume, of the liquid.In addition to the semi-stable 129Xe NMR has been used in a wide range of simple ILs.o-Ps preferentially locates in the hydrophobic domains of self-assembled amphiphiles, given the diffusion length of o-Ps in liquids is ∼10 nm.129Xe NMR measurements (s) is approximately 9 orders of magnitude greater than those of PALS measurements (ns) meaning 129Xe NMR measurements give insight into the dynamic free volume of the fluid over a relatively large timescale whereas PALS probes the free volume in very short snap shots, essentially capturing free volume as accessible and static as long as motions of the liquid molecules through that volume are slower than 0.3 GHz.129Xe NMR measurements on [C4C1im][Me2PO4]x[NTf2]x1– and [C4C1im][OTf]x[NTf2]x1– mixtures to establish the relationship between the spatial arrangement of these mixtures, their free volume and their thermodynamics of mixing. These mixtures have been chosen as we have previously reported their physicochemical properties  and examined their structures through NMR and computational studies.4C1im]+ cation for which we have previously undertaken physicochemical property and structural studies to further probe the relationship between these properties and spatial effects. The intention of these studies is to provide insight into the interplay between microscopic structural and spatial effects and the macroscopic properties of IL mixtures.This work aims to combine SAXS, PALS and The structures of the IL ions examined are given in S of 0.004 to 0.29 Å–1 (S = 1/d). 2D scattering patterns were recorded using a Pilatus 2M detector and silver behenate (which has a well-defined lattice parameter of 58.38 Å) was used to calibrate all X-ray scattering data. The temperature of the capillaries was maintained at 353 K through the use of a water recirculator fitted to the capillary holder, with the temperature determined by a thermocouple inserted into the capillary holder. The scattering images were integrated to give 1D scattering plots using custom software developed in National Instruments LabVIEW. The position and shape of the scattering peaks were analysed using the Origin data analysis package. Scattering profiles were fitted to three Pearson VII profiles corresponding to peak I, peak II and peak III (vide infra).Samples for X-ray scattering experiments were loaded into 1.5 mm thin-walled special glass X-ray capillary tubes . Scattering experiments were carried out on beamline I22 at Diamond Light source with an X-ray energy of 18 keV (wavelength of 0.69 Å) and a sample to detector distance of 1.2 m giving access to 22NaCl positron source sealed in Mylar was immersed in approximately 1 mL of IL. Lifetimes were collected at 298 K for a minimum of 24 h (5 files of 1 × 106 integrated counts) and analysed using LT v9 software.72PALS experiments were conducted on an EG&G Ortec spectrometer. A 30 μCi τ1 was fixed to 0.125 ns and attributed to p-Ps annihilation and τ2 was approximately 0.4 ns due to the annihilation of the free positron with electrons within the sample. τ3, the lifetime of o-Ps, was fit as a discrete lifetime. Previous analyses of IL samplesτ2 and τ3 with a distribution as opposed to discrete values to improve the fit statistics indicating a broad range of cavity sizes present within the ILs. However, for the ILs examined here, it was found that the reduced chi-squares fit was improved and the uncertainties were smaller when a discrete fit was performed, therefore this method has been used.The data were fitted to 3 lifetimes, where –1. The density meter was calibrated using degassed distilled water and the accuracy was intermittently monitored by the use of degassed distilled water as a standard. Prior to each measurement, ‘dry’ IL samples were further dried at 323 K for at least 16 h. All density measurements were obtained in triplicate to ensure reproducibility.Density measurements were performed in an Anton Paar ‘DMA 38’ vibrating tube density meter at 298 K with a stated accuracy of ±0.001 g mL–2 Torr) until equilibrium was obtained, and then subjected to several freeze–pump–thaw cycles. At least 10 cycles were necessary to completely remove dissolved gas. The IL was then subjected to dynamic vacuum overnight to remove any residual gas traces.IL (300 μL) was transferred into a Wilmad NMR tube . The tube was immediately connected to a Schlenk manifold using a glass tube connection, with the seal maintained by an O-ring. The experimental setup is depicted in the ESI Fig. S3–S5.3), enabling the determination of the quantity of Xe present. The NMR tube was then opened and immersed in liquid nitrogen leading to the deposition of solid Xe in the NMR tube. The NMR tube was then flame-sealed, taking care to ensure that the flame does not cause the sublimation of Xe, burn the IL or damage the glass tube. The sample was then carefully removed from liquid nitrogen and allowed to warm to room temperature.Xe gas (approximately 170 Torr) was inserted into the manifold, with the pressure determined using an inline pressure gauge. The volume of this section of the manifold was accurately known  and equipped with a 5 mm broadband inverse probehead. Typical settings: 1024 scans, 50 s relaxation delay, 710 ppm spectral width. 129Xe spectra were referenced utilising the approach of Lim and King,129Xe chemical shifts were referenced by setting the value of 129Xe in benzene to 188.1 ppm.1H signals of methanol as a standard.The 4C1im]+ cation. These are [C4C1im]Clx[NTf2]x1–, [C4C1im]Clx[OTf]x1–, [C4C1im]Clx[SCN]x1–, [C4C1im][OTf]x[NTf2]x1– and [C4C1im][Me2PO4]x[NTf2]x1–. These IL mixtures were chosen as we have previously studied their structures using computational and NMR techniques.14SAXS has been extensively used as a technique to characterise the spatial arrangement and order of ILs.S value (peak I) is assigned to the presence of an intermediate range ordering, typically the formation of polar and non-polar domains.74 This correlation length is based on the inter-ion distance in the direction of the alkyl chain for ILs with ions that possess a sufficiently long alkyl chain.i.e. cation–cation and anion–anion, within the polar domains. The final feature (peak III) is a combination of a number of different contributions including the distance between oppositely charged ions (i.e. cation–anion) as well as intramolecular correlations.In general there have been three primary features observed in the SAXS patterns of ILs . The fead) was determined using the relationship d = 1/S. Given the greater physical meaning of d relative to S, trends will be examined in terms of their effect on d for the subsequent discussion. For the mixtures which did not contain [C4C1im]Cl, i.e. [C4C1im][OTf]x[NTf2]x1– and [C4C1im][Me2PO4]x[NTf2]x1–, SAXS patterns were also obtained at 298 K as all compositions of these mixtures were liquid at this temperature. While the absolute values of the correlation distances obtained at 25 °C varied from those at 353 K, identical trends were observed, as detailed in the ESI  < [C4C1im][Me2PO4] (12.8 Å) ≈ [C4C1im][OTf] (13.1 Å) < [C4C1im][SCN] (14.1 Å) < [C4C1im]Cl (15.3 Å). These values increase with decreasing anion size, a result which has been found previously for amphiphilic imidazolium ILs bearing the [NTf2]–, [PF6]–, [BF4]– and Cl– anions.2]– may lead to coiling of the alkyl chain to maximise dispersive interactions with the anion which in turn would reduce the observed correlation length by enabling the closer approach of ions along the alkyl chain axis. In contrast to the peak position, the relative intensity of peak I across the simple ILs increases with the basicity rather than size of the anion. This supports the rationalisation that the absence of a clear peak for [C4C1im][NTf2] is due to the weakly interacting nature of the anion.For the simple ILs, the correlation length corresponding to peak I increases in the order: [C4C1im]Cl and [C4C1im][SCN] compared to the more intense peaks observed for [C4C1im][Me2PO4], [C4C1im][OTf] and [C4C1im][NTf2]. While it could be envisaged that this trend could be due to variation of the X-ray scattering cross sections of the anions, this is not borne out by close consideration of their respective cross-sections  < [C4C1im]Cl (6.6 Å) < [C4C1im][OTf] (6.8 Å) ≈ [C4C1im][Me2PO4] (6.8 Å) < [C4C1im][NTf2] (7.5 Å). The reproducibility of the peak position for peak II was found to be approximately ±0.1 Å. While the general trend is approximately in order of anion size, the anion–anion correlation length obtained for [C4C1im][SCN] is smaller than that of [C4C1im]Cl despite the greater size of the [SCN]– anion. Furthermore, the magnitude of the correlation length does not vary proportionally with the size of the anion, for example, there is a considerable size difference between the [Me2PO4]– and Cl– anions despite the similar correlation distances of these ILs. This trend implies that these values are indicative of a more subtle interplay between the IL conformation and anion size. DFT calculations have identified that more basic anions exhibit a stronger preference for in-plane interactions with the imidazolium ring due to hydrogen bonding, which in turn favours the π+–π+ interaction of the imidazolium rings. In contrast, larger, more diffuse anions were found to preferentially occupy positions above the plane of the ring due to the more favourable anion–π+ interactions.+–π+ stacking, which would account for the larger correlation distance for [C4C1im]Cl relative to [C4C1im][SCN]. For larger anions such as [OTf]– and [Me2PO4]–, the effect of π+–π+ interactions on anion–anion distances would be reduced as the anions are now of comparable size or larger than the imidazolium ring. Hence, the like charge correlation distance is a factor of both the anion size and the predominant solution conformation arising from the geometry and basicity of the IL anion.The position of peak II shifts subtly between each IL with the correlation length increasing in the order: [C4C1im]Cl (3.84 Å) < [C4C1im][SCN] (4.07 Å) < [C4C1im][Me2PO4] (4.47 Å) < [C4C1im][OTf] (4.56 Å) < [C4C1im][NTf2] (4.66 Å). The reproducibility of these values was found to be approximately ±0.02 Å. The correlation distance of peak III increases with increasing anion size with the exception of the [Me2PO4]– and [OTf]– anions for which this order is reversed. Given the complexity of the correlations that result in peak III it is difficult to assign this trend to a single underlying phenomenon. However, the smaller correlation length for [C4C1im][Me2PO4] relative to [C4C1im][OTf] and the relatively large difference between the values for [C4C1im]Cl and [C4C1im][SCN] suggests that more basic anions (such as [Me2PO4]– and Cl–) yield relatively shorter correlation distances for peak III than would be anticipated by their size. This is consistent with the major contribution to this peak being cation–anion alternation,Peak III is well-defined for all of the ILs examined with the correlation distance increasing across the set of simple ILs in the order: [C– or [NTf2]–) leads to a decrease in the correlation distance for peak I below that of either of the simple ILs and considerably below that which would be expected from a linear relationship between these ILs. This trend could not be clearly identified for the [C4C1im]Clx[SCN]x1– or [C4C1im][Me2PO4]x[NTf2]x1– mixtures as the correlation distances for most of the compositions of these mixtures were within experimental error.Examining the trends within the mixtures , it is e4C1im]Clx[NTf2]x1– mixtures exhibiting the greatest decrease with smaller decreases for the other mixtures. These results indicate that the difference in ion size is the major contributing factor to this phenomenon, with similar ion sizes leading to a small or undiscernible effect (e.g. [C4C1im]Clx[SCN]x1– and [C4C1im][Me2PO4]x[NTf2]x1–) with a large effect observed in mixtures containing ions of drastically different sizes (e.g. [C4C1im]Clx[NTf2]x1–).The magnitude of the decrease in correlation distance varies markedly across the mixtures where it is observed, with the Clx[NTf2]x1–. These observations imply that dispersive interactions between the alkyl chain and the less basic anion positioned above the imidazolium ring are favourable. Dispersive interactions would encourage the alkyl chain to coil towards the less basic anion positioned above the imidazolium ring, reducing the volume of the alkyl chain excluded from the polar region and hence greatly reduce the observed correlation distance for peak I. This effect would be strongest where both the size and basicity of the anions differed greatly, as is observed for [C4C1im]Clx[NTf2]x1–. For mixtures containing relatively large anions such as [C4C1im][Me2PO4]x[NTf2]x1–, the steric bulk of the more basic [Me2PO4]– anion would reduce the conformational flexibility of the alkyl chain, minimising the magnitude of the decrease in correlation distance along the alkyl chain axis.The trends depicted in 4C1im][Me2PO4]x[NTf2]x1– and [C4C1im][OTf]x[NTf2]x1–), the peak II correlation distances exhibit an almost linear relationship with x. There are subtle variations from linearity with [C4C1im][OTf]x[NTf2]x1– mixtures displaying slightly increased correlation distances relative to a linear fit between the simple ILs whereas the [C4C1im][Me2PO4]x[NTf2]x1– mixtures show slightly lower distances. The subtle contraction of the structure of the [C4C1im][Me2PO4]x[NTf2]x1– mixtures compared to the [C4C1im][OTf]x[NTf2]x1– mixtures can be rationalised in terms of the π+–π+ stacking of the imidazolium cations which has been found to be promoted in the former but not the latter case.14The correlation distances for peak II for all of the IL mixtures studied are depicted in – anion display more complex behaviour than those containing only larger anions. Both the [C4C1im]Clx[NTf2]x1– and [C4C1im]Clx[OTf]x1– mixtures exhibit no real change in correlation distance with increasing Cl– content until x = 0.50 whereupon it increases with increasing Cl– content before a dramatic reduction in the correlation distance for x = 0.89 (Clx[NTf2]x1–) or x = 0.83 (Clx[OTf]x1–). Such behaviour was not observed in MD simulations of the [C4C1im]Clx[OTf]x1– mixtures, which showed only very subtle monotonic changes in anion–anion distances with composition , is the longest positronium lifetime detected, corresponding to o-Ps ‘pick-off’ annihilation; R (Å) is the radius of the hole; and R0 = R + ΔR where ΔR = 1.66 Å and is an empirically determined value that refers to the thickness of an electron layer at the boundary of the infinite potential well within which the o-Ps is confined.The analysis of the PALS data was conducted by calculating the average radius of holes using the Tao–Eldrup equation (eqn (2)).vh), obtained from the PALS analysis by using the assumption of the presence of spherical voids, are depicted in τ3 and the fitted intensity (I3). An important consideration for the analysis of PALS data is the so-called ‘knee’ temperature (Tk), above which the rate of structural reorientation of the liquid is faster than the o-Ps annihilation rate, such that the volume obtained is constant and no longer reflects the changes in free volume structure of the liquid due to increasing temperature.50Tk has been determined for an array of [C4C1im]+ ILs.4C1im]Cl, Tk values obtained for the [C4C1im]+ ILs were in the range 270–285 K and no decrease in the hole volume (vh) due to solvent reorganisation effects was observed for these ILs at temperatures below 300 K. As the vh values here were measured at 298 K which lies within this stable region (above the Tk and below 300 K), no significant temperature dependence of vh would be expected. Measurements in this window of stability ensures both that the vh values observed are within the stable region across all of the IL mixtures and are therefore comparable and that these values can be compared with 129Xe NMR as they are reflective of the free volume of the liquid on the PALS timescale rather than being artificially reduced from the rapid reorganisation of the solvent.The hole volumes (vh obtained for [C4C1im][OTf] and [C4C1im][NTf2] (224 Å3 and 250 Å3 respectively) are in agreement with those reported previously.vh for [C4C1im][Me2PO4] (162 Å3) which has not been determined previously is substantially smaller than for the other simple ILs examined here and is closest to that obtained at Tk for [C4C1im][BF4] of the imidazolium ILs that have been previously studied. The [Me2PO4]– anion is larger than both the [BF4]– and [OTf]– anions despite the low vh value determined .vh to the occupied volume has been related to macroscopic properties such as viscosity and conductivity whereby the greater this ratio, the lower the viscosity and greater the conductivity of the IL as would be expected by virtue of the relatively larger free volume available for relatively smaller entities to diffuse through. Consequently, the small value of vh obtained for [C4C1im][Me2PO4] despite its larger occupied volume compared to [C4C1im][OTf] suggests it will be more viscous and less conductive, which is consistent with the experimental observations of these properties.2PO4]– and [OTf]– ions despite the larger size of the [Me2PO4]– indicates that the viscosity of these fluids cannot be accounted for by hard-sphere models, as has been proposed for ILs containing more diffuse anions.4C1im][Me2PO4] must arrive from the strength of interaction between ions. For example, the anisotropy of the charge distribution over the O atoms of the [Me2PO4]– anion may induce a stronger cation–anion interaction, reducing the free volume. This is supported by consideration of this result in light of the small vh values that have been obtained previously for [C4C1im]Cl, which suggests that more basic anions reduce the free volume of the liquid which correspondingly leads to their increased viscosity.The values of vh does not vary linearly with anion composition and both mixtures display free volumes which are larger than would be anticipated from linear interpolation of the simple ILs. vh’, (vEh) i.e. the difference between vh and the vh predicted by a linear relationship between the simple ILs.From 4C1im][Me2PO4]x[NTf2]x1– mixtures is far greater than for [C4C1im][OTf]x[NTf2]x1–. This trend is qualitatively the same as that which had been determined previously for the excess volume of mixing for these mixtures.VE) of these mixtures and vEh exists, the density of the mixtures that had been analysed with PALS was measured  increase in free volume rather than any changes to the occupied volume. As exemplified by the relatively small 129Xe NMR spectra of the same [C4C1im][Me2PO4]x[NTf2]x1– and [C4C1im][OTf]x[NTf2]x1– mixtures that were analysed by PALS are depicted in δ = 0.00 ppm). This referencing procedure is valid as similar Xe pressures were used for each IL or IL mixture  of each of the mixtures was calculated, where: δE = δ – x1δ1 – x2δ2; x1,2 refers to the mole fraction of IL 1 and IL 2 respectively; and δ1,2 is the solvated Xe chemical shift in IL 1 and 2. These values are depicted in vEh  that give rise to their observed physical properties.Moreover, the Supplementary informationClick here for additional data file.


Surface water samples collected from the East China Sea (ECS) in August 2013 were analyzed for 129I, 127I and their inorganic chemical species in the first time. The measured 129I/127I ratio is 1–3 orders of magnitude higher than the pre-nuclear level, indicating its dominantly anthropogenic sources. Relatively high 129I levels were observed in the Yangtze River and its estuary, as well as in the southern Yellow Sea, and 129I level in seawater declines towards the ECS shelf. In the open sea, 129I and 127I in surface water exists mainly as iodate, while in Yangtze River estuary and some locations, iodide is dominated. The results indicate that the Fukushima nuclear accident has no detectable effects in the ECS until August 2013. The obtained results are used for investigation of interaction of various water masses and water circulation in the ECS, as well as the marine environment in this region. Meanwhile this work provides essential data for evaluation of the possible influence of the increasing NPPs along the coast of the ECS in the future.Anthropogenic  The Fukushima nuclear accident happened in March 2011 has released a large amount of radioactive substance to the atmosphere and seas. The measurement of 129I in the Fukushima offshore seawater has shown a significant Fukushima derived 129I signals9129I, as a fission product, can be used to monitor and evaluate the impact of the nuclear facilities; this requires a baseline value of 129I. However, no such 129I baseline in seawater along the coastal region of China is available.129I 1.7 Ma is −) and iodate (IO3−) with minor organic iodine in seawater. Iodate is thermodynamically stable and predominant in the open sea113− to I− in seawater is often related to biological activity such as phytoplankton129I can provide critical information for understanding the marine environment and process.Iodine is one of redox sensitive elements; it occurs mainly as iodide  was observed in the open sea area, which is 8–18 times higher than the lowest values (3.1–7.1 μg/L) in the Yangtze River mouth  were observed the Yangtze River estuary  and the offshore seawater . Further increased concentrations of 127I ranging in 45.1–51.8 μg/L occurred in the southern ECS . In the middle and southeast of ECS , the highest concentrations of 127I (50.15–54.74 μg/L) were measured  were observed in the Yangtze River mouth (stations 52–54) and the Yangtze River estuary , as well as in the southern Yellow Sea (stations 43–46). The 129I concentrations decrease gradually from the Yangtze River estuary towards east and southeast to the ECS shelf. Low 129I concentrations (0.8–1.1) × 107 atoms/L were measured in the border of the YS and ECS (stations 47–49). In addition, 129I concentrations ((0.7–1.2) × 107 atoms/L) in the eastern ECS  and coastal area in the southern ECS  are also low. While, intermediate level ((1.0–1.5) × 107 atoms/L) occurred in the middle and southern ECS  (129I/127I atomic ratios vary significantly from 3.2 × 10−11 to 1.2 × 10−9 (129I concentrations. The highest 129I/127I atomic ratios of (90–120) × 10−11 occurred in the Yangtze River mouth (stations 52–54), which are more than 10 times higher than those in the ECS (<10 × 10−11). The lowest 129I/127I ratios ((3.1–6.4) × 10−11) were observed in the eastern and southern ECS . While, relative high 129I/127I ratios were measured in the southern YS (stations 42–46) (8.9–13.3) × 10−11) and in the Yangtze River estuary  (7.2–13.3) × 10−11).129I and 127I in iodide (I−) and iodate (IO3−) species  and station 4 with 127I−/127IO3− ratios of 1.3–28.9, iodide/iodate molar ratios for both 129I and 127I were less than 1 in open sea water (129I−/129IO3− ratios (12.0–20.2) were observed in waters at the Yangtze River mouth (stations 53 and 54). While a considerable decrease of 129I−/129IO3− ratio (1.0–1.6) were measured in the Yangtze River estuary towards to the ECS at sampling stations 1 and 51. Low 129I−/129IO3− molar ratios was observed in waters from the south of the Hangzhou Bay and in the open sea waters further to 30°N in the center of the ECS (stations 16–31) and the southern ECS (stations 27–41).Some of surface seawater samples in the ECS were analyzed for  species . To bettstations . Except ea water . The hig129I concentrations ((0.73–3.99) × 107 atoms/L) and the 129I/127I atomic ratios ((3.1–120) × 10−11) in the surface water in the ECS is 1–3 orders of magnitude higher than the pre-nuclear value (1.5 × 10−12 for 129I/127I ratio and 0.043 × 107 atoms/L for 129I concentration)129I in the surface seawater in the ECS. The highest 129I level ((1.2–4.0) × 107 atoms/L for 129I concentrations and (10.1–119) × 10−11 for 129I/127I atomic ratios) was observed in Yangtze River mouth and a relative high 129I levels of (1.0–2.5) × 107 atoms/L for 129I concentrations and (7.0–12) × 10−11 for 129I/127I ratios were measured in the southern Yellow Sea, and the 129I levels decline towards to the ECS shelf, the lowest 129I levels ((3.1–6.4) × 10−11 for 129I/127I ratios) in the investigated area were measured in the ECS shelf and southern ECS.129I level in surface water in the ECS is more than one order of magnitude lower than that in contaminated seawater. In the North Sea, Baltic Sea, Norwegian Sea and Arctic, 129I/127I ratios of 10−8–10−6 were reported because of huge amount of 129I discharged from the European nuclear fuel reprocessing plants (NFRPs)at Sellafield (UK) and La Hague (France) has dispersed with sea current to these area4171819129I/127I ratios reached to 2.2 × 10−9 (6.3 × 108 atoms/L for 129I concentration) due to the releases from the Fukushima accidentThe measured 129I level in the ECS is comparable to those observed in adjacent sea around Japan before Fukushima accident, where 129I concentrations of (1.2–2.1) × 107 atoms/L (with 129I/127I ratios of 4.64–7.2 × 10−11) were report129I concentrations of (4.0–12.7) × 107 atoms/L and 129I/127I ratios of (2–57) × 10−10)129I level in the North Atlantic results from the leakage and southwards dispersion of the high reprocessing derived 129I water in the English Channel. In addition, the 129I in the ECS is also comparable to those observed in the Indian Ocean (40–50°S), where the 129I concentrations were (0.60–1.52) × 107 atoms/L  × 107 atoms/L for 129I concentrations and (0.28–2.9) × 10−11 for 129I/127I ratios)  2324. Thi129I in the ECS might originate from 1) releases from nuclear facilities in coastal region of the ECS; 2) fallout of the atmospheric NWTs in 1945–1980; 3) releases from NFRPs and long distance transport; 4) nuclear accidents (e.g. Chernobyl and Fukushima accidents).The dominantly anthropogenic 129I has been produced during the operation of the nuclear power plants (NPPs)129I in the seawater surround a Chinese NPP in the coastal region of the ECS has confirmed no excessive 129I in the seawater in 10 km range of this NPP129I in the ECS is negligible.Although huge amount of 129I has been released to the atmosphere from the atmospheric NWTs in 1945–1980129I signal in 1972–1976, which was attributed to the contribution of Chinese NWTs in this period129I signal of Chinese NWTs in the corresponding layer of this sediment core and these tests were implemented more than 30 years ago, the direct contribution of the Chinese NWTs to the 129I in the ECS, especially the present surface seawater, should be insignificant. However, the global fallout of 129I from all atmospheric NWTs might be one of 129I sources in the ECS because of the large releases. A relative high 129I signal of the global fallout peaked in 1962–1963 has been observed in the sediment core collected in the Jiaozhou Bay129I in the ECS in early time of atmospheric NWTs, the leaching of 129I from the earth surface by rainwater and subsequent transport to the ECS through rivers might be one of important source and pathway of 129I in the ECS.It has been estimated that 50–150 kg of 129I at the PPG can be also transported to the ECS in early time of the NWTs at PPG. The 129I in the sediment core collected in Jiaozhou Bay in the YS has recorded a small but obvious PPG derived signal of 129I in 1957129I peak occurred in 1957–1958 in coral samples collected in the coastal region in Philippine has also been reported129I might have a contribution to 129I in the ECS. However, the distribution of 129I in the ECS shows the lowest 129I level in the east edge of the ECS, i.e. the KC belt, indicating no significant amount of 129I in the ECS was transported from the PPG through the KC. This might be attributed that the NWTs at PPG has terminated in 1962, and transit time from PPG to the ECS is less than 2 years, the high water soluble 129I released from these NWTs did not significantly deposited and remained in the sediment in the PPG area, instead it was quickly diluted and dispersed to a large area with sea currents. Therefore, unlike plutonium, the contribution of the PPG to the 129I in the present surface water in the ECS is insignificant.105 NWTs have been conducted in the Pacific Proving Grounds (PPG) in the Marshall Islands (11°N) in 1946–1962, where is about 5000 km southeast of the ECS. Besides those injected to the stratosphere and contributed to the global fallout, large amount of radioactive substances from these NWTs were deposited in the southern Pacific Ocean surrounding the Marshall Islands . A number of investigations have reported that the close-in fallout of plutonium in the PPG has being carried by the north equatorial current, which is followed by the Kuroshio Current (KC) and continuously transported to the South China Sea and ECS2828129I in the environment123129I level observed in the precipitation collected in 1979–2003 in Ishigaki-shima 129I level in the ECS. The NFRP in northwest China is far away from the ECS (>5000 km) and the catchment of the rivers (>2000 km) flowing into the ECS. 129I in the sediment core collected in the YS and surface soil collected in southern China did not show any contribution of Chinese NFRP. Therefore, there is no significant contribution of Chinese NFRP to 129I in the ECS.Releases from the NFRPs are so far the largest source of 129I to the seas, which was dispersed to a large area in northern North Atlantic Ocean by sea currents129I still remains at a high level of about 250 kg/y. Re-emission of the reprocessing derived 129I from the contaminated seawater to the atmosphere has become a major source of 129I in the air in recent years129I record in the sediment core from the YS has demonstrated that the atmospheric releases of the European NFRPs and re-emission of the reprocessing derived 129I in the sea is a major source of 129I in the present sediment in the YC129I in the Japan Sea was also attributed to the European NFRPs129I in the ECS besides the global fallout of the NWTs.Besides atmospheric releases, the two NFRPs at Sellafield (UK) and La Hague (France) have discharged more than 6500 kg of 129I level observed in Yangtze River mouth compared to the stations in the ECS indicates that riverine input might be an important source of 129I in the ECS. Yangtze River is the longest river (6300 km length) in China with a catchment area of 1,808,500 km2 located in southern China (25–34°N). The fallout of 129I on the soil of the catchment has being continuously leached by rain and entered to river water, causing a high 129I level in the Yangtze River. The high level 129I in the southern YS indicate a higher deposited and riverine input of 129I from the Yellow River. The YS and the catchment area of the Yellow River are located in north China (32.5–40°N), and therefore received higher fallout of 129I compared to the ECS, because most of NWT sites and NFRPs are located in the middle and higher latitude in the Hemisphere16129I levels in soil and seaweed in the north China and the YS is higher than that in the south China and the ECSThe significantly higher China 25–°N. The f2 catchment area and 688 km length), Min River  and Ou River . Considering total surface area of 1,940,000 km2, the riverine input from the terrestrial deposition is one of major contribution of 129I in the ECS. This is also supported by investigation on the distributions of another important fallout radionuclide 137Cs in the sediment in the Yangtze River estuary, which showed that the major part of 137Cs in the estuary originated from riverine transport and input36Besides the Yangtze River, there are more than 40 rivers flowing into the ECS, among them the relative big ones include Qiantang River  are relative low (30–33 μg/L). 129I absent ground seepage water input along the shore might occur here. A hypoxic condition observed at sampling station 48  might indicate a branch of the KC in the ECS in this region. The KC formed from the subtropical mode water of the central North Pacific129I  at about 31°N in the northern ECS.Low ific129I . The dis129I level was also observed along the south coast of the ECS at stations 24, 25 and 32. This might indicate that the high 129I level Yangtze River water does not move southerly along the coast. Considering 127I concentrations are also lower in this region and there is not big river flowing into the ECS in this region, it can be assumed that the groundwater seepage in this region might also occur.Low 127I mainly exists as iodate with iodide/iodate molar ratios of 0.04–0.60 in most sampling stations in the ECS, this is similar to those observed in the North Sea (0.11–0.48)4127I−/127IO3− molar ratio of 0.04 was observed at sampling station 41, close to the Mien Hwa Canyon (MHC) with a northwestwards KC intrusion branch. This might be attributed to the Kuroshio subsurface water (KSW) upwelling in this region44127I−/127IO3− and 129I−/129IO3− ratios (R2 = 0.007) was observed for all seawater samples investigated. Meanwhile 129I−/129IO3− ratios are normally higher than 127I−/127IO3− ratios. This is attributed to the different sources of 127I and 129I in these waters. 129I in the ECS mainly originates from the atmospheric fallout and riverine input, whereas 127I originates from the sea currents which flow into the ECS.129I and 127I originated from the rainwater leaching of soil in the catchment, the similar high iodide/iodate ratios of 12 for both 127I and 129I were measured at station 54. This indicates iodide is the dominant species of iodine isotopes. This is similar as that observed in the Elbe River in the North Sea127I−/127IO3− ratios of 0.6–1.5 were measured in the downstream water at stations 52 and 53. This might be attributed to the tidal action and sea water intrusion showed by an elevated salinity (0.5) at sampling station 52. The similar declined 129I−/129IO3− ratio (1.5) was measured at station 52, whereas a much higher value up to 20 at sampling station 53. Compared to 127I, 129I−/129IO3− ratios might reflect the water circumstance in this region, because the contribution from the seawater intrusion is minor. The high 129I−/129IO3− ratio at sampling station 53 indicates the reductive circumstance of the Yangtze River water remained at this station. The declined 129I−/129IO3− ratio at station 52 close to the Yangtze River estuary indicates the effluence of the seawater, and the reductive circumstance is changed, causing oxidation of 129I− to 129IO3−.In the Yangtze River mouth, both of 127I−/127IO3− ratios (7–29) were measured at sampling stations 1 and 51 in the ECS, which are even higher than that in the Yangtze River mouth. It has been reported that a red tide (phytoplankton blooming) occurred in the estuary of the Yangtze River in August 2013, and the measured concentrations of chlorophyll were 5.6–37.7 mg/m3 14High 127I and 2.9 for 129I), which are comparable or even higher than that in the Yangtze River mouth (stations 52). An oxygen depleted condition was measured in the water at sampling station 3, 4 and 48 during sampling campaign in August 2013. The dominant iodide specie in this region (sampling sate 3–4) might results from the anoxic condition in this region, which reduce iodate to iodide.High iodide/iodate ratios were also observed at sampling station 4  which was transported from the Yangtze River estuary northwards through the sampling station 6–9. This also indicates that the reduction of iodate in the anoxic region or biologically active region, especially phytoplankton blooming region, is a relative fast process.Since the total The ECS is the largest marginal sea in the northwestern Pacific, bounded by China, Japan, Ryukyu archipelago, and Korea . It recein situ and stored in 2.5 L polyethylene plastic containers in dark under normal laboratory conditions until analysis.Surface seawater samples (<2 m depth) were collected during the R/V “Dongfanghong 2” cruise in August 2013 using a built-in seawater sampling system in the laboratory of the vessel at 54 sampling stations in the ECS and its adjacent region . The temet al.127I− and 127IO3−. The detailed procedure is presented in the Supporting information.A modified procedure from Hou et al.129I. For separation of iodide, after addition of 0.20 mg of 127I− carrier and 200 Bq125I− tracer to 1200 ml seawater, NaHSO3 solution was added to final concentration of 0.3 mmol/L, and then HCl was added to adjust pH to 5.1, 50 mg Ag+ (as 0.10 mmol/L AgNO3) was slowly added to the sample under stirring to coprecipitate iodide as AgI-AgCl. For total inorganic iodine, 500 ml seawater was taken to a beaker, 0.20 mg of 127I− carrier, 200 Bq 125I− (NaI) and 2.0 mmol NaHSO3 solution were added. HCl was then added to adjust pH < 2, iodate was reduced to iodide in this condition. 150 mg Ag+ was slowly added to the solution under stirring to coprecipitate the formed iodide as AgI-AgCl. The precipitate was washing with 1–7.5% ammonium to obtain a final precipitate of 1–3 mg for AMS measurement of 129I.A modified co-precipitation method from Luo Procedure blanks for iodide and total inorganic iodine were prepared using the same procedure as for the samples. In this case, deionized water was used instead of the samples.127I was obtained.Iodine in the separated iodate and iodide fractions and original seawater was measured used ICP-MS  after 10 times dilution using 1% ammonium solution. The detection limit of 0.02 ng/mL for 129I/127I atomic ratios in the prepared targets were measured by AMS using 3MV Tandem AMS system (HVEE) in the Xi’an AMS center. 127I5+ was measured as charges (current) using a Faraday cup and 129I5+ was measured using a gas ionization detector. All samples were measured for 6 cycles and 5 min per sample in each cycle. The procedure background of 129I/127I ratio was measured to be 1.0 × 10−13. For the samples with 129I/127I ratio of 10−10, the measurement uncertainty is less than 1.75%129I has been reported elsewhereThe separated AgI–AgCl coprecipitate was dried in an oven at 60–70 °C, the dried precipitate was ground to fine powder and mixed with niobium powder  in a mass ratio of 1:5, which was finally pressed into copper holder. The How to cite this article: Liu, D. et al.129I and its species in the East China Sea: level, distribution, sources and tracing water masses exchange and movement. Sci. Rep.6, 36611; doi: 10.1038/srep36611 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Here, we show that spinosad resistance in the tomato leaf miner, Tuta absoluta is associated with aberrant regulation of splicing of Taα6 resulting in a novel form of insecticide resistance mediated by exon skipping. Sequencing of the α6 subunit cDNA from spinosad selected and unselected strains of T. absoluta revealed all Taα6 transcripts of the selected strain were devoid of exon 3, with comparison of genomic DNA and mRNA revealing this is a result of exon skipping. Exon skipping cosegregated with spinosad resistance in survival bioassays, and functional characterization of this alteration using modified human nAChR α7, a model of insect α6, demonstrated that exon 3 is essential for receptor function and hence spinosad sensitivity. DNA and RNA sequencing analyses suggested that exon skipping did not result from genetic alterations in intronic or exonic cis‐regulatory elements, but rather was associated with a single epigenetic modification downstream of exon 3a, and quantitative changes in the expression of trans‐acting proteins that have known roles in the regulation of alternative splicing. Our results demonstrate that the intrinsic capacity of the α6 gene to generate transcript diversity via alternative splicing can be readily exploited during the evolution of resistance and identifies exon skipping as a molecular alteration conferring insecticide resistance.Many genes increase coding capacity by alternate exon usage. The gene encoding the insect nicotinic acetylcholine receptor ( Alternative splicing enables a single gene to code for multiple proteins and is thus a key driver of proteome diversity. Despite its importance, the regulation of alternative splicing is complex and not fully understood.Drosophila melanogaster is theoretically capable of producing >30 000 different subunit variants  are neurotransmitter‐gated ion channels that play a vital role in nerve signalling at the postsynaptic membrane of both vertebrates and invertebrates. The nAChRs of insects are a subject of considerable fundamental interest, in part because they exhibit an extraordinary capacity to generate a diversity of mRNA products from single genes as a result of alternate exon splicing, exon exclusion or A‐to‐I pre‐mRNA editing. Indeed, the nAChR Dα6 of Tuta absoluta, the tomato leaf miner, is an economically important pest of tomatoes which, if not controlled, can cause up to 100% yield loss  were collected in 2010 from tomato fields in Spain (Murcia), Italy (Sicily) and Portugal (Lisboa), respectively. The strain GA is a mixed field population collected in 2008 from different areas of Brazil (São Paulo and Minas Gerais states) and reared since then without insecticide selection. The Spin‐parent population was collected from Cilha Queimada, Alcochete, Portugal, in 2012 after reported control failure using spinosad (Spin). The SpinSel population was selected by placing larvae (n > 100) on spinosad‐coated tomato leaves for 72 h and taking the survivors to the next generation. Insects were selected for 11 generations, the concentration of spinosad increasing with time to a final concentration of 120 mg/L. All strains were reared on tomato under conditions of 26 °C temperature and 16‐h light.Three populations of http://www.irac-online.org/content/uploads/Method_022_Tuta.pdf). For synergist bioassays, larvae were exposed to piperonyl butoxide (PBO), diluted with acetone to a concentration of 75 mg/L, for 2 h prior to leaf‐dip bioassay. A range of PBO doses were trialled in preliminary experiments and this was the highest PBO dose which did not cause control mortality. Probit analysis and the calculation of LC50 values were performed with GenStat 15th Edition  was used to clone PCR products as required.Total RNA was extracted from three pools of 10–20 larvae using the ISOLATE II RNA Mini Kit (Bioline) and reverse‐transcribed to cDNA using Superscript III reverse transcriptase (Invitrogen). DreamTaq green (Thermo Fisher Scientific) was used in nested PCR to amplify cDNA using the primers listed in Table S7 (Supporting information). Rapid amplification of cDNA ends (RACE) using the SMARTerTM RACE cDNA Amplification kit (Clontech Laboratories) was used to obtain the 5′ end of Taα6 gene encompassing exon 3 was PCR‐amplified as above and PCR products were direct sequenced using the primers listed in Table S7 (Supporting information). The significance of the association between exon skipping and survival to the discriminating dose of spinosad was examined using Fisher's exact test and quantified by calculating the odds ratio.To examine the association of exon skipping and spinosad resistance, 50 male pupae of the SpinSel strain were collected and placed in a cage containing a tomato plant with 50 female pupae of the Spin strain. Reciprocal crosses were set up in a second cage in the same way. F1 adults were then pooled and allowed to randomly mate to generate F2 larvae. At the L2 stage, approximately eight larvae were placed on a single tomato leaf treated (dipped) with 120 mg/L spinosad, with a total of 15 replicates carried out. Controls (four replicates) comprised L2 larvae placed on single leaves dipped in diluent minus spinosad. Mortality was scored at 72 h as above. Larvae that survived spinosad exposure and control (those exposed to diluent minus spinosad) larvae were snap‐frozen in liquid nitrogen and RNA extracted and cDNA synthesized as above. To genotype individual larvae, the region of the ® Plant Mini Kit (Qiagen). The Universal GenomeWalker™ 2.0 kit (Clontech Laboratories) was used to obtain sequence of the large intron downstream of exon 3a and long PCR mix (Thermo Fisher Scientific) was used to amplify genomic DNA. After this initial characterization, a 4000‐bp region encompassing and flanking exons 3a and 3b was sequence characterized from the Spin, SpinSel and TA4 strains using the primers listed in Table S7 (Supporting information). PCR products were direct sequenced in the first instance and cloned and sequenced using the StrataClone PCR cloning kit (Agilent Genomics) if direct sequencing failed to provide clean sequence data.Genomic DNA was extracted from pools of 10–20 larvae using the DNeasyet al. hα7 flanking exon 3 using the QuikChange II XL site‐directed mutagenesis kit (Agilent Technologies). Exon 3 was excised and plasmid DNA treated with Mung bean nuclease (New England Biolabs) to remove the 5′ and 3′ overhangs prior to religation using T4 ligase. The absence of exon 3 and the preservation of the reading frame were verified by nucleotide sequencing (Eurofins Genomics).Functional expression of insect only nAChRs in heterologous systems has proven technically difficult. In contrast, the vertebrate nAChR α7 readily expresses as a functional receptor in vitro and so human nAChR α7 in pSP64GL (Broadbent Xenopus sp. oocytes (stages V–VI) purchased from the European Xenopus Resource Centre, University of Portsmouth, were treated with 2 mg/mL collagenase (Sigma‐Aldrich) for 30 min in calcium‐free Barth's solution  followed by three rinses with clean Barth's solution. Following manual defolliculation, the oocytes were injected with cRNA (25 ng/oocyte) using a Drummond variable volume microinjector. The oocytes were incubated at 18 °C in calcium‐containing (77 mm) Barth's solution with 4 μg/mL kanamycin, 50 μg/mL tetracycline, 100 U/mL penicillin and 100 μg/mL streptomycin. Two‐electrode voltage‐clamp electrophysiology recordings were performed 3–5 days after injection when the oocytes, continuously perfused with frog Ringer's solution , were challenged with various concentrations of ligands freshly prepared in frog Ringer's solution. The current required to maintain the membrane potential at −60 mV following drug application was measured.Capped RNA was synthesized using the mMessage mMachine SP6 RNA transcription kit (Life Technologies) following the manufacturer's protocol. T. absoluta, RNA was extracted from eggs, larvae, pupae and adults of the TA1 strain using the Bioline Isolate II RNA mini kit . RNA was pooled and sent to Eurofins Genomics, Germany, for preparation of a random‐primed normalized cDNA library (with an insert size of 150–450 bp) to ensure the detection of transcripts expressed at even low levels. This was sequenced to high coverage on a single lane of an Illumina HiSeq 2000 using 100‐bp paired‐end sequencing. To identify changes in gene expression between the Spin and SpinSel strains, RNA was extracted from three replicates of 10 larvae from each as above, sent to The Genome Analysis CentreTM (TGAC) and multiplexed for sequencing on one lane of an Illumina HiSeq 2000 using 100‐bp paired‐end sequencing. FastQC was used to check the quality of the raw reads obtained  and deseq2 (version 1.12.4)  between SpinSel and TA4 are also differentially methylated (and in the same direction) between SpinSel and Spin.Genomic DNA was extracted from the Spin, SpinSel and TA4 strains as detailed above and sent to Zymo Research for bisulphite conversion, PCR and sequencing. DNA was bisulphite converted using the EZ DNA Methylation‐LightningTM Kit (Zymo Research) according to the manufacturer's instructions. A total of 30 amplicons in the case of the SpinSel and TA4 strains and 26 amplicons in the case of the Spin strain, encompassing a 4000‐bp region flanking exons 3a and 3b, were PCR‐amplified using primers listed in Table S8 (Supporting information). Products were barcoded for sequencing on an Illumina MiSeq with >350 000 read pairs obtained for each strain. Reads were aligned to the reference and methylation called at CpG sites using Bismark  compared to the most susceptible laboratory strain, ‘TA3’ . PBO was selected as an inhibitor of cytochrome P450s and esterases as previous biochemical characterization of spinosad resistance in T. absoluta using next‐generation sequencing of a normalized cDNA library prepared from the TA1 strain (susceptible to spinosad) to identify initial nAChR α6 transcripts, followed by PCR and rapid amplification of cDNA ends (RACE) to obtain full‐length cDNA sequences. The complete coding mRNA sequence of the Taα6 subunit obtained comprises 1530 bp encoding 510 amino acids and shows high levels of sequence identity with other lepidopteran nAChR α6 gene sequences . As reported previously for other insects, Taα6 transcripts were identified containing either of the two mutually exclusive exons 3a and 3b and one of three mutually exclusive exons of exon 8 .The spinosad target site, the nAChR α6 subunit, was identified from Taα6 transcripts from cDNA prepared from three replicate pools of Spin and SpinSel larvae revealed a deletion of 45 bp encoding 15 amino acids (at positions 65–80) in all transcripts obtained from SpinSel. This deletion precisely corresponds to the position and size of exon 3a/3b . In the Spin strain, 50% of clones sequenced represented transcripts with exon 3a, 33% incorporated exon 3b and 17% lacked both exons 3a and 3b, indicating that Taα6 transcripts modified in this way were almost certainly present in the parental strain at low frequency prior to selection. In contrast, 100% of the clones sequenced from SpinSel lacked exon 3a/3b. A point mutation causing an amino acid substitution (G275E) in exon 9 of Taα6 has been recently associated with spinosad resistance in populations of T. absoluta in Brazil , indicating that the loss of these exons in mRNA results from exon skipping rather than a deletion of the exons in genomic DNA. Thus, in the Spin strain, most mRNA transcripts contain one or other of the two mutually exclusive exons 3a or 3b depending on which of the two alternate 3′ splice sites upstream of each exon is recognized. In the case of SpinSel transcripts, both exons 3a and 3b are skipped with only the 5′ splice site of the intron downstream of exon 2 and the 3′ splice site in the intron upstream of exon 4 recognized . The functional significance, if any, of these findings is not clear. It has been suggested that truncated nAChR subunits may act as an ‘acetylcholine sponge’ in a manner similar to that of the molluscan ACh‐binding protein  association between exon 3 skipping and survival to spinosad exposure supporting a causal role in resistance.To further examine the association between exon skipping and spinosad resistance, the Spin and SpinSel strains were reciprocally crossed and F1 progeny pooled and allowed to mate randomly to generate a hybrid F2 population containing all possible phenotypes/genotypes. A subset of this population was then exposed to a discriminating dose of spinosad for 72 h, which, based on the recessive inheritance of loss‐of‐function mutations in the nAChR α6 gene of other insects  ruling out the possibility of a mutation in an exonic splicing regulator binding site, such as an exon splicing enhancer or silencer (ESE/ESS), which promote exon inclusion or exclusion. The Splicing Regulation Online Graphical Engine (SROOGLE) was used to identify splice/branch sites and polypyrimidine tracts flanking exons 3a and 3b . The TA4 strain is susceptible to spinosad  which exhibited a methylation ratio of 30% in SpinSel vs. 0% and 0.5% in TA4 and Spin, respectively. This CpG site is in intronic sequence 21 bp downstream of exon 3a  only contained reads from Spin and SpinSel, while the second (assembly 2) combined these reads with those of another strain TA1 which was sequenced previously (see Methods). Assembly statistics are shown in Table S9 (Supporting information). A total of 289 transcripts, that were assigned a functional description by BLAST (see Methods), were identified as significantly differentially expressed (76 overexpressed and 213 underexpressed in SpinSel when compared to Spin) using the Spin/SpinSel assembly as a reference, and a total of 254 annotated transcripts identified as differentially expressed (68 overexpressed and 186 underexpressed) using the Spin/SpinSel/TA1 assembly as a reference .Alternative exons like exon 3a/b in et al. et al. et al. et al. et al. Interrogation of these gene lists identified a number of transcripts encoding potential splice factor proteins including suppressor of white apricot (SWAP), RNA‐binding protein 1 (RBP1), gem‐associated protein 5 (Gemin5) and u11 u12 small nuclear ribonucleoprotein 48 kDa Table . Importaet al. et al. et al. To validate the results of RNAseq analyses, the expression of the candidate genes identified was examined in Spin and SpinSel by qPCR. Surprisingly, only Gemin5 was confirmed as significantly downregulated in the SpinSel strain Fig. . FurtherBeyond splice factors, transcriptome profiling of the Spin and SpinSel strains revealed very few genes overexpressed in the SpinSel strain belonging to the major superfamilies of enzymes frequently implicated in metabolic resistance in insects. For example, no transcripts encoding cytochrome P450s (P450s), carboxylesterases (CEs) or UDP‐glucuronosyltransferase (UGTs) were identified as overexpressed in reads mapped to either assembly 1 or 2 . A single transcript encoding a glutathione S‐transferase (GST) (comp155108_c1_seq10) was identified as upregulated (17‐fold by DESeq2) in the SpinSel strain in reads mapped to assembly 2 . However, this transcript encodes a conserved GST enzyme belonging to the zeta class that corresponds to GST zeta 1 (GSTZ1), an enzyme catalysing maleylacetoacetate to fumarylacetoacetate in the penultimate step of the phenylalanine/tyrosine degradation pathway  or from quantitative or qualitative changes in trans‐acting proteins such as splicing factors. In addition, a growing body of work has provided evidence of the importance of epigenetic mechanisms such as changes in DNA methylation in the regulation of alternative splicing. This study provides evidence of a role for the latter processes with exon skipping associated with a change in the level of methylation downstream of exon 3a and the expression of at least one important splice factor. Further detailed exploration of the relative contribution of these mechanisms in exon skipping and insecticide resistance is now required. In this regard, recent advances in (epi)genome editing using the CRISPR/Cas9 system has made it possible to both modify candidate gene expression and carry out epigenetic editing at specific loci .Raw Sequence Data obtained in this study has been deposited in the National Center for Biotechnology Information Sequence Read Archive under the following Accession nos.: Fig. S1 (A) cDNA and predicted protein sequence of the Tuta absoluta nAChR α6 subunit (with exon 3a and 8a). (B) Alternative exons 3a and 3b and exons 8a, 8b and 8c of the Tuta absoluta nAChR α6 subunit.Click here for additional data file.Fig. S2 Comparison of the amino acid sequence of the Tuta absoluta α6 subunit with that of Bombyx mori (GenBank ABL67934.1) and Plutella xylostella (GenBank GU207835.1).Click here for additional data file.Fig. S3 Alignment of a 4000 bp genomic sequence encompassing the Taα6 exon 3 cluster from the Spin, SpinSel and TA4 strains.Click here for additional data file.Fig. S4 Amino acid alignment of the exon 2–4 region of Taα6 from 21 sequenced clones generated from four different Tuta absoluta spinosad susceptible strains .Click here for additional data file.Fig. S5 Alignment of contigs encoding suppressor of white apricot from two de novo Tuta absoluta transcriptome assemblies.Click here for additional data file.Table S1 Exon three usage in sequenced clones of Taα6 in the Spin and SpinSel strains.Click here for additional data file.Table S2 Larval mortality of F2 hybrids of the Spin and SpinSel strains to a discriminating dose of spinosad (120 mg/L).Click here for additional data file.Table S3 Table of CpG sites and corresponding methylation ratios in the exon 3 cluster for the SpinSel, Spin and TA4 Tuta absoluta strains as revealed by bisulphite sequencing.Click here for additional data file.Table S4 Transcripts identified by RNAseq as significantly differentially expressed between the Spin and SpinSel Tuta absoluta strains using assembly one as a reference (see methods).Click here for additional data file.Table S5 Transcripts identified by RNAseq as significantly differentially expressed between the Spin and SpinSel Tuta absoluta strains using assembly two as a reference (see methods).Click here for additional data file.Table S6 Expression of isoforms encoding suppressor of white apricot in the Tuta absoluta transcriptome.Click here for additional data file.Table S7 Sequences of primers used in this study.Click here for additional data file.Table S8 Sequence of primers used for Bisulphite PCR.Click here for additional data file.Table S9 Assembly statistics of Tuta absoluta transcriptomes.Click here for additional data file.


We observed for the first time a shift of ∆Tc ~ 1 K by oxygen isotope substitution of 16O → 18O in SrRuO3 by precise measurements of DC and AC magnetizations. The results surprisingly lead to the noteworthy inverse isotope effect with negative coefficient α = −∂ lnTc/∂ lnM. The Raman spectra indicate that the main vibration frequency of 16O at 363 cm−1 shifts to 341 cm−1 following oxygen isotope substitution 18O. This shift is remarkably consistent with the Debye frequency being proportional to ∝ 1√M where M is the mass of an oxygen atom. The positive isotope shift of ∆Tc can be understood by taking account of the electron-phonon interaction.The oxygen isotope effect of the ferromagnetic transition in itinerant ferromagnet strontium ruthenate SrRuO Strongly correlated electron systems (SCES) exhibit many interesting quantum phenomena such as unconventional superconductivity and metal–insulator transitions. SCES include high-temperature superconductors12356710x1−CaxMnO3 has been investigated12x1−CaxMnO3 upon 18O substitution with the largest Curie Temperature (Tc) shift of Tc(16O) = 222.7 K to Tc(18O) = 202.0 K observed when x = 0.20. This shift may be caused by strong electron–lattice coupling and has some relation with large magnetoresistance14x1−CaxMnO3 is caused by the double-exchange interaction17The isotope effect of the ferromagnetic transition in Laift of TcO = 222.7x1−CaxMnO3, which is also a material that exhibits colossal magnetoresistance, has been investigatedTc of Prx1−CaxMnO3 was lowered upon isotope substitution 16O → 18O; for example, Tc(16O) = 112 K shifted to Tc(18O) = 106 K when x = 0.2. It is expected that this isotope effect arises from strong electron–phonon coupling21Tc has also been examined for the ferromagnetic superconductor RuSr2GdCu2O8Tc of ~0.35 K was obtained through the isotope effect, which also influenced superconducting transition temperature. As for strontium ruthenates, an anomalous isotope effect was observed for the superconducting transition temperature of the spin-triplet superconductor Sr2RuO43 films showed anomalous temperature dependence near the ferromagnetic transition temperature3. There has been an attempt to measure the isotope effect in the weak itinerant ferromagnet ZrZn2Tc was not determined because the shift of Tc was small and there was uncertainty arising from different impurity levels. To date, a distinct isotope effect of itinerant ferromagnets has not been observed.The isotope effect in the ferromagnetic insulating state of PrTc of the itinerant ferromagnet SrRuO3, which is a metal27Tc of ~160 K293 is increased about 1 K upon 18O isotope substitution. This positive isotope effect needs a new analogy to explain the ferromagnetic interaction in itinerant electron systems. A softening of the oxygen vibration modes is induced by isotope substitution (16O → 18O), which is clearly indicated by Raman spectroscopy. The Raman spectra also confirm that almost all the oxygen atoms (more than 80%) are substituted.In this article, we report the isotope effect of ωD. In fact, the Raman spectra of SrRuO3 before and after oxygen isotope substitution clearly indicate that the main vibration frequency of 16O at 372 cm−1 is lowered to 351 cm−1 for 18O. This shift is consistent with ωD being proportional to 1/√M, where M is the mass of an oxygen atom. Thus, our experiments confirm that the isotope shift of Tc is induced by the decrease of the frequency of the oxygen vibration mode. Our results reveal a small increase of Tc on 18O substitution, which is in contrast to the typical oxygen isotope effects of materials showing colossal magnetoresistance. The oxygen isotope effect in SrRuO3 arises from the electron–phonon interaction described by the electron–phonon coupled field theoryTc in itinerant ferromagnets by considering the spin-fluctuation theory for ferromagnetsωD increases the relative strength of the Coulomb interaction U. This results in a positive isotope shift of Tc in accordance with our experimental results.The increase of atomic mass caused by isotope substitution leads to a decrease of the Debye frequency 3 was synthesized by the conventional solid-state reaction method from a stoichiometric mixture of SrCO3 and RuO2. The starting materials were calcined at 800 °C for 24 h and then sintered at 1000 °C for 48 h in air, resulting in a dense pellet. Oxygen isotope substitution was performed by annealing. To ensure oxygen isotope substitution, samples were first annealed under the following conditions. Samples were places in two independent quartz tubes in a furnace and exposed to 16O2 or 18O2 gas at atmospheric pressure. Samples in both 16O2 and 18O2 gas were annealed at 1000 °C for 91 h, cooled in the furnace to room temperature and then annealed again at 1000 °C for 90 h. Raman spectroscopy of the samples confirmed that >80% of the oxygen atoms were substituted  up to 5 Tesla at temperatures T from 5 to 300 K. AC magnetic susceptibility was measured using a Hartshorn bridge circuit. The AC magnetic field produced by the primary coil was 1 Oe and the frequency of the AC signal was 180 Hz.DC and AC magnetization measurements were conducted to determine 18O-substituted sample with 16O to produce a sample indicated as 16OR. The Raman spectrum of 16OR is also shown in The oxygen phonon modes are observed at room temperature. A softening of the oxygen vibration modes was confirmed by Raman spectroscopy. We resubstituted an 3 samples substituted with oxygen 16O, 18O, and 16OR at T = 5 K are presented in The magnetic field dependence of magnetization of SrRuO3 samples substituted with 16O, 18O and 16OR are illustrated in 18O substitution is clearly observed. The shift shows that ∆Tc is ~1 K. Therefore, the ferromagnetic transition temperature of SrRuO3 was increased by about 1 K by 18O substitution. The temperature dependence of magnetization at 1 kOe for SrRuO3 following oxygen isotope substitution is depicted in 3 is increased by 18O substitution. Again, the shift indicates that ∆Tc is ~1 K. This agrees with the shift estimated from the AC magnetic susceptibility measurements.The temperature dependences of AC magnetic susceptibility of SrRuOωD increases the relative strength of the Coulomb interaction U. This leads to the positive shift of Tc in accordance with experiments. This is stated theoretically as follows. In the study of magnetism, the Hubbard model is of fundamental importance323334353638Tc is determined through solution of the gap equation. The Hamiltonian is given byLet us examine the isotope effect theoretically. An important point is that the decrease of ckσ and c†kσ are Fourier transforms of the annihilation and creation operators ciσ and c†iσ at site i, respectively. Here, niσ = c†iσciσ is the number operator, and ξk = εk− μ is the dispersion relation measured from the chemical potential μ. The electron field ψσ and the phonon field ϕ are defined, respectively, as follows:where V is the volume of the system. We denote the number of electrons with spin σ as nσ =  is the density of states at the Fermi level. The vertex correction is of the order of ωD/EF in accordance with the Migdal theorem4041n↑ − n↓, we obtain the equation up to the order of ∆ of, Ueff = U + Ugρ(μ0)(ωD/2εF)ln(εF/ωD). With the help of the Sommerfeld expansion, Tc in the mean-field approximation is given bywhere R is a constant. It has been suggested that the vertex correction like that shown in Tc U. Thus, from the formula of Tc in Tc: ∂Tc/∂M > 0. The positive isotope shift of Tc is also obtained by taking the spin fluctuation effect into account3243α = −∂ ln Tc/∂ ln M is given aswhere Tc increases with M.This indicates that α is negative and Tc in itinerant ferromagnet SrRuO3. First, Tc was estimated from DC and AC magnetization measurements. We found that Tc increases upon oxygen isotope substitution of 16O for 18O. This results in an inverse isotope effect of ∂ ln Tc/∂ ln M < 0. This finding is in contrast to the results obtained for Mn oxides that show colossal magnetoresistance. The isotope effect of Tc occurs through the electron–phonon interactions, especially the electron–phonon vertex correction.We examined the isotope effect of 18O for 16O. This technique would be helpful in searching new functional materials.We summarize the results and significance of our work as follows. (1) The inverse isotope effect of the Curie temperature was observed clearly in an itinerant ferromagnetic material for the first time. (2) Our result shows that the electron-phonon interaction is ubiquitous in the world, even in ferromagnetic materials. (3) The positive shift of the Curie temperature can be understood based on a theoretical model of the Coulomb interaction and the electron-phonon interaction. (4) We have established the experimental procedure to substitute isotope oxygen How to cite this article: Kawanaka, H. et al. Enhancement of ferromagnetism by oxygen isotope substitution in strontium ruthenate SrRuO3. Sci. Rep. 6, 35150; doi: 10.1038/srep35150 (2016).


The data presented in this article are related to the research article entitled “The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical”   Specifications TableValue of the data●To facilitate the reader's understanding of this study.●Extend readers' knowledge about the free radical reaction of carbohydrates.●To lay a foundation for further study on the mechanism of polysaccharide degradation.1Eight figures related to the research article entitled “The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical”  2Data provided in this article are based on computation performed applying Gaussian 09 at B3LYP/6–31+G level and are treated using ChemBioDraw Ultra 12.0, Origin 7.5 or BIOVIA Discovery Studio Visualizer 2016.The chemical structures and the scheme of reaction pathways are shown below.SuA6, TS(III)SuA6, TS(II)SuB1 and TS(II)MaB6 were illustrated in