We characterized an exponential response in N2O production to decreasing oxygen between 1 and 30 μmol O2 l−1 within and below the oxycline using 15NO2−, a relationship that held along a 550 km offshore transect in the North Pacific. Differences in the overall magnitude of N2O production were accounted for by archaeal functional gene abundance. A one-dimensional (1D) model, parameterized with our experimentally derived exponential terms, accurately reproduces N2O profiles in the top 350 m of water column and, together with a strong 45N2O signature indicated neither canonical nor nitrifier–denitrification production while statistical modelling supported production by archaea, possibly via hybrid N2O formation. Further, with just archaeal N2O production, we could balance high-resolution estimates of sea-to-air N2O exchange. Hence, a significant source of N2O, previously described as leakage from bacterial ammonium oxidation, is better described by low-oxygen archaeal production at the oxygen minimum zone's margins.Oceanic oxygen minimum zones are strong sources of the potent greenhouse gas N Understanding the production processes behind oceanic sources of nitrous oxide (N2O), a potent greenhouse gas, is of critical importance. Here, the authors reveal an archaeal-mediated N2O production pathway in the North Pacific, which increases exponentially with decreasing oxygen. There is clearly therefore a need to improve our understanding of the production of this atmospherically potent N2O at both the margins of OMZs and beyond in hypoxic, coastal waters2O (oversaturation relative to atmospheric equilibration) and O2 in surface waters to indicate bacterial nitrification as the predominant source of N2O , though its exponential form has not, to the best of our knowledge, been characterized experimentally in the ocean below 30 μmol O2 l−1 , if classic oxygen-stressed, bacterial-nitrifier N2O production was active, but this is not the case2O production has routinely been used for the purposes of mass balance or to simply rationalize water column distributions of N2O without any supporting experimental evidence9The documented thickening of OMZs across the worldl−1 ref. . In addi2O production in the ocean, it is now evident that the Archaea are widespread in the ocean, playing significant roles in key processes such as nitrification and potentially the production of N2O  where N2O accumulates2In addition to the poorly substantiated mechanistic basis for bacterial-nitrifier NN2O refs , 16, 17.N2O refs , are com2O production to decreasing oxygen between 1 to 30 μmol l−1 and to archaeal gene abundance, together with a 15N pattern in the N2O that is reconciled by an archaeal mode of production.Here we provide experimental evidence, with samples from the eastern tropical North Pacific (ETNP), which clearly links an exponential increase in N2 l−1 at its base but remained above the limit of detection for the Seabird Sensor to approximately 340 m  we measured a broad, characteristic peak in N2O of up to 104 nmol N2O l−1 , within the wider boundary of the OMZ as a wholeWe set our observations for N15N2O in incubations with 15NO2− at two depths at each of our six offshore sites (n=12 groups of experiments). To generate natural variation in ambient water column oxygen and nitrous oxide concentrations, each depth was either within or beneath the oxycline (n=70) for the production of N2O as a function of oxygen  and was maximal in waters degassed with nitrogen , at a level far above  that expected for denitrification  of 4% to 35% for all the levels) across the 12 groups of experiments , which allowed the overall magnitude of total N2O production to vary randomly between the 12 groups of experiments, while keeping the response to oxygen (b) constant. The mixed-effects model captures the data well (2O with oxygen decreasing below 30 μmol O2 l−1 is not only consistent with N2O accumulating below 30 μmol O2 l−1 in the water column (2O over time at two oxygen concentrations (−3 d−1) and representative of the 12 main experiments , it was approximately linear over the first 18 h and then decreased over time. If anything, our single time point incubations may have underestimated N2O production slightly. Overall, however, we conclude that our experiment captured the regulation of N2O production by oxygen in the ocean. None of the incubations produced any 15N labelled N2, not even at 1 μmol O2 l−1.Ambient controls were used to represent Neriments . To accog oxygen . The mosata well . The over column  but alsor column . We alsotrations  to check2 l−1, significant oxidation of both ammonium and nitrite , and net nitrification of up to 8.2 nmol N l−1 d−1  to vary randomly between the 12 groups of experiments the mixed-effects model was able to derive an overall ‘population' estimate for the response to decreasing oxygen. The magnitude of any deviation from this estimate, that is, the random intercept, can be used to further explore relationships with other explanatory variables that could potentially account for that random variation in the magnitude of N2O production. For example, there was a clear effect of ambient oxygen concentration in the water column: with samples collected from above 30 μmol O2 l−1 producing less N2O, on average, to those collected from below 30 μmol O2 l−1 . We also quantified archaeal nirK  and bacterial nirK and nirS , genes coding for the potential to reduce NO2−  for median N2O production (58 nmol m−3 d−1), below 30 μmol O2 l−1. Accordingly, 2 and 5 attomol N2O per copy per hour for the two genes, respectively, is representative of published rates  nonlinear mixed-effects models failed to converge and to explore the production of Ngression . Oxygen al genes . To confour ref. ).2O, coded in R , parameterization with M2 maintained the initial steady state conditions (−1) of N2O, whereas, with M5, we saw significant N2O accumulation. Note that with M2 the vast majority of the N2O is assumed to come from a 1:1 coupling . The sea-surface N2O concentration in our model was fixed (Dirichlet boundary conditions) based on the average concentration from our observations (9.3 nmol N2O l−1). Sea-air exchange (efflux to the atmosphere) was therefore implicit in our model and this efflux of N2O was sustained by a positive concentration gradient with depth, implying an upward flux of N2O. Since the model water column was losing N2O at the surface, it is implicit that this upward flux of N2O should compensate for the gas exchange loss term . Therefore, we calculated the flux over the upper 2 and 5 m of the model water column to derive the model sea-air flux. These depths were chosen to represent the turbulent layer near the sea surface given the relatively low average wind speed  and water column profile data , with an equivalent exchange for N2O of 17 μmol N2O m−2 d−1 ; the latter agreeing well with the 1D model estimate for sea to air exchange of 16.0–17.9 μmol N2O m−2 d−1. Our experimental manipulation of oxygen, combined with a mixed-effects modelling approach has enabled us to parameterize a simple, single process 1D model that reproduces the pattern of N2O observed in the top 350 m of the tropical North Pacific.We used the coefficients from our nonlinear mixed-effects models , which is typical of 15N tracer work for 15N-gas production633Here we were following up on our previous work in the Arabian Seatributed , then weroughout . Put sim15N-NO2− pool occurred via oxidation of 14N-NH4+ to 14N-NO2− and 15N-NO2− to 15N-NO3−, as this would increase the chance of any subsequent denitrification producing 45N2O, relative to 46N2O. For this to be plausible, however, 14N-NO2− production would need to make ∼20 μmol l−1 at the start of the incubation. We did measure significant nitrification activity , from ammonia oxidation222O production in the oligotrophic North PacificamoA or γ-amoA. Given this apparent absence of any bacterial, nitrifier–denitrifier genomic potential, along with the overestimation of N2O production in our model . Although we did not quantify NO3− reduction, if its activity were comparable to our measured rates of NO2− oxidation it would have the same negligible effect on the 15N labelling of the NO2− pool.One possibility is that dilution of the activity  similar odel M5, , throughl−1 ref.  with com2O production is best explained by low oxygen together with a high abundance of both AnirK and AamoA. Hence, the patterns in the genomic potential for both the reduction of NO2− (our source of 15N) and oxidation of ammonium  does not necessarily confer a direct role on that gene for a measured process. Yet, similar positive relationships between Thaumarchaeota cell abundances and nitrification potentials are present in the low-oxygen waters of the Baltic, which, along with exponential increases in AamoA abundance below 100 μmol O2 l−1 in the Atlantic, suggest maintenance of active populations of Thaumarchaeota in low-oxygen waters13Our oxygen experiments, combined with statistical modelling, indicate that highest N2O production122OH and NO in Thaumarchaeotal ammonia oxidation provide support for a 1:1 coupling in N2O production in these organisms222O production occurs via an ammonia oxidizing bacterial type biochemical leak2OH and NO) in hybrid N2O formation might represent a genuine route of energy conservationNor or its equivalent in the archaea, we have to assume that our exogenous N2O  was produced through canonical denitrification operating as far as N2O but it remains to be proven whether the ability to metabolize NO further to N2O is truly absent from archaea in the ocean. Furthermore, modelling of N2O production in the ocean2O production primarily to bacterial ammonium oxidation (nitrification). Here we show that this is not the case and a major driver of N2O production in the ocean is likely to be archaeal hybrid N2O formation.Overall, our data agree with the growing body of evidence for archaeal-mediated N2O production in the ocean is widely accepted , its exponential form has not, to the best of our knowledge, been characterized experimentally in the ocean below 30 μmol O2 l−1  and can struggle to capture the full dynamics of N2O production in low-oxygen waters2O/AOU that allow the yield to change as a function of oxygen do a better job but still struggle at the transition  from production to consumption of N2O  below the pycnocline, whereas with our single process variant the model only gained 1.2%. This suggests that the dynamics of N2O production in the two studies were fundamentally different. Indeed, we did not measure any production of 15N2 in any of the oxygen treatments, that is, none of the 15N2O from the reduction of 15NO2− was further reduced to 15N2, even at 1 μmol O2 l−1, which is a key feature of the Babbin model. That, along with the non-binomial distribution of 15N in our N2O, relative to the NO2− pool, discounts denitrification as the primary source of N2O here and it is redundant in our model.The original version of the model by Babbin 2O m−2 d−1, on average from the N2O–CO2 field data and a 1D directly parameterized model) agreed very well with each other and with those in the literature—despite different approaches. For example, 13 μmol N2O m−2 d−1 has previously been taken as representative of the ETNP−2 d−1 has been estimated for the tropical south Pacific22O budget is the respective area of OMZ used for any extrapolation. The latter is partly defined by the concentration chosen for oxygen at which the microbiology either produces or consumes N2O and this is contentious52O production with decreasing oxygen between 1 to 30 μmol O2 l−1 and apply this to regions of the ocean defined as OMZs by oxygen minima below 20 μmol O2 l−1  and the entire global extent of OMZs (30.4 × 1012 m2) generates 2.1 Tg N y−1 and 5.1 Tg N y−1, respectively, as N2O. The latter of which agrees very well with estimates of approximately 5.8 Tg N y−1 derived from the oxygen-sensitive model of Nevison et al.15The two methods that we used to estimate sea to air exchange  was used to collect and characterize the water at each site between 5 and 4,000 m. The distribution of N2O, CO2 and NO2− was measured as described previously2 after rapid equilibration and reduction to CH4 .A standard conductivity–temperature–depth rosette (24 Niskin (20 litres) and full Sea-Bird 24 electronics . It is important to appreciate that all published work to date that uses 15N to trace the production of either N2O or N2 applies a ‘tracer' at concentrations in excess of apparent Km values for these processes, that is, typically 5–10 μmol NO2− l−1 spike, compared with 1–2 μmol NO2− l−1km and, as such, should be considered as potentials634ø=47 mm, 0.2 μm pore size filters). The filters were immediately frozen in 2 ml cryovials, in liquid nitrogen, and stored at −80 °C for later extraction of nucleic acids (see below). Production of 15N2O and 15N2 was measured in the three remaining vials against reference samples for each of the treatments, or natural abundance, by mass spectrometry  and, in addition, general bacterial and archaeal Marine Group I 16S rRNA genes . We did, however, measure N2O production at 2, 4, 9, 18, 36 and 72 h at two sites, for two oxygen treatments during a subsequent cruise to check that our single time point incubation was not overestimating production.We measured the production of ix sites . Seawateix sites . Seawate15N2 analysis, or a helium-filled 20 ml gas-tight vial  for 15N2O analysis. The 20 ml vials ended up with 10 ml of seawater and 10 ml of helium headspace to which we added a carrier of 3 nmol N2O, as sparging with the compressed air, N2 and O2 treatments effectively removed all of the natural N2O from the samples. These were then analysed for enrichment of both single- and dual-labelled 45N2O and 46N2O, respectively, against seawater samples (collected on the cruise) sparged with the five treatment gases, or, in the case of the ambient treatment, reference samples of seawater, using a trace gas pre-concentrator unit 2O , and it was linear (r2=0.998) over the range 0 to 20.72 nmol N2O absolute .All the samples were transferred under constant temperature back to the home laboratory in London and were brought to 22 °C before processing. Two subsamples of the 1 litre vials were forced out under helium and transferred to either a helium-filled 12 ml gas-tight vial , for 2 gas to equilibrate between the water phase and headspace. Samples of the headspace (100 μl) were then analysed for enrichment in 15N2 by injection  into an elemental analyzer , interfaced with the continuous flow isotope ratio mass spectrometer (CF-IRMS)2 absolute . Values for the production of 29N2 and/or 30N2 were calculated as excess over the production in the time zero ‘reference' samplesAfter bringing the remaining 12 ml gas-tight vials to 22 °C, a helium headspace (1 ml) was added and the vials shaken by hand and left overnight on rollers (Spiramix) to allow N15NO2− to trace the production of N2O as per our previous work in the Arabian Sea2O in response to oxygen. In the first method, given that the archaea appear to lack Nor, we assume that they cannot make N2O purely from exogenous NO2− and that any measured production of p46N2O  must be due to canonical denitrification reducing NO2− as far as N2O. Then, any production of N2O that we cannot account for by canonical denitrification with exogenous NO2− we assign to hybrid N2O formation, as in the most recent models for Thaumarchaeotal ammonia oxidation2O is due to a classic bacterial-type mode of nitrifier–denitrification, with random isotope pairing of 14NO and 15NO upstream of the production of N2O.We used 2O that we assume to be owing to canonical denitrification of exogenous NO2− according to:We calculate the overall production of NNO2− is the fraction of 15N in the NO2− pool  plus 1% (w/v) acid-washed polyvinylpolypyrrolidone, 500 μl of Tris-equilibrated phenol (pH 8.0), and 50 μl of 20% (w/v) sodium dodecyl sulfate. The extraction process involved bead beating and passing the samples through hydroxyapatite and Sephadex G-75 spin columns, to separate nucleic acids from proteins and salts−1 Tris, 1 mM EDTA [pH 8.0]) and stored at −80 °C.In the home laboratory, each Supor filter was cut in half and one half was placed into a 2 ml sterile screw-cap tube, containing −1 of each primer and 1 μl of 10 times diluted DNA. The conditions for all reactions were as follows: 95 °C for 3 min; 40 cycles of 95 °C for 0.05 min and 60 °C for 0.30 min; 95 °C for 0.05 min; 65 °C for 0.05 min and a final step of 95 °C for 0.5 min. Absolute quantification of the targeted genes was performed with a series of 10-fold standard dilutions, using the CFX Manager version 2.0 software (Bio-Rad). Standards for bacterial 16S rRNA, nirS and nosZ genes were derived from Pseudomonas brenneri DSM15294; environmental PCR products were used for bacterial amoA, AamoA, nirK, AnirK and MG1 16S. Samples with Cq values that were the same or greater than those of the no template controls were assumed to be below the limit of detection (LOD). In each of these cases, the calculated LOD for the particular qPCR plate was used as the value for that sample (maximum LOD=171 copies ml−1). Specificity of the AnirK PCR was assessed by sequencing product from a number of sites. All showed that the PCR assay was specific for its target gene (data not shown).The extracted DNA was used for quantification of functional genes  from the second depth at each site ; nitrite oxidation from net accumulation of 15NO3− over 3, 6, 12, 24, 48 or 96 h from 15NO2− and overall net nitrification from the accumulation of total 15NOx− after 96 h from 15NH4+. The samples were fixed (50 μl 50% (w/v) ZnCl2) and production of 15NOx−, 15NO2− or 15NOx− measured with a sulphamic acid assay at the University of Southern Denmark.To account for any possible turnover of the ach site . Water w2O (1 m depth resolution), coded in R. The model is largely based on the parameterizations given by Babbin et al.2O) as well as biological production of N2O. Vertical transport was parameterized according to Fickian diffusion with a diffusivity Kz of 4 × 10−4 m2 s−1 at the surface, decreasing linearly to 4 × 10−5 m2 s−1 at 10 m and remained constant thereafter apart from the pycnocline (20–48 m depth) where Kz was 1 × 10−5 m2 s−1. This Kz profile effectively simulated near surface turbulence while the remaining water column was dominated by diffusive processes. An upwelling velocity (wup) of 8 × 10−7 m s−1 and a particle sinking velocity  values for the ETNP were taken from the literature44−1, a sub-surface maximum of 5 μmol l−1 at 32 m and decreasing thereafter to 1.3 μmol l−1 at 400 m. Model POC remineralization (POCrem) was parameterized as a first-order process with a rate constant of 5 × 10−7 s−1. POC production at the surface was implicit via the fixed boundary concentration as in Babbin et al.pPOCZ) at depth (Z) as a function of the upwelling NO3− flux and light attenuation:The model resolved the upper 400 m of the water column at 1 m depth resolution and 1 min time intervals. Boundary conditions at the surface and at 400 m were fixed and prescribed by the respective averages from our profiles. This average profile also described initial conditions for NOF is the ratio of upwelled NO3− used by primary producers (0.2), [NO3−] is the concentration of NO3− at depth Z, Kd is the light attenuation coefficient (0.09 m−1) and rN:Cremin is the N:C ratio production/remineralization (rN:Cremin=16/106). The value of Kd was chosen as it gave a subsurface pPOCZ maximum which was consistent with the positions of the subsurface POC- and chlorophyll-concentration maxima at the base of the mixed layer as observed during our cruise. NO3−, PO43− and O2 were linked to POC production/remineralization according to Redfield stoichiometry, as in Babbin et al.2 consumption followed a respiratory ratio of ∼1.4 (rO:Crem=150:106).where 2O was parameterized for two separate variants of the model: (i) according to Babbin et al.2 concentration as described here. All processes except those producing N2O were identical in both the models. In our second variant, we parameterized model N2O production (pN2O in nmol m−3 d−1) using the estimates for a and b from our nonlinear mixed-effects models . Mixing ratios of CO2 and water in the marine air and equilibrator headspace were determined by non-dispersive infrared dection . Measurements were referenced against secondary calibration gases  with known CO2 mixing ratios  in synthetic air mixtures (21% oxygen and 79% nitrogen). All calibration gases were calibrated against certified primary standards from the National Oceanic and Atmospheric Administration (244.9 and 444.4 μmol CO2 per mole). The pCO2 system described here showed high consistency with a similar pCO2 system and pCO2 calculated from independent TA, DIC and pH during ‘at sea' inter-comparisonpCO2 and wind data and resultant efflux estimates.High temporal resolution measurements of pCO2 (pCO2sw) data were available but in which N2O was not directly quantified, we predicted molar seawater N2O concentrations (N2Osw) using the linear relationship between N2Osw and the molar seawater concentration of CO2  and the corresponding N2O flux estimated from the high-resolution CO2 fluxes calculated using the average 12 h wind speed:Then, for the samples for which high-resolution seawater 2 CO2sw; . To do tR  in each incubation bottle and total production of N2O is that measured in each bottle at the end of its incubation. For the 12 sets of experiments analysed using nonlinear mixed-effects models, we either fitted both the intercept  and sensitivity  as random effects, or, a and b, each individually, and compared model fit in each case with the Akaike Information Criterion (AIC). Relationships between these ‘random' elements, that is, variance not explained by experimental oxygen and other possible explanatory variables  were explored visually  and then more rigorously using multiple regression and the entire, linearized and centred (xc) data set . Here, we judge the simplest model  against more complex models  also using likelihood ratio testing.Where The data that support the findings of this study are available from the authors on reasonable request, see author contributions for specific data sets.How to cite this article: Trimmer, M. et al. Nitrous oxide as a function of oxygen and archaeal gene abundance in the North Pacific. Nat. Commun.7, 13451 doi: 10.1038/ncomms13451 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-8, Supplementary Tables 1-4 and Supplementary References


Aim of this study was to comparatively evaluate the accuracy of respiration–correlated (4D) and uncorrelated (3D) cone beam computed tomography (CBCT) in localizing lipiodolized hepatocellular carcinomas during stereotactic body radiotherapy (SBRT).4D–CBCT scans of eighteen HCCs were acquired during free–breathing SBRT following trans–arterial chemo–embolization (TACE) with lipiodol. Approximately 1320 x–ray projections per 4D–CBCT were collected and phase–sorted into ten bins. A 4D registration workflow was followed to register the reconstructed time–weighted average CBCT with the planning mid–ventilation (MidV) CT by an initial bone registration of the vertebrae and then tissue registration of the lipiodol. For comparison, projections of each 4D–CBCT were combined to synthesize 3D–CBCT without phase–sorting. Using the lipiodolized tumor, uncertainties of the treatment setup estimated from the absolute and relative lipiodol position to bone were analyzed separately for 4D– and 3D–CBCT.Qualitatively, 3D–CBCT showed better lipiodol contrast than 4D–CBCT primarily because of a tenfold increase of projections used for reconstruction. Motion artifact was observed to subside in 4D–CBCT compared to 3D–CBCT. Group mean, systematic and random errors estimated from 4D– and 3D–CBCT agreed to within 1 mm in the cranio–caudal (CC) and 0.5 mm in the anterior–posterior (AP) and left–right (LR) directions. Systematic and random errors are largest in the CC direction, amounting to 4.7 mm and 3.7 mm from 3D–CBCT and 5.6 mm and 3.8 mm from 4D–CBCT, respectively. Safety margin calculated from 3D–CBCT and 4D–CBCT differed by 2.1, 0.1 and 0.0 mm in the CC, AP, and LR directions.3D–CBCT is an adequate alternative to 4D–CBCT when lipoid is used for localizing HCC during free–breathing SBRT. Similar margins are anticipated with 3D– and 4D–CBCT. Trans–arterial chemoembolization (TACE) is a common first line treatment for patients with unresectable hepatocellular carcinomas (HCC). However, the tumor response after TACE alone has been frequently incomplete even for repetitive treatments. By combining TACE with hypofractioned stereotactic body radiotherapy (SBRT), improved tumor response and overall survival have been achieved ,2,3. Oneet al., motion blurring in free–breathing CBCT can impact the target localization and increase the inter–observer's variability in thoracic SBRT [Despite the potential of lipiodol contrast, motion mitigation during image guidance (IG) using on–board cone beam CT (CBCT) is important to reduce the uncertainty of ambiguous target definition when tumor at different positions over the respiratory cycles is compressed onto a single 3D image during reconstruction. As indicated by Sweeney cic SBRT . Single cic SBRT ,9.et al.[Recently, the potential of using four–dimensional (4D)–CBCT reconstructions to reduce motion blurring artifacts has been investigated in SBRT ,10,11. Uet al. has demoet al. demonstrated for two liver cases that motion–compensated 4D–CBCT can further reduce motion blurring and streak artifacts due to sparse projection data and hence resulting in improved visibility of implanted fiducials and tissue contrast between anatomical structures [et al.[Previous comparative studies of clinical accuracy of 3D and 4D–CBCT have largely focused on thoracic lesion ,10. Zhanructures . Yet, ths [et al., showingThis study was approved by the local ethics committee of the New Territories West Cluster of Hospital Authority Hong Kong. The need of written informed consent from the participants was waived because this study was not treatment-based. For protection of patient confidentiality, the patient information was anonymized.Eighteen patients with non–resectable HCC were de–identified and included in this retrospective analysis. The tumor volume ranged from 10 to 1100 cc median = 186 cc). One to two cycles of first–line TACE was given to these patients one week prior to 4D–CT simulation . Individ6 cc. Oneet al. the tumor at the mid–ventilation may not correspond to its time–weighted average position (MidP) due to motion hystersis [4D–CT imaging was performed in helical mode with 2 mm axial slices on a Philips Brilliance BigBore scanner . A phase–based approach was used to sort the 4D–CT raw data into ten series of 3D images each sharing equal time percentage of the breathing cycle. At our institution, treatment planning was based on the mid–ventilation (MidV) planning target volume (PTV) concept and followed the method described in Ref.  for motiystersis . The meaystersis .x, y, and z directions corresponding to the left–right (LR), cranio–caudal (CC) and antero–posterior (AP) directions, respectively. The safety margin to ensure dose coverage to 90% GTV was calculated by the van Herk’s recipe [Σ and σ are the components of systematic and random errors including tumor delineation, 4D–CT/CBCT registration, intra–fractional baseline drift, β = 1.03 for the 85% isodose level (IDL) and σp = 3.2 mm for penumbra in water. For 4D–CT/CBCT registration, both Σ and σ were corrected to effective Σeff and σeff accounting for the finite number of SBRT fractions [For all patients, the lipiodol retention on the MidV images was used as contrast medium to assist delineation of the gross tumor volume (GTV) and to provide the tumor motion trajectory over the breathing cycle. The tumor motion was then used to formulate the individual safety margin in the s recipe  M=2.5Σ+βractions , with av3 v.9.2, Philips Radiation Oncology Systems, Fitchburg, USA) using 1–2 partial arcs and mixed energies of 6 and 10 MV.Volumetric–modulated arc radiotherapy was individually optimized with SmartArc  including the lipiodol contour. Rotational errors were corrected in the same manner as described in the 4D registration protocol, i.e. using the spinal vertebra. Finally, necessary adjustments were made manually based on the visual inspection of the lipiodol by the same clinician.r).For inclusion of the lipiodol retention in the analysis, either homogeneous accumulation or partial defect in both the simulation 4D–CT and the setup verification 4D–CBCT scans was considered as acceptable. A total of 105 scans were analyzed, 4–7 scans for each patient including the first and last scans, and other 2–5 scans in between. Inter–fractional changes of absolute and relative lipiodol position to the bony vertebrae were analyzed from the results of the 3D– and 4D–registration of each scan as the difference of the lipiodol centroid position on the 3D– and 4D–CBCT at MidP, compared with that at MidV on the planning 4D–CT. The agreement of inter–fractional change of the lipiodol position between 3D– and 4D–CBCT correction was assessed by Pearson’s correlation coefficient  to investigate its correlation with D was measured as the distance from the dome of the diaphragm to the last seen GTV contour in the cranial direction on the 4D–CT simulation scan. Other predictive factors of The 4D–CBCT was taken as the reference treatment setup in our online correction protocol. The difference between the clinical 4D–CBCT and the retrospective reconstructed 3D–CBCT registrations provides the tumor prediction error r motion , we measΣeff) and random errors (σeff) were also calculated. Population statistics were also obtained for the tumor prediction error for estimating the margin with the 3D–CBCT based correction protocol.The individual mean (μ) and standard deviation (SD) of the inter–fractional changes of the lipiodol position were estimated separately for 3D– and 4D–CBCT. Group means (GM) were calculated by averaging the individual patient means. The pooled population effective systematic (t–test. Differences of the tumor prediction errors and their variability (1 SD) were tested by one–way ANOVA. All tests were performed in SPSS .The Pearson’s correlation coefficient was used to quantify the correlation of the lipiodol position between 3D and 4D–CBCT and the difference of the absolute group means from zero was evaluated by one sample r) of 0.927 , 0.931 , and 0.873 . On average, the lipiodol was clearly visualized on both 3D– and 4D–CBCT scans, resulting in similar absolute lipiodol position . Borderline statistical significance was found in the correlation between D and tumor size, with Pearson’s correlation coefficients (r) of 0.45 (p = 0.064) and 0.48 (p = 0.054), respectively. When the outliner of D produced a significant r of 0.62 (p = 0.008). The variability of r = 0.50) but significant (p = 0.035) increase with increasing D, but not for p > 0.05). There were no differences between The absolute tumor prediction errors irection . The LR,et al. [Combining tumor delineation uncertainties, 4D–CT /–CBCT registration error of 1 mm (systematic and random), and intra–fractional tumor stability  into thThe tumor prediction error resulting from offset between 3D– and reference 4D–CBCT contributed 10.7 (LR), 11.2 (CC), and 10.2 (AP) mm to the PTV for motion up to 20 mm. Using our online lipiodol–guided correction protocol, an additional 1.0 mm margin was required for 3D–CBCT with respect to the reference 4D–CBCT. For comparison to offline bone–alignment correction by 4D–CBCT, the possible gain in margin reduction from online lipiodol–guided correction decreased from 3.4 (LR), 7.9 (CC) and 3.4 mm (AP) using 4D–CBCT to 2.3 (LR), 6.9 (CC) and 2.5 (AP) mm with 3D–CBCT.et al. [The potential advantage of 4D–CBCT may be to mitigate motion–related artifacts and thus potentially allowing more accurate tumor targeting at the time–weighted average position during SBRT. However, systematic comparison with 3D–CBCT was only available for lung tumors so far, but not for liver tumor such as HCC. This may well be due to the fact that without contrast injection, liver tumors are generally not visible because of limited contrast with the parenchyma. As demonstrated by Jones et al. , intraveBased the analysis of registration errors on clearly visible lipiodol from TACE, this study was for the first time able to assess the tumor positioning results for liver tumors from CBCT with and without motion–correlation. For our cohort of 18 patients, we found that registration errors of 3D– and 4D–CBCT were highly correlated in the three principal motion axes. As a result, the systematic and random errors of these IG–techniques were very similar. Only the systematic error in the CC direction was found to be slightly smaller with 3D–CBCT and the difference was within 1 mm from 4D–CBCT . This coet al. [et al. including ten HCC patients, which concluded that target localization accuracy using the liver contour was found to be unaffected by large breathing amplitudes with 4D–CBCT as seen with 3D–CBCT [Despite the difference in anatomical region, our results were largely consistent with other studies of SBRT for lung tumors. For example, systematic and random differences between 3D– and 4D–CBCT was found to be < 1 mm and the variability between 3D– and 4D–CBCT was largest in the CC direction . Howeveret al.  for lung 3D–CBCT . A lobe–As shown in et al. [Although 3D– and 4D–CBCT resulted in similar setup errors in the lipiodol position, 4D–CBCT has the advantage of reducing motion artifacts. Noteworthy though is, that the 3D–CBCT images in this study were synthesized using the projection data that they were acquired with the clinical 4D–CBCT protocol. In fact, the actual clinical 3D–CBCT acquisition protocol takes only 652 projections which were almost half of the 4D–CBCT protocol. As low contrast visibility and structural noise become worse as the number of projections decreases, the image quality of a real 3D–CBCT would be actually worse compared to 4D–CBCT . Nonetheet al.  suggesteet al. [Current 4D–CBCT reconstruction algorithms on commercial image–guidance platforms are known to produce sub–optimal image quality without further application of motion–compensation by on–line or prior motion model ,26. Greaet al. , showinget al. , which pet al. . A recenet al. . Future 3D–CBCT is an adequate alternative to 4D–CBCT when lipiodol after TACE is used for target localization in free–breathing SBRT of hepatocellular carcinomas. Despite motion artifacts similar safety margins are anticipated with 3D– and 4D–CBCT.


The structural effects that result from the uptake of NH3 were studied by powder X‐ray diffraction (PXD), FTIR spectroscopy and SEM. NH3 release at elevated temperatures was followed by in situ PXD. The cycling capabilities and air stability of the systems were also explored. NH3 is released from the hexaammines in a three‐step process to yield the diammine, monoammine and NiX2 dihalides respectively and (re)ammoniation occurs readily at room temperature. The hexaammines do not react with air after several hours of exposure.Although major difficulties are experienced for hydrogen‐ storage materials to meet performance requirements for mobile applications, alternative fuel cell feedstocks such as ammonia can be stored in the solid state safely at high capacity. We herein describe the NiX The current dilemma that faces designers of hydrogen‐storage materials is to balance a high gravimetric and volumetric hydrogen density with facile and reversible uptake and release.−3 (at 10 bar),One approach to the significant challenges associated with the storage of hydrogen, especially for mobile applications, is to consider “indirect sources” of hydrogen or the complete replacement of hydrogen with an alternative high‐energy‐density sustainable fuel. Ammonia is one such possibility with the capacity to be cracked catalytically to hydrogen and nitrogen or, as it has an energy density of 13.6 GJ m2 and has the advantage of an existing production and distribution infrastructure. Its current inexpensive (but unsustainable) production in the Haber–Bosch process makes it a cheap alternative to the direct use of hydrogen and alternative sustainable methods of generation offer hope for future “greener” large‐scale production.3‐storage materials offer a potentially satisfactory solution.Ammonia contains 17.6 wt % H3 chemically in the solid state. Alkaline earth metal halides are particularly suitable, and the highest reversible gravimetric capacities achievable (safely) are for the MgCl2‐NH3 system (51.8 wt % for [Mg(NH3)6]Cl2).3‐storage materials. They combine a strongly salt‐like character, which is important to form complex cations, with the affinity of transition metals towards ammonia as a ligand to form ammine coordination complexes.Solids that contain ammonia or its salts have a long history of chemical use that dates back to Arabic alchemists, and the first European Sal Ammoniac factory was established in Edinburgh as early as 1765.2 . We used a combination of powder X‐ray diffraction (PXD) and spectroscopy to confirm the structures of each of the respective halide hexa‐ and diammines. We combined these data with those from thermal analysis to determine the decomposition conditions and reaction pathways in each halide system from the fully ammoniated compounds to the respective dihalides.We describe here a systematic study of the ammonia uptake and release behaviour of the nickel(II) halides, NiX2, over 50 % of the total theoretical ammonia uptake (44.0 wt %) occurred within 5 min, and >90 % of the gravimetric capacity was achieved in ≈25 min under 1 bar NH3 . After 120 min, the chloride absorbed 42.9 wt % NH3 . The uptake thus compares favourably with other metal ammines and also the best porous materials under similar conditions.2: yellow, NiBr2: brown, NiI2: black) to pale violet (for NiCl2 and NiBr2) or white (NiI2) as a result of ammoniation. Microanalyses of the different products confirmed that the hydrogen composition of the powders matched that expected for the stoichiometric hexaammine halides (H [wt %] for [Ni(NH3)6]X2: X=Cl: found: 7.7(7), calcd: 7.77; X=Br: found: 5.5(3), calcd: 5.62; X=I: found: 4.4(3), calcd: 4.33).It is perhaps slightly unexpected to observe how readily the nickel halides react with dry ammonia at room temperature. By way of example for NiClρ) across the series from X=Cl through Br to I with the single exception of the symmetrical deformation mode. It has been noted previously that the δs mode is coupled strongly to the deformation of the H−N−Ni bond angle.δs frequency from X=Cl through Br to I implies a decrease of the Ni−N bond strength .3 rocking mode frequency from X=Cl through Br to I is symptomatic of progressively weaker H⋅⋅⋅X hydrogen bonds.The FTIR spectra of the ammoniated halides were ostensibly very similar and are in good agreement with previous studies of ammines .3)6]I2 is perhaps the most unexpected. Although ammonia uptake resulted in notable volume changes for all of the halides, for the iodide in particular, the colour change was accompanied by a dramatic volume increase upon reaction. A combination of evidence from SEM imaging and peak broadening in the PXD patterns reveals very small crystallite sizes in the hexaammine powders. The size distribution in the samples is far from monodisperse, and varies from sub‐micron crystallites (which measure hundreds of nanometres or less) to agglomerations of particles in the micron range, as shown by SEM imaging  analysis of the ammines gave a Ni/X elemental ratio of approximately 1:2 as expected (for [Ni(NH3)6]I2, Ni/I=1:1.9(1)) and confirmed the absence of any impurities. Nitrogen was detectable by EDX methods but could not be quantitatively determined reliably.Each of the ammines were pale in colour, and the white colour of [Ni(NH3)6]X2 crystals using POWDERCELL confirmed the identity of the ammoniated compounds.A comparison of the PXD patterns of the reaction products with documented crystal structures of the corresponding [NiNH6]X2 crysx, y, z; 192 l) site. Whereas realistic N−H bond lengths would be expected to be ≈1.01 Å, as found in gaseous or solid ammonia,any rotational position because of the symmetrical arrangement of the four chloride anions around any ammonia molecule . This leads to the absence of an energetic minimum of any significance for any rotational arrangement . The arrangement of the Cl atoms thus leads to a low energetic rotation barrier for the ammonia ligands. A definitive model for the hydrogen disorder and a more complete description of the hydrogen bonding network is only likely to be forthcoming from powder neutron diffraction experiments with deuterated materials, and we expect to report the outcome of such experiments in due course.The fitting of hydrogen positions in these compounds is not straightforward as the electron density is nearly uniformly distributed in a circle around the triangular molecular positions.3)6]X2  are built from isolated complex [Ni(NH3)6]2+ cations and halide anions =1.67 Å, r(Br−)=1.82 Å, r(I−)=2.06 Åρ(NH3) band in the FTIR spectra and weaker hydrogen bonding interactions from N−H⋅⋅⋅Cl to N−H⋅⋅⋅I. The long distance between each anion and the central atom of the cationic complex  confirms that the interaction is essentially electrostatic. Although this might not be unexpected for the packing of charged transition metal complexes and counter anions in the solid state, what is perhaps more surprising is the degree of structural rearrangement that occurs upon ammoniation under mild conditions. The starting (de‐ammoniated) materials NiX2  are composed of layers of edge‐connected [NiX6] octahedra in which a strong central atom–ligand  interaction exists ,The structures of X2 samples 6]Cl2 to approximately 353 K for both [Ni(NH3)6]Br2 and [Ni(NH3)6]I2. In all cases de‐ammoniation is complete below 600 K, and the final reaction step to the respective expected dihalides finishes at progressively lower temperatures from X=Cl (580 K) to X=I (549 K). The MS spectra taken from the evolved gas  in each case. Given that a solid oxide direct ammonia fuel cell, for example, would crack ammonia to N2 and H2 at the anode, neither of these decomposition products would present any issues from their presence in the feed gas stream. The mass spectra show two peaks that appear to correspond to a two‐step desorption process with Ni(NH3)2X2 as the only apparent intermediate compound for each X. Accordingly, the DTA signal shows two endothermic peaks synchronous to the two weight‐loss steps. We saw no evidence under these conditions  to support a de‐ammoniation step to the equivalent monoammines from the TG, DTA, d(TG)/dT or the MS profiles for any of the halide diammines.From the TG profiles, ammonia release appears to proceed in two steps. The onset temperatures for the first desorption step lie in the range from close to room temperature (≈303 K) for Cl2 at variable heating rates. We were thus able to derive the activation energy for each of the decomposition steps by applying the Kissinger method.−1 for the decomposition of the hexaammine, diammine and monoammine, respectively.Given that a further de‐ammoniation step (to the monoammine) has been described previously and that the monoammines Ni(NH−1) provide vital evidence for the formation of an intermediate monoammine before the decomposition to NiX2. Given that the monoammine decomposition step has a lower activation energy than the equivalent diammine ammonia loss, the latter process is effectively rate‐determining. At 15 and 20 K min−1, the rate of the diammine decomposition becomes comparable to that of the monoammine, and the complete three‐step decomposition mechanism is, therefore, observed. These observations concur with the three‐step mechanism observed by George and Wendtland−1. Hence, overall one can conclude that the de‐ammoniation of [Ni(NH3)6]X2 for X=Cl follows the three‐step process shown in Equations 1–Hence TG‐DTA‐MS experiments conducted at higher heating rates . The hexaammine phase observed at 298 K is completely transformed into the diammine phase at 360 K, which in turn decomposes gradually to NiCl2. The formation of the poorly crystalline NiCl2 phase can be observed from 460 K and is completed by 480 K 2X4/2] chains (see below). This again is in accordance with the fact that the δs mode is the most sensitive to deformations of the Ni−N bond.SEM images and FTIR spectra were also acquired for the intermediate diammines. The morphology  of the diammines is retained from the respective hexaammines and a broad particle size distribution is again prevalent. The IR spectra of the diammine samples  can all be assigned according to the bands expected from previous studies (Table S2).3)2X2  as the β‐type polymorphs with the Cd(NH3)2Cl2 structure type (orthorhombic space group Cmmm), in which the metal atom is surrounded by octahedra made up of two trans NH3 ligands and four X atoms (see Supporting Information). The individual octahedra share edges that contain X atoms to form 1∞[Ni(NH3)2X4/2] chains. Although the PXD pattern quality from the employed experimental configuration was not ideal, we were able to refine the structures for Ni(NH3)2X2 for X=Br, I. The refinement for X=Cl, however, did not reach convergence if the thermal displacement parameters were varied and permitted a structural model to be obtained only if the temperature factors were fixed to generic values. This is a facet of the relatively low quality of the powder data and the more uniform electron density distribution compared to that of the ammines that contained the electron‐rich Br− and I− anions. Therefore, the refined structure for Ni(NH3)2Cl2 should be viewed as a partial model.In accordance with the results of Leineweber and Jacobs for nickel chloride and bromide diammines,3)2I2. However, the distances between the central Ni atom and the surrounding ligands refined from our in situ data are considerably longer than the distances refined by Leineweber et al. 2Br2 type; orthorhombic space group Pbam), which is reported to form during annealing at 573 K.Notably, at no point did our in situ experiments show any evidence of a phase‐transition of the diammines into the α‐type structure (Mg(NH1∞[Ni(NH3)2X4/2] chains. Half of the chains in the α‐type are rotated by 90° compared to that of the β‐type. The inter‐chain relationships are different in the two structure types. If we consider the Ni(NH3)2Br2 polymorphs as examples, although each chain in the β‐type structure is surrounded by four nearest‐neighbour chains (d(Ni−Ni)=5.8584 Å), the chains in the α‐type have only two nearest neighbours (d(Ni−Ni)=5.8650 Å).d(Ni−Ni)=6.5541 Å) than those in the β‐type (two chains with d(Ni−Ni)=8.2730 Å and two chains with d(Ni−Ni)=8.2970 Å), which stabilises the α‐type over the β‐type structure at higher temperatures. This is also consistent with the hydrogen bonding situation and the lower density of the α‐type that overall underlines the fact that the α‐phase is the thermodynamically disfavoured phase. The relationships between the stability of the phases as a function of ammonia partial pressure will be the subject of further studies. The NiX2 phases formed upon further heating are poorly crystalline under the synthesis conditions employed here. The levels of crystallinity were comparable with those observed in diffraction patterns for the equivalent halide starting materials supplied commercially.The two diammine structure types are closely related to each other and only differ significantly through the packing of the 3)6]X2 product from the surface of the dihalide starting material. Conversely, however, the hexaammine desorption reaction would appear to be consistent with a transformation process that invokes isolated [Ni(NH3)6]2+ complex cations and halide anions rather than extended 1∞[Ni(NH3)6]X2} chains in which the individual component octahedra are separated from each other by significant distances (edge‐to‐edge distance 4.099(2) Å for X=Cl).3)6] octahedral edges and 3.128(2) Å away from the nearest [Ni(NH3)6] octahedron face. Given the distances involved and the implications with regard to the strength of the chain, it might seem unlikely that the reaction takes place by the combination of these hypothetical 1∞[Ni(NH3)6]X2} species. Alternatively, one might conceive of a model in which the metal–ligand recombination during the hexaammine to diammine ammonia desorption is rather governed by filling vacancies in the complex octahedra left by desorbing ammonia. The only possibility to fill these vacancies is by the approach and incorporation of the surrounding halide anions into the ligand coordination sphere. The so‐induced distortion in the ligand sphere could then provoke the subsequent loss of further ammonia molecules and their replacement by chloride anions in the manner of a self‐catalysing reaction until the stable 1∞[Ni(NH3)2Cl4/2] configuration is reached.Sørensen and co‐workers have established a model to explain the absorption–desorption mechanism for similar metal dihalide ammonia reactions by applying a model of chain abstraction from the surface.3)6]Cl2 with air under ambient conditions by time‐resolved PXD over 60 h to assess possible hydrolysis/decomposition and the kinetics of any such processes in view of the outcomes if a tank and associated storage system were compromised 6]Cl2 reacts over the duration of the experiment to yield a new phase, which can be indexed to a cell with approximate cell parameters of a≈7.90 Å, b≈7.80 Å, c≈3.80 Å, α=β=γ=90°. This is most likely a compound that contains both water and NH3 as ligands in a nickel‐centred complex because the hexaammine phase remains stable both under ammonia and an inert atmosphere. This hitherto unknown phase is only detectable after 5–10 h, and even after 60 h of exposure to air, the sample still contains detectable amounts of [Ni(NH3)6]Cl2. One could assume, therefore, that in the event of a hypothetical tank rupture under ambient conditions, any formation of ammonia would be slow and not considerable over hour timescales. Our further investigations of the formation and identity of the aqua‐ammine phase will be presented elsewhere.An important characteristic of potential fuel storage materials in terms of safety is their chemical stability in air. We were able to demonstrate recently that ligated water in nickel complex materials can be replaced easily by ammonia.d Figure . Cl2. This characteristic is especially important in the case of a rupture of a potential ammonia store.Nickel dihalide ammines are suitable materials for the reversible storage of significant amounts of NH3 is released upon heating. Our diffraction results demonstrate that only the respective diammines are readily observed in situ in each system. The diammine intermediate phases, Ni(NH3)2X2, adopt the Cd(NH3)2Cl2 structure type.Thermal desorption can be resolved into three reaction steps, and NH2O)6]Cl2  was dehydrated by heating at 250 °C for 12 h in air before use. NiBr2 , NiI2  and NH3  were used as received from the supplier. All the anhydrous nickel dihalides were characterised before use by using PXD.Br2 were obtained by using the same geometry and an air‐sensitive sample holder with Mylar windows. For variable‐temperature experiments, an Anton Paar HK1200N high‐temperature cell with Kapton windows was used to heat the samples to the necessary temperatures under a constant flow of Ar gas . The samples were heated at 5 K min−1 to the respective target temperatures in steps of 20 K  and then held at the various measurement temperatures while data were collected. The patterns were recorded in the 2 θ range of 10–90° with a step size of 0.05° for 1.25 h. The data for the Rietveld refinement of the diammines were obtained from scans measured from 2 θ=10–85° at 373 K for X=Cl, Br and 393 K for X=I. The ambient reactivity experiment was performed with a flat plate‐mounted sample. Measurements from 2 θ=10–45° (step size of 0.05°) were performed sequentially with a scan duration of 30 min in each case over a cumulative period of 63 h. A structure‐less profile was fitted for refineable data using the Le Bail method within Jana2006.R0=1.308 and b=0.37.PXD patterns were collected for samples  of the various products mounted in capillaries , which were prepared in a glovebox. Diffraction data were collected by using a Bruker D8 Advance powder diffractometer with CuK2 were performed by using a Rubotherm IsoSORP magnetic suspension balance and gas dosing system. NiCl2 powder (251.4 mg) was contained in a glass sample holder within the reaction chamber. The sample was held under dynamic vacuum for 30 min before ammonia uptake to minimise the possibility of water uptake. The sample was exposed to a static atmosphere of ammonia gas at 1 bar, and the mass change of the sample was monitored.Thermogravimetric ammonia uptake measurements for NiCl−1 and a heating rate of 4 K min−1 for cycling studies. Additional thermal measurements for the Kissinger analyses were performed with heating rates of 2, 10, 15 and 20 K min−1. [Ni(NH3)6]Cl2 (15±1 mg) was used in each case in these variable heating rate experiments. The STA instrument was located in an Ar‐filled glovebox . This configuration enables even samples of extreme air sensitivity to be analysed without contact with air. TG‐DTA–MS samples (≈25 mg) were loaded in cylindrical Al2O3 sample holders.TG‐DTA–MS experiments were conducted under flowing Ar  by using a Netzsch 409 PC STA instrument coupled to a Hiden Analytical HPR 20 mass spectrometer at an initial heating rate of 5 K minCombustion analyses were conducted on samples (≈2 mg) by using an Exeter Analytical CE‐440 elemental analyzer to obtain values for hydrogen content. Contact with air during the sample preparation was minimised (<1 min) to avoid sample degradation.−1 in the range of 4000–600 cm−1. The solid‐state samples (≈10 mg) were measured by attenuated total reflectance (ATR) by pressing the samples on to the diamond crystal of a MIRacle (PIKE) sample holder. The inevitable air exposure time of the sample transfer to the FTIR spectrometer was minimised to ≈5 s.FTIR spectroscopy was conducted by using a Shimadzu FTIR‐8400S spectrometer by accumulating 24 spectra at a resolution of 2 cm2 silicon drifted EDX detector  and calibrated with Cu. High‐magnification images of the hexaammines were taken by using a ΣIGMA field‐emission SEM (Carl Zeiss). An acceleration voltage of 15 kV was applied using a target‐to‐sample distance of 8.5 mm. Samples were coated with gold before the measurement. Unavoidable air contact upon transfer was minimised to less than 1 min to preserve the sample from degradation.Images of the diammines were obtained by using a XL 30 ESEM (Philips) SEM with a tungsten filament using a 15 kV acceleration voltage. The EDX spectra were recorded by using a 10 mmAs a service to our authors and readers, this journal provides supporting information supplied by the authors. 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Helicoverpa armigera resistance to Bacillus thuringiensis (Bt) cotton producing Cry1Ac is progressing in northern China, and replacement of Cry1Ac cotton by pyramided Bt cotton has been considered to counter such resistance. Here, we investigated four of the eight conditions underlying success of the refuge strategy for delaying resistance to Cry1Ac+Cry2Ab cotton, a pyramid that has been used extensively against H. armigera outside China. Laboratory bioassays of a Cry2Ab‐selected strain (An2Ab) and a related unselected strain (An) reveal that resistance to Cry2Ab (130‐fold) was nearly dominant, autosomally inherited, and controlled by more than one locus. Strong cross‐resistance occurred between Cry2Ab and Cry2Aa (81‐fold). Weaker cross‐resistance (18‐ to 22‐fold) between Cry2Ab and Cry1A toxins was also present and significantly increased survival of An2Ab relative to An on cotton cultivars producing the fusion protein Cry1Ac/Cry1Ab or Cry1Ac. Survival on Cry1Ac+Cry2Ab cotton was also significantly higher in An2Ab than in An, showing that redundant killing on this pyramid was incomplete. Survival on non‐Bt cotton did not differ significantly between An2Ab and An, indicating an absence of fitness costs affecting this trait. These results indicate that a switch to three‐toxin pyramided cotton could be valuable for increasing durability of Bt cotton in China.Evolution of  We also investigated the genetic basis of resistance to Cry2Ab  in the An2Ab strain.We specifically considered four of the eight conditions  was used to test 96 F2 neonates from each line at the discriminating concentration of 2 μg Cry2Ab per cm2 diet, which was adopted from Mahon et al. . Survivors from the ten lines with the highest survival (from 6.3% to 16.7%) were pooled to produce the An2Ab strain, which was further selected for resistance to Cry2Ab for 37 consecutive generations. During selection, an average of 1,440 neonates were selected per generation and concentrations of Cry2Ab were chosen to yield 60%–80% mortality. After completion of diet bioassays, survivors were reared to pupation on untreated diet to propagate the An2Ab strain.The susceptible An strain of n et al. . This diLarvae were reared on artificial diet, and adults were maintained as described previously , provided the Cry2Ab protoxin used for selection and in experiments. The Cry1A activated toxins  and Cry2Aa protoxin used in experiments were provided by Dr. Marianne P. Carey . Cry2Ab was produced as outlined in Wei et al. , and Cry2.3H. armigera responses to the Bt toxins. Cry1A toxins were solubilized in 100 mM Na2CO3 buffer  and Cry2A protoxins in 50 mM Na2CO3 buffer  to produce toxin stock suspensions (1 mg/ml) for each Bt toxins. Stock suspensions were further diluted with a 10 mM, pH 7.4 phosphate‐buffered solution (PBS) to obtain appropriate concentrations used in bioassays. Liquid artificial diet (900 μl) was dispensed into each well of a 24‐well plate. After the diet cooled and solidified, 100 μl of the PBS solution with the desired concentration of Bt toxin was applied evenly to the diet surface of each well of a 24‐well plate and allowed to air dry at room temperature. A single larva was placed in each well of the plate and covered with two layers of black cloth to prevent escape. Forty‐eight larvae (two replicates) were tested for each strain and toxin concentration, including a control with PBS and no toxin. At the end of the bioassay period, response of larvae was scored as dead if they died or weighed <5 mg.We used diet overlay bioassays for selection of resistance to Cry2Ab and evaluation of H. armigera  were used and the response of larvae was recorded after 7 days. This method was established in Australia for testing Cry2Ab against 2.41 hybrids were backcrossed to the An strain because resistance to Cry2Ab was incompletely dominant. At least 50 adults of each sex were used in mass crosses. Responses to Cry2Ab of An2Ab, An, and F1 hybrids from the two reciprocal crosses and the backcross progeny were determined using the bioassay described earlier.Male moths from the An2Ab strain were mass‐crossed with virgin females of the An strain and vice versa. Fh, which varies from 0 (completely recessive) to 1 (completely dominant) /. We used the EC50 values  of An2Ab, An, and F1 progeny to calculate the dominance parameter D , where MF1 and MAn are mortalities of the F1 offspring and An strain, respectively. In the second method, we assumed that effects of alleles at all loci are equal and additive on a logarithmic scale and independent segregation of resistance loci to calculate expected responses of the backcross progeny  is ne = (μP2 − μP1)2/(8σs2) ≤ n, where μP2 and μP1 are the log (EC50) for resistant and susceptible strains, respectively. The extra genetic variance segregating in the backcross generation beyond that of F1 hybrids, σs2, was estimated using equation (2) in Tabashnik, Schwartz, Finson, and Johnson (1 × An), F1, An, and An2Ab strains  direct test of one‐locus model  were put on a cotton leaf kept in a 115‐ml glass tube. To preserve leaf turgor, the petiole of each leaf was inserted into 20 ml of 1% agar gel at the bottom of the tube. After transfer of neonates, each tube was covered with two layers of black cloth to prevent insects from escaping.Feeding experiments were carried out in growth chambers using cotton material from field‐grown plants. Neonates (up to 24 hr old) of the An and An2Ab strains were initially fed cotton leaves (top second or third leaf on the main stem) for 5 days. For each combination of strain and cultivar, groups of five neonates (n = 10 cups per group), and each group was located at an arbitrarily selected location on a shelf in a growth chamber. Because survival after 5 days on non‐Bt cotton was high (near 100%), for each strain, survivors from each of six arbitrarily sampled tubes  were transferred to cups with non‐Bt cotton plant material (n = 6 cups per strain). The six cups were arbitrarily divided into three groups (n = 2 cups per group), and each group placed at an arbitrarily selected location on a shelf in a growth chamber.After the initial 5‐day period, survivors (either second or third instars) from tubes were transferred to a 330‐ml clear plastic cups covered with two layers of black cloth. Larvae were supplied with cotton stems bearing leaves, buds, and bolls until they reached the fourth instar. Plant stems were inserted into 40 ml of 1% agar gel at the bottom of cups. For each combination of strain and Bt cultivar, 30 cups with plant material were arbitrarily divided into three groups  were arbitrarily taken across dates. These samples were analyzed for concentration of Cry1A and Cry2Ab toxins using toxin‐specific enzyme‐linked immunosorbent assays (ELISA). As in Greenplate et al. , the 95% fiducial limits of the EC50, the slope of the concentration‐response line, and the standard error of the slope. We considered two EC50 values significantly different if their 95% fiducial limits did not overlap, which is a conservative criterion  was close to that of the An2Ab strain (130‐fold), with D and h values near 1 (Table Relative to the EC1 × An) differed significantly from expected mortality . With method 2, observed mortality was significantly different from expected mortality for the model with one locus, but there was no significant difference between observed and expected mortality for models with two and five loci (Table S1). The average absolute difference between observed and expected mortality was highest for the one‐locus model (53.1%) and declined for the two‐locus (24.2%) and five‐locus (12.1%) model (Table S1). Both model  and concentration  significantly affected the absolute difference between observed and expected mortality. Linear contrasts revealed that deviations between observed and expected mortality were significantly higher in the one‐locus than two‐locus (p = .0012) and five‐locus model (p < .0001). Deviations were also significantly higher in the two‐locus than five‐locus model (p = .05).In direct tests of the one‐locus model (method 1), observed mortality in the backcross progeny  and lower (18‐ to 22‐fold) between Cry2Ab and the Cry1A toxins , cultivar , and the interaction between these factors . Survival to pupation was significantly higher in An2Ab than in An on cultivars producing a single Cry1A toxin (GK19 and 33B), showing that cross‐resistance between Cry2Ab and these toxins  and plant structures , but the interaction between these factors was not significant . After accounting for differences among plants structures, the concentration of Cry1A toxins was significantly higher in the Cry1Ac cultivar (33B) than in the Cry1Ac+Cry2Ab (Bollgard 2) and Cry1Ac/Cry1Ab cultivar (GK19) , intermediate in buds , and smallest in bolls . Linear contrasts revealed significant differences between Cry1A concentration of leaves and bolls (p < .005), but Cry1A concentration did not differ significantly between leaves and buds or buds and bolls (p > .12). As in previous studies .The concentration of Cry1A toxins differed significantly among Bt cultivars  and weaker but significant cross‐resistance to the Cry1A toxins (18‐ to 22‐fold). Such cross‐resistance between Cry2Ab and Cry1A increased survival of the An2Ab strain relative to the An strain on the Cry1A cultivars, showing that cross‐resistance conferred a selective advantage to Cry2Ab‐resistant individuals on Cry1A cotton. Results with Cry1Ac+Cry2Ab plants show that redundant killing was incomplete, as survival on this pyramid was significantly higher in An2Ab than in An. However, survival did not differ significantly between An and An2Ab on non‐Bt cotton, indicating an absence of fitness costs affecting this trait. These results indicate that none of the four conditions evaluated here, which are expected to influence success of the refuge strategy for Cry1Ac+Cry2Ab cotton, were met in the An2Ab strain of H. armigera from northern China.Our results indicate that resistance to Cry2Ab in the An2Ab strain of 2 screen method, whereby a discriminating concentration of Cry2Ab was used to isolate 10 field‐derived lines carrying resistance alleles, which were then pooled and selected in the laboratory for resistance to Cry2Ab. Although such approach increases the likelihood of isolating field‐derived resistance alleles, further work will be needed to identify genes that confer resistance to Cry2Ab in An2Ab and confirm that mutations in these genes are present in field populations. The genetic basis of resistance to Cry1Ac has been well studied  was tightly linked with resistance to Cry2Ab . Results from this analysis provide strong evidence that more than one locus confer resistance to Cry2Ab in the An2Ab stain of H. armigera. By contrast, methods 2 and 3 relied on the assumptions of equal and additive effects of alleles and independently segregating loci to calculate expected responses of the backcross progeny or estimate the minimum number of independently segregating resistance genes, respectively  than Cry1Ac  . Similar to results obtained with P. gossypiella from Arizona  on Cry1Ac+Cry2Ab cotton  − ], with 0 indicating no redundant killing and 1 complete redundant killing  in individuals feeding on presquare cotton and lowest (0.81) in individuals feeding on cotton with bolls  should be considered for sustaining efficacy of pyramided Bt crops (Bates, Zhao, Roush, & Shelton, For pests with low inherent susceptibility to Bt toxins such as http://dx.doi.org/10.5061/dryad.fc8np.Data available from the Dryad Digital Repository:  Click here for additional data file.


Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous FAD2Cs genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop  Classical breeding relies on the availability of alleles in natural accessions, but also in the different subgenomes in the case of polyploid crop species. These limitations could be overcome by genetic modifications that allow the dominant expression of a transgene carrying the desired trait or allele, or alternatively the dominant down‐regulation of endogenous genes by silencing. Down‐regulation strategies, albeit efficient, may pose problems in the long run since the endogenous genes are still expressed, leading to potential instability of the desired trait after several generations  or by high‐fidelity homologous recombination that requires the presence of a template, usually provided by the other chromosomal strand. Imperfect repair of the DNA break leads, at a variable frequency, to nucleotide addition or deletion, thereby creating a stable mutation. The creation of specific DNA breaks can be obtained by the expression of nucleases such as zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) and, more recently, the Cas9 protein associated with type II clustered regulatory interspaced short palindromic repeats (CRISPR) (Hsu Nicotiana benthamiana), sorghum and sweet orange . The sgRNA#1 and sgRNA#2 were cloned downstream of the Camelina U3 and U6 promoters, respectively, in a modified version of the pDE‐Cas9 vector  were used to develop allele‐specific primers for simple allele‐discriminating PCR . Mutations could be identified in 20/53 (37.7%) and 36/44 (81.8%) of the sgRNA1 and sgRNA2 T2 lines, respectively (Table FAD2 copy (Table S2). A total of 21 different mutant alleles were identified, ranging from insertions or deletions of 1 to a few nucleotides, to larger deletions that would be predicted to impact FAD2 protein structure differently  of the T1 lines transformed with the sgRNA2 construct already showed the presence of at least one mutation among the 6 homeologous copies of 6% of theCsFAD2 genes showed significant differences in several lines. While the wild‐type OAI varied between 14% and 18%, the progeny of several T2 lines showed an OAI between 25% and 31%, with one line reaching 41% . The increase in 18:1 was mainly associated with a reduction in 18:2. Several seeds from each T2 progeny were grown, and OAI was determined from leaves of each T3 plants. Individual lipid phenotyping of T3 plants allowed rapid screening of the different lines and confirmed the extent of 18:1 increase coupled to 18:2 and 18:3 reduction . An oleic acid index (OAI) corresponding to the relative levels of monounsaturated oleic acid (18:1) among 18‐carbon saturated and polyunsaturated fatty acids  was used to rapidly compare the different lines for FAD2 activity. Fatty acid analysis of pooled seeds from T2 plants harbouring or not mutations in CsFAD2 homeologous loci  was analysed further since this residue was always included in several deletions involving 2, 3, 6 or 11 amino acids  at 5 of the 6 possible CsFAD2 positions, 2A6.6 showed the following deletions for, respectively, CsFAD2‐1, CsFAD2‐2 and CsFAD2‐3: TGG580 (W194∆), –GTGGCC583 (WP195∆) and a 40 bp (−23 to +17 around the PAM). The line 2F4.29 carried 5 frameshift mutations leading to potentially inactive FAD2 desaturases, while 2A6.6 carried 1 frameshift mutation for CsFAD2‐3 and 4 in frame amino acid deletion involving W194 leading to a FAD2 desaturase potentially still active. However, both lines showed very similar OAI. 2F4.29 showed a lower OAI in T4 seeds compared to 2A6.6 (52.9% and 59.8% respectively) but higher in T3 leaves (33.7% and 31%) (Table S2). The other deletion alleles involved 2 or more amino acids always including W194. These results indicated that W194 is probably essential for FAD2 activity and that oleic acid accumulation in the different lines resulted mostly from the combinatorial distribution of equivalent strong alleles rather than the effect of specific alleles.The other lines showed combinations of alleles with homozygous, heterozygous or biallelic combinations for the different ly Table . Of the ly Table . Tryptopfad2 alleles on seed and oil lipid profile  were included in the analysis, since the third line yielded almost no seeds (2B4‐9). The oleic content was proportional to the number of mutated CsFAD2 alleles, and a combination of 5–6 mutated alleles was required for achieving the highest OAI . It was reported that Camelina subgenome 3 (CsG3) was globally more expressed than the two other subgenomes  to 91% (triple mutant) to determine the total fatty acid profile in seeds Figure C. As expCsFAD2 genes by CRISPR‐Cas9 was highly effective in Camelina. We showed efficient mutant recovery at the three CsFAD2 genes, allowing a large diversity of genetic combinations with single, double and triple mutants that were obtained in only 3 generations . Even with a large collection of natural accessions representing the different alleles used in this study, classical breeding could not have achieved the same result so quickly and so easily. Our results showed that mutagenesis was variable between generations with mutations detected for instance in leaves and not present in the seed progeny indicating the existence of somatic mutations. Mutations taking place later in development are most likely to be transmitted to the next generation if they occur in germinal tissues. Cases where mutation could be detected in T2 but not in T1 have already been reported in Arabidopsis in previous studies   . The two sgRNA sequences were eventually selected for their consensus sequence with the three Camelina FAD2 genes . The genomic sequence of Camelina sativa cv. Céline CsFAD2‐1, CsFAD2‐2 and CsFAD2‐3 was named according to the previous work . The research of the guide RNA was based on the genome sequence of Arabidopsis using the TEFOR website  and guide RNA 2 (5′gccaagtacaagggccacccg3′) were synthesized, respectively, with Camelina U3 and Camelina U6 promoter sequences and flanked by Gateway (Thermofisher Lifetech) attb1 and attb2 recombination sites. Camelina U3 and Camelina U6 promoters used in this study  were identified by Basic Local Alignment Search Tool (http://www.phytozome.net/physcomitrella_er.php) using the Arabidopsis U6‐26 and U3B snRNA sequences (X52528 and X52629 respectively) as queries.Specificity of the sgRNA for the et al., SpeI and PmeI) with a DsRed cassette (AvrII and HpaI).Those cassettes were synthesized by GenScript and cloned individually into pDONR207 (Life Technologies) by Gateway recombination, resulting in a set of pDONR207‐sgRNA vectors in which the guide RNA cassettes could be later recombined into pDE‐Cas9 (Fauser m), MgCl2 (12.6 mm) and acetosyringone (100 μm) in a vacuum under low pressure (50 mbar). Transgenic lines were selected by the detection of the DsRed marker fluorescence in seeds  was transformed following an improved method of the traditional Arabidopsis floral‐dip method  according to the manufacturer's instructions. The presence of mutations was first detected by sequencing PCR amplicons using consensus primers for the three FAD2 genes (CDSFAD2_For145‐CsFAD2‐FN‐Rev). To determine the nature , each FAD2 gene was amplified at either sgRNA1 or sgRNA2 sites by the simple allele‐discriminating PCR (SAP) . At least one specific primer in forward  or reverse direction  was used with a reverse (CsFAD2_Enz_Rev) or forward (CsFAD2_Enz_For) primers common to the three copies. Sequencing was carried out with CDSFAD2_For145/CsFAD2_553_Rev primers for ARNg1 and CsFAD2_396_For/CDSFAD2_Rev812 primers for ARNg2. All primers used are described in Table S1 and Figure S2. Mixed Sequences Reader program (http://msr.cs.nthu.edu.tw/) was used to analyse mixed sequence caused by heterozygous mutations.DNA was extracted from Camelina leaves using the DNeasyet al. (Camelina leaves were immediately frozen after sampling at −80 °C and then lyophilized. For the quantification of the fatty acid methyl esters (FAMEs), around 2 mg of dry leaves or 20 seeds were used, and FAMEs extraction followed by GC‐MS analysis was performed according to Li et al. . The ratet al. .Figure S1. Structure of Camelina and Arabidopsis FAD2 genes.Figure S2. Camelina CsFAD2 coding sequences and the different primers used in the study.Figure S3. Combinatorial fad2 alleles associated with C18 content in T2 and T3 CsFAD2 CRISPR lines.Figure S4. OAI for the different allelic combinations at the three CsFAD2 loci.Figure S5. Expression levels of CsFAD2 genes.Figure S6. Fatty acid content of oil and cake fractions of selected CsFAD2 CRISPR lines analyzed in Figure Figure S7. Camelina U3 and U6 promoters used in this study.Table S1. Primer sequences used for amplification and sequencing.Click here for additional data file.Table S2. Summary of the different genotypes of CsFAD2 CRISPR T2 and T3 lines.Click here for additional data file. Click here for additional data file.