The rph1 Gene Is a Common Contributor to the Evolution of Phosphine Resistance in Independent Field Isolates of Rhyzopertha Dominica

Phosphine is the only economically viable fumigant for routine control of insect pests of stored food products, but its continued use is now threatened by the world-wide emergence of high-level resistance in key pest species. Phosphine has a unique mode of action relative to well-characterised contact pesticides. Similarly, the selective pressures that lead to resistance against field sprays differ dramatically from those encountered during fumigation. The consequences of these differences have not been investigated adequately. We determine the genetic basis of phosphine resistance in Rhyzopertha dominica strains collected from New South Wales and South Australia and compare this with resistance in a previously characterised strain from Queensland. The resistance levels range from 225 and 100 times the baseline response of a sensitive reference strain. Moreover, molecular and phenotypic data indicate that high-level resistance was derived independently in each of the three widely separated geographical regions. Despite the independent origins, resistance was due to two interacting genes in each instance. Furthermore, complementation analysis reveals that all three strains contain an incompletely recessive resistance allele of the autosomal rph1 resistance gene. This is particularly noteworthy as a resistance allele at rph1 was previously proposed to be a necessary first step in the evolution of high-level resistance. Despite the capacity of phosphine to disrupt a wide range of enzymes and biological processes, it is remarkable that the initial step in the selection of resistance is so similar in isolated outbreaks.

Phosphine resistance in Sitophilus oryzae (L.) from eastern Australia: Inheritance, fitness and prevalence

The inheritance and fitness of phosphine resistance was investigated in an Australian strain of the rice weevil, Sitophilus oryzae (L.), as well as its prevalence in eastern Australia. This type of knowledge may provide insights in to the development of phosphine resistance in this species with the potential for better management. This strain was 12.2 × resistant at the LC50 level based on results for adults exposed for 20 h. Data from the testing of F1 adults from the reciprocal crosses (R♀ × S♂ and S♀ × R♂) showed that resistance was autosomal and inherited as an incompletely recessive trait with a degree of dominance of -0.88. The dose-response data for the F1 × S and F1 × R test crosses, and the F2 progeny were compared with predicted dose-response assuming monogenic recessive inheritance, and the results were consistent with resistance being conferred by one major gene. There was no evidence of fitness cost based on the frequency of susceptible phenotypes in hybridized populations that were reared for seven generations without exposure to phosphine. Lack of fitness cost suggests that resistant alleles will tend to persist in field populations that have undergone selection even if selection pressure is removed. Discriminating dose tests on 107 population samples collected from farms from 2006 to 2010 show that populations containing insects with the weak resistant phenotype are common in eastern Australia, although the frequency of resistant phenotypes within samples was typically low. The prevalence of resistance is a warning that this species has been subject to considerable selection pressure and that effective resistance management practices are needed to address this problem. Crown Copyright © 2014.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Study of surface acidity of oxide catalysts by photoacoustic spectroscopy

Photoacoustic spectroscopy has been employed to estimate quantitatively the acid sites on oxide catalysts. The technique involves the measurement of the ratio of intensities of absorption bands due to conjugate bases and acids of indicators adsorbed on the catalyst surface as a function of the amount of added n-butylamine. Basic sites in sodium-impregnated alumina samples have been examined by adsorbing phenolphthalein on these surfaces.

Growth of tin oxide single crystals in porcelain tunnel kilns

Single crystals of tin oxide have been grown under conditions obtained in oil fired porcelain tunnel kilns. It was noted that the reducing conditions in the kilns help in the growth of SnO2 crystals at much lower temperatures (1300°C). The growth seems to more pronounced in presence of silicon carbide. The crystals grow as long fibres of 0.1 to 0.5 mm dia. and 10 to 50 mm length. The crystals exhibit rutile structure and the direction of growth seems to be favoured in any one of the major axes a and c.

Large-chamber methane and nitrous oxide measurements are comparable to the backward Lagrangian stochastic method

Measurement of individual emission sources (e.g., animals or pen manure) within intensive livestock enterprises is necessary to test emission calculation protocols and to identify targets for decreased emissions. In this study, a vented, fabric-covered large chamber (4.5 × 4.5 m, 1.5 m high; encompassing greater spatial variability than a smaller chamber) in combination with on-line analysis (nitrous oxide [N2O] and methane [CH4] via Fourier Transform Infrared Spectroscopy; 1 analysis min-1) was tested as a means to isolate and measure emissions from beef feedlot pen manure sources. An exponential model relating chamber concentrations to ambient gas concentrations, air exchange (e.g., due to poor sealing with the surface; model linear when ≈ 0 m3 s-1), and chamber dimensions allowed data to be fitted with high confidence. Alternating manure source emission measurements using the large-chamber and the backward Lagrangian stochastic (bLS) technique (5-mo period; bLS validated via tracer gas release, recovery 94-104%) produced comparable N2O and CH4 emission values (no significant difference at P < 0.05). Greater precision of individual measurements was achieved via the large chamber than for the bLS (mean ± standard error of variance components: bLS half-hour measurements, 99.5 ± 325 mg CH4 s-1 and 9.26 ± 20.6 mg N2O s-1; large-chamber measurements, 99.6 ± 64.2 mg CH4 s-1 and 8.18 ± 0.3 mg N2O s-1). The large-chamber design is suitable for measurement of emissions from manure on pen surfaces, isolating these emissions from surrounding emission sources, including enteric emissions. © © American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

Stable Dispersions of Metal Oxide Nanowires and Nanoparticles in Water, Dimethylformamide and Toluene

In view of the important need to generate well-dispersed inorganic nanostructures in various solvents, we have explored the dispersion of nanostructures of metal oxides such as TiO2, Fe3O4 and ZnO in solvents of differing polarity in the presence of several surfactants. The solvents used are water, dimethylformamide (DMF) and toluene. The surfactant-solvent combinations yielding the best dispersions are reported alongwith some of the characteristics of the nanostructures in the dispersions. The surfactants which dispersed TiO2 nanowires in water were polyethylene oxide (PEO), Triton X-100 (TX-100), polyvinyl alcohol (PVA) and sodium bis(2-ethylhexyl) sulphosuccinate (AOT). TiO2 nanoparticles could also be dispersed with AOT and PEO in water, and with AOT in toluene. In DMF, PVA, PEO and TX-100 were found to be effective, while in toluene, only AOT gave good dispersions. Fe3O4 nanoparticles were held for long periods of time in water by PEO, AOT, PVA and polyethylene glycol (PEG), and by AOT in toluene. In the case of ZnO nanowires, the best surfactant-solvent combinations were found to be, PEO, sodium dodecyl sulphate (SIDS) and AOT in water and AOT, PEG, PVA, PEO and TX-100 in DMF In toluene, stable dispersions of ZnO nanowires were obtained with PEO. We have also been able to disperse oxide nanostructures in non-polar solvents by employing a hydrophobic silane coating on the surface.

Do Phosphine Resistance Genes Influence Movement and Dispersal Under Starvation?

Phosphine resistance alleles might be expected to negatively affect energy demanding activities such as walking and flying, because of the inverse relationship between phosphine resistance and respiration. We used an activity monitoring system to quantify walking of Rhyzopertha dominica (F.) and a flight chamber to estimate their propensity for flight initiation. No significant difference in the duration of walking was observed between the strongly resistant, weakly resistant, and susceptible strains of R. dominica we tested, and females walked significantly more than males regardless of genotype. The walking activity monitor revealed no pattern of movement across the day and no particular time of peak activity despite reports of peak activity of R. dominica and Tribolium castaneum (Herbst) under field conditions during dawn and dusk. Flight initiation was significantly higher for all strains at 28 degrees C and 55% relative humidity than at 25, 30, 32, and 35 degrees C in the first 24 h of placing beetles in the flight chamber. Food deprivation and genotype had no significant effect on flight initiation. Our results suggest that known resistance alleles in R. dominica do not affect insect mobility and should therefore not inhibit the dispersal of resistant insects in the field.

Developing effective fumigation protocols to manage strongly phosphine-resistant Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae)

BACKGROUND The emergence of high levels of resistance in Cryptolestes ferrugineus (Stephens) in recent years threatens the sustainability of phosphine, a key fumigant used worldwide to disinfest stored grain. We aimed at developing robust fumigation protocols that could be used in a range of practical situations to control this resistant pest. RESULTS Values of the lethal time to kill 99.9% (LT99.9, in days) of mixed-age populations, containing all life stages, of a susceptible and a strongly resistant C. ferrugineus population were established at three phosphine concentrations (1.0, 1.5 and 2.0 mg L−1) and three temperatures (25, 30 and 35 °C). Multiple linear regression analysis revealed that phosphine concentration and temperature both contributed significantly to the LT99.9 of a population (P < 0.003, R2 = 0.92), with concentration being the dominant variable, accounting for 75.9% of the variation. Across all concentrations, LT99.9 of the strongly resistant C. ferrugineus population was longest at the lowest temperature and shortest at the highest temperature. For example, 1.0 mg L−1 of phosphine is required for 20, 15 and 15 days, 1.5 mg L−1 for 12, 11 and 9 days and 2.0 mg L−1 for 10, 7 and 6 days at 25, 30 and 35 °C, respectively, to achieve 99.9% mortality of the strongly resistant C. ferrugineus population. We also observed that phosphine concentration is inversely proportional to fumigation period in regard to the population extinction of this pest. CONCLUSION The fumigation protocols developed in this study will be used in recommending changes to the currently registered rates of phosphine in Australia towards management of strongly resistant C. ferrugineus populations, and can be repeated in any country where this type of resistance appears.

Impact of nitrification inhibitor (DMPP) on soil nitrous oxide emissions from an intensive broccoli production system in sub-tropical Australia

Vegetable cropping systems are often characterised by high inputs of nitrogen fertiliser. Elevated emissions of nitrous oxide (N2O) can be expected as a consequence. In order to mitigate N2O emissions from fertilised agricultural fields, the use of nitrification inhibitors, in combination with ammonium based fertilisers, has been promoted. However, no data is currently available on the use of nitrification inhibitors in sub-tropical vegetable systems. A field experiment was conducted to investigate the effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on N2O emissions and yield from broccoli production in sub-tropical Australia. Soil N2O fluxes were monitored continuously (3 h sampling frequency) with fully automated, pneumatically operated measuring chambers linked to a sampling control system and a gas chromatograph. Cumulative N2O emissions over the 5 month observation period amounted to 298 g-N/ha, 324 g-N/ha, 411 g-N/ha and 463 g-N/ha in the conventional fertiliser (CONV), the DMPP treatment (DMPP), the DMMP treatment with a 10% reduced fertiliser rate (DMPP-red) and the zero fertiliser (0N), respectively. The temporal variation of N2O fluxes showed only low emissions over the broccoli cropping phase, but significantly elevated emissions were observed in all treatments following broccoli residues being incorporated into the soil. Overall 70–90% of the total emissions occurred in this 5 weeks fallow phase. There was a significant inhibition effect of DMPP on N2O emissions and soil mineral N content over the broccoli cropping phase where the application of DMPP reduced N2O emissions by 75% compared to the standard practice. However, there was no statistical difference between the treatments during the fallow phase or when the whole season was considered. This study shows that DMPP has the potential to reduce N2O emissions from intensive vegetable systems, but also highlights the importance of post-harvest emissions from incorporated vegetable residues. N2O mitigation strategies in vegetable systems need to target these post-harvest emissions and a better evaluation of the effect of nitrification inhibitors over the fallow phase is needed.

Atomic Layer Deposition of Multicomponent Oxide Materials

Atomic layer deposition (ALD) is a method for thin film deposition which has been extensively studied for binary oxide thin film growth. Studies on multicomponent oxide growth by ALD remain relatively few owing to the increased number of factors that come into play when more than one metal is employed. More metal precursors are required, and the surface may change significantly during successive stages of the growth. Multicomponent oxide thin films can be prepared in a well-controlled way as long as the same principle that makes binary oxide ALD work so well is followed for each constituent element: in short, the film growth has to be self-limiting. ALD of various multicomponent oxides was studied. SrTiO3, BaTiO3, Ba(1-x)SrxTiO3 (BST), SrTa2O6, Bi4Ti3O12, BiTaO4 and SrBi2Ta2O9 (SBT) thin films were prepared, many of them for the first time by ALD. Chemistries of the binary oxides are shown to influence the processing of their multicomponent counterparts. The compatibility of precursor volatilities, thermal stabilities and reactivities is essential for multicomponent oxide ALD, but it should be noted that the main reactive species, the growing film itself, must also be compatible with self-limiting growth chemistry. In the cases of BaO and Bi2O3 the growth of the binary oxide was very difficult, but the presence of Ti or Ta in the growing film made self-limiting growth possible. The application of the deposited films as dielectric and ferroelectric materials was studied. Post-deposition annealing treatments in different atmospheres were used to achieve the desired crystalline phase or, more generally, to improve electrical properties. Electrode materials strongly influenced the leakage current densities in the prepared metal insulator metal (MIM) capacitors. Film permittivities above 100 and leakage current densities below 110-7 A/cm2 were achieved with several of the materials.

Metal-insulator-semiconductor capacitors with bismuth oxide as insulator

Metal-insulator-semiconductor capacitors using aluminum Bi2O3 and silicon have been studied for varactor applications. Reactively sputtered Bi2O3 films which under suitable proportions of oxygen and argon and had high resistivity suitable for device applications showed a dielectric constant of 25. Journal of Applied Physics is copyrighted by The American Institute of Physics.