Status of resistance to phosphine in insect pests of stored products in Vietnam

In response to numerous reports of failures to control insect pests of stored products with phosphine in Vietnam, a national survey for resistance to this key fumigant was undertaken in 2009–2011. Data from a more limited survey undertaken by the authors in 2002 in northern Vietnam are also presented. Samples collected in the 2002 survey (Sitophilus oryzae, n=8; Tribolium castaneum, n=8) were tested using a full dose- response assay, while for the 2009–11 survey, F1 generations were tested for resistance with two discriminating dosages of phosphine to detect frequency of weak and strong resistance phenotypes. Compared with a susceptible reference strain, in 2002, resistance to phosphine was indicated in six T. castaneum samples but only two of S. oryzae. Resistance factor, however, did not exceed 2.8-fold in T. castaneum and 1.7 in S. oryzae indicating relatively low frequency and weak expression of resistance. In 2009–11 survey, 176 samples were collected from a range of food and feed storages along the supply chain and from all major regions of Vietnam (125 sites). Rhyzopertha dominica and S. oryzae were the most common species found infesting stored commodities. Resistance was detected at high frequency in all the species. Weak and strong resistance phenotype frequencies were, respectively: Cryptolestes ferrugineus (37 and 58%, n=19), R. dominica (1.5 and 97%, n=65), S. oryzae (34 and 59%, n=82) and T. castaneum (70 and 30%, n=10). Strong resistance phenotype was detected in all the major regions and all parts of the supply chain but frequency was the highest in central storages and animal feed establishments. The increase in frequency and strength of resistance to phosphine in the eight years between the two surveys has been rapid and dramatic. The survey demonstrates the threat of resistance to grain protection in Vietnam and highlights the need for training of fumigators, and the development and adoption of phosphine resistance management tactics nationally.

Resistance to phosphine in Sitophilus oryzae in Australia: A national analysis of trends and frequencies over time and geographical spread

In Australia, along with many other parts of the world, fumigation with phosphine is a vital component in controlling stored grain insect pests. However, resistance is a factor that may limit the continued efficacy of this fumigant. While strong resistance to phosphine has been identified and characterised, very little information is available on the causes of its development and spread. Data obtained from a unique national resistance monitoring and management program were analysed, using Bayesian hurdle modelling, to determine which factors may be responsible. Fumigation in unsealed storages, combined with a high frequency of weak resistance, were found to be the main criteria that led to the development of strong resistance in Sitophilus oryzae. Independent development, rather than gene flow via migration, appears to be primarily responsible for the geographic incidence of strong resistance to phosphine in S. oryzae. This information can now be utilised to direct resources and education into those areas at high risk and to refine phosphine resistance management strategies.

Influence of phosphine resistance genes on flight propensity and resource location in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): the landscape for selection

Phosphine resistance in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has evolved through changes to enzymes involved in basic metabolic pathways. These changes impose metabolic stress and could affect energy-demanding behaviours. We therefore tested whether phosphine resistance alleles impact the movement of these insects in their quest for new resources. We measured walking and flight parameters of four T. castaneum genotypes: (1) a field-derived population, (2) a laboratory cultured, phosphine-susceptible reference strain, (3) a laboratory cultured, phosphine-resistant reference strain, and (4) a resistant introgressed strain that is almost identical genetically to the susceptible population. The temporal pattern of flight was identical across all populations, but resistant beetles took flight significantly less, walked more slowly, and located resources less successfully than did susceptible beetles. Also, the field-derived beetles (proved not to be carrying resistance genes) walked significantly faster and more directly towards food resources, and had a higher propensity for flight when compared to the susceptible laboratory beetles. These negative effects suggest survival of beetles with the resistance alleles will be compromised should they leave phosphine application sites. The field for selection therefore extends beyond the site at which phosphine fumigant imposed its effect, and other mutations are also likely to be affected in this way.

Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera : Tenebrionidae) populations from Turkey

Fumigation with phosphine gas is the primary method of controlling stored grain pests. In Turkey, phosphine has been used extensively since the 1950's. Even though high levels of phosphine resistance have been detected in several key stored products pests across the world, it has never been studied in Turkey despite this long history of phosphine use. High-level phosphine resistance has been detected and genetically characterised previously in the rust red flour beetle, Tribolium castaneum in other countries. Since this pest is also a common problem in stored grain environment in Turkey, the current study was undertaken for the first time, to investigate the distribution and strength of phosphine resistance in T. castaneum. Four strains of T. castaneum were tested through bioassays for determining the weak and strong phosphine resistance phenotypes on the basis of the response of adults to discriminating phosphine concentrations of 0.03 mg/L and 0.25 mg/L, for 20 hour exposures respectively. Phenotype testing showed all strains exhibited some level of phosphine resistance with a maximum level of 196 fold. Sequencing and genetic testing of seven field-collected strains showed that all of them carried a strong resistance allele in at the rph2 locus similar to the one previously reported. Our results show that strong resistance to phosphine is common in Turkish strains of T. castaneum.

Random Mating Between Two Widely Divergent Mitochondrial Lineages of Cryptolestes ferrugineus (Coleoptera: Laemophloeidae): A Test of Species Limits in a Phosphine-Resistant Stored Product Pest

Effective pest management relies on accurate delimitation of species and, beyond this, on accurate species identification. Mitochondrial COI sequences are useful for providing initial indications in delimiting species but, despite acknowledged limitations in the method, many studies involving COI sequences and species problems remain unresolved. Here we illustrate how such impasses can be resolved with microsatellite and nuclear sequence data, to assess more directly the amount of gene flow between divergent lineages. We use a population genetics approach to test for random mating between two 8 ± 2% divergent COI lineages of the rusty grain beetle, Cryptolestes ferrugineus (Stephens). This species has become strongly resistant to phosphine, a fumigant used worldwide for disinfesting grain. The possibility of cryptic species would have significant consequences for resistance management, especially if resistance was confined to one mitochondrial lineage. We find no evidence of restricted gene flow or nonrandom mating across the two COI lineages of these beetles, rather we hypothesize that historic population structure associated with early Pleistocene climate changes likely contributed to divergent lineages within this species.

Effect of diet on phosphine toxicity, rate of development and reproduction of the rice weevil Sitophilus oryzae (Linnaeus)

One of the loci responsible for strong phosphine resistance encodes dihydrolipoamide dehydrogenase (DLD). The strong co-incidence of enzyme complexes that contain DLD, and enzymes that require thiamine as a cofactor, motivated us to test whether the thiamine deficiency of polished white rice could influence the efficacy of phosphine fumigation against insect pests of stored grain. Three strains of Sitophilus oryzae (susceptible, weak and strong resistance) were cultured on white rice (thiamine deficient), brown rice or whole wheat. As thiamine is an essential nutrient, we firstly evaluated the effect of white rice on developmental rate and fecundity and found that both were detrimentally affected by this diet. The mean time to reach adult stage for the three strains ranged from 40 to 43 days on brown rice and 50–52 days on white rice. The mean number of offspring for the three strains ranged from 7.7 to 10.3 per female over a three day period on brown rice and 2.1 to 2.6 on white rice. Growth and reproduction on wheat was similar to that on brown rice except that the strongly resistant strain showed a tendency toward reduced fecundity on wheat. The susceptible strain exhibited a modest increase in tolerance to phosphine on white rice as expected if thiamine deficiency could mimic the effect of the dld resistance mutation at the rph2 locus. The strongly resistant strain did not respond to thiamine deficiency, but this was expected as these insects are already strongly resistant. We failed, however, to observe the expected synergistic increase in resistance due to combining thiamine deficiency with the weakly resistant strain. The lack of interaction between thiamine content of the diet and the resistance genotype in determining the phosphine resistance phenotype suggests that the mode of inhibition of the complexes is a critical determinant of resistance.

meso-Porphyrinylphosphine Oxides: Mono- and Bidentate Ligands for Supramolecular Chemistry and the Crystal Structures of Monomeric [10,20-Diphenylporphyrinatonickel(II)-5,15-diyl]-bis-[P(O)Ph2] and Polymeric Self-Coordinated ([10,20-Diphenylporphyrinatozinc(II)-5,15-diyl]-bis-[P(O)Ph2])

A series of porphyrins substituted in one or two meso-positions by diphenylphosphine oxide groups has been prepared by the palladium catalysed reaction of diphenylphosphine or its oxide with the corresponding bromoporphyrins. Compounds {MDPP-[P(O)Ph2]n} (M = H2, Ni, Zn; H2DPP = 5,15-diphenylporphyrin; n = 1, 2) were isolated in yields of 60-95%. The reaction is believed to proceed via the conventional oxidative addition, phosphination and reductive elimination steps, as the stoichiometric reaction of η1-palladio(II) porphyrin [PdBr(H2DPP)(dppe)] (H2DPP = 5,15-diphenylporphyrin; dppe = 1,2-bis(diphenylphosphino)ethane) with diphenylphosphine oxide also results in the desired mono-porphyrinylphosphine oxide [H2DPP-P(O)Ph2]. Attempts to isolate the tertiary phosphines failed due to their extreme air-sensitivity. Variable temperature 1H NMR studies of [H2DPP-P(O)Ph2] revealed an intrinsic lack of symmetry, while fluorescence spectroscopy showed that the phosphine oxide group does not behave as a "heavy atom" quencher. The electron withdrawing effect of the phosphine oxide group was confirmed by voltammetry. The ligands were characterised by multinuclear NMR and UV-visible spectroscopy as well as mass spectrometry. Single crystal X-ray crystallography showed that the bis(phosphine oxide) nickel(II) complex {[NiDPP-[P(O)Ph2]2} is monomeric in the solid state, with a ruffled porphyrin core and the two P=O fragments on the same side of the average plane of the molecule. On the other hand, the corresponding zinc(II) complex formed infinite chains through coordination of one Ph2PO substituent to the neighbouring zinc porphyrin through an almost linear P=O---Zn unit, leaving the other Ph2PO group facing into a parallel channel filled with disordered water molecules. These new phosphine oxides are attractive ligands for supramolecular porphyrin chemistry.

[P,P-Di-tert-butyl-N-trimethylsilyl- P-(trimethylsilylamino)phosphineimidato-N,N]bis(pyridine-N)- lithium(I)

In the title compound, [Li(C14H36N2PSi2)(C5H5N)2], the bulky chelating monoanionic P,P-di-tert-butyl-N-trimethylsilyl-P-(trimethylsilylamino)phosphine imidate ligand and two pyridine ligands bind to Li in a pseudo-tetrahedral arrangement with twofold symmetry. The Li-N [phosphine]distance is 2.048 (5) Å, while the LiP distance is 2.520 (6) Å

Crowded Diphosphinomethane Ligands in Catalysis: [(R2PCH2PR′2-κ2P)-NiR″]+ Cations for Ethylene Polymerization without Activators

The preparation of a series of nickel dichloride complexes with bulky diphosphinomethane chelate ligands R2PCH2PR′2 is reported. Reaction with the appropriate Grignard reagent leads to the corresponding dimethyl and dibenzyl complexes. Cationic monomethyl and mono-η3-benzyl complexes are generated from these dialkyl complexes by protonation with [H(OEt2)2]+[B(3,5-(CF3)2C6H3)4]−, while the complex [(dtbpm κ2P)Ni(η3-CH(CH2Ph)Ph]+[B(3,5-(CF3)2C6H3)4]−is obtained from protonation of the Ni(0) olefin complex (dtbpm-κ2P)N(η2-trans-stilbene). Crystal structures of examples of dichlorides, dimethyl, dibenzyl, cationic methyl, and cationic η3-benzyl complexes are reported. Solutions of the cations polymerize ethylene under mild conditions and without the necessity of an activating agent, to form polyethylene having high molecular weights and low degrees of chain branching. In comparison to the Ni methyl cations, the η3-benzyl cation complexes are more stable and somewhat less active but still very efficient in C2H4 polymerization. The effect on the resulting polyethylene of varying the substituents R, R′ on the phosphine ligand has been examined, and a clear trend for longer chain PE with less branching in the presence of more bulky substituents on the diphosphine has been found. Density functional calculations have been used to examine the rapid suprafacial η3 to η3 haptotropic shift processes of the[(R2PCH2PR′2)Ni] fragment and the η3−η1 change of the coordination mode of the benzyl group required for polymerization in those cations.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Phosphorus-based functional groups as hydrogen bonding templates for rotaxane formation

We report on the use of the hydrogen bond acceptor properties of some phosphorus-containing functional groups for the assembly of a series of [2]rotaxanes. Phosphinamides, and the homologous thio– and selenophosphinamides, act as hydrogen bond acceptors that, in conjunction with an appropriately positioned amide group on the thread, direct the assembly of amide-based macrocycles around the axle to form rotaxanes in up to 60% yields. Employing solely phosphorus-based functional groups as the hydrogen bond accepting groups on the thread, a bis(phosphinamide) template and a phosphine oxide-phosphinamide template afforded the corresponding rotaxanes in 18 and 15 % yields, respectively. X-Ray crystallography of the rotaxanes shows the presence of up to four intercomponent hydrogen bonds between the amide groups of the macrocycle and various hydrogen bond accepting groups on the thread, including rare examples of amide-to-phosphonamide, -thiophosphinamide and -selenophosphinamide groups. With a phosphine oxide-phosphinamide thread, the solid state structure of the rotaxane is remarkable, featuring no direct intercomponent hydrogen bonds but rather a hydrogen bond network involving water molecules that bridge the H-bonding groups of the macrocycle and thread through bifurcated hydrogen bonds. The incorporation of phosphorus-based functional groups into rotaxanes may prove useful for the development of molecular shuttles in which the macrocycle can be used to hinder or expose binding ligating sites for metal-based catalysts.

A Bayesian hurdle model for analysis of an insect resistance monitoring database

Motivated by the analysis of the Australian Grain Insect Resistance Database (AGIRD), we develop a Bayesian hurdle modelling approach to assess trends in strong resistance of stored grain insects to phosphine over time. The binary response variable from AGIRD indicating presence or absence of strong resistance is characterized by a majority of absence observations and the hurdle model is a two step approach that is useful when analyzing such a binary response dataset. The proposed hurdle model utilizes Bayesian classification trees to firstly identify covariates and covariate levels pertaining to possible presence or absence of strong resistance. Secondly, generalized additive models (GAMs) with spike and slab priors for variable selection are fitted to the subset of the dataset identified from the Bayesian classification tree indicating possibility of presence of strong resistance. From the GAM we assess trends, biosecurity issues and site specific variables influencing the presence of strong resistance using a variable selection approach. The proposed Bayesian hurdle model is compared to its frequentist counterpart, and also to a naive Bayesian approach which fits a GAM to the entire dataset. The Bayesian hurdle model has the benefit of providing a set of good trees for use in the first step and appears to provide enough flexibility to represent the influence of variables on strong resistance compared to the frequentist model, but also captures the subtle changes in the trend that are missed by the frequentist and naive Bayesian models.

Mathematical modelling of fumigant transport in stored grain

Computational fluid dynamics, analytical solutions, and mathematical modelling approaches are used to gain insights into the distribution of fumigant gas within farm-scale, grain storage silos. Both fan-forced and tablet fumigation are considered in this work, which develops new models for use by researchers, primary producers and silo manufacturers to assist in the eradication grain storage pests.

Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum : a new approach

Reported homocysteine (HCY) concentrations in human serum show poor concordance amongst laboratories due to endogenous HCY in the matrices used for assay calibrators and QCs. Hence, we have developed a fully validated LC–MS/MS method for measurement of HCY concentrations in human serum samples that addresses this issue by minimising matrix effects. We used small volumes (20 μL) of 2% Bovine Serum Albumin (BSA) as surrogate matrix for making calibrators and QCs with concentrations adjusted for the endogenous HCY concentration in the surrogate matrix using the method of standard additions. To aliquots (20 μL) of human serum samples, calibrators or QCs, were added HCY-d4 (internal standard) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) as reducing agent. After protein precipitation, diluted supernatants were injected into the LC–MS/MS. Calibration curves were linear; QCs were accurate (5.6% deviation from nominal), precise (CV% ≤ 9.6%), stable for four freeze–thaw cycles, and when stored at room temperature for 5 h or at −80 °C (27 days). Recoveries from QCs in surrogate matrix or pooled human serum were 91.9 and 95.9%, respectively. There was no matrix effect using 6 different individual serum samples including one that was haemolysed. Our LC–MS/MS method has satisfied all of the validation criteria of the 2012 EMA guideline.