Comparative Toxicity of Fumigants and a Phosphine Synergist Using a Novel Containment Chamber for the Safe Generation of Concentrated Phosphine Gas

BACKGROUND: With the phasing out of ozone-depleting substances in accordance with the United Nations Montreal Protocol, phosphine remains as the only economically viable fumigant for widespread use. However the development of high-level resistance in several pest insects threatens the future usage of phosphine; yet research into phosphine resistance mechanisms has been limited due to the potential for human poisoning in enclosed laboratory environments. PRINCIPAL FINDINGS: Here we describe a custom-designed chamber for safely containing phosphine gas generated from aluminium phosphide tablets. In an improvement on previous generation systems, this chamber can be completely sealed to control the escape of phosphine. The device has been utilised in a screening program with C. elegans that has identified a phosphine synergist, and quantified the efficacy of a new fumigant against that of phosphine. The phosphine-induced mortality at 20°C has been determined with an LC(50) of 732 ppm. This result was contrasted with the efficacy of a potential new botanical pesticide dimethyl disulphide, which for a 24 hour exposure at 20°C is 600 times more potent than phosphine (LC(50) 1.24 ppm). We also found that co-administration of the glutathione depletor diethyl maleate (DEM) with a sublethal dose of phosphine (70 ppm, <LC(5)), results in a doubling of mortality in C. elegans relative to DEM alone. CONCLUSIONS: The prohibitive danger associated with the generation, containment, and use of phosphine in a laboratory environment has now been substantially reduced by the implementation of our novel gas generation chamber. We have also identified a novel phosphine synergist, the glutathione depletor DEM, suggesting an effective pathway to be targeted in future synergist research; as well as quantifying the efficacy of a potential alternative to phosphine, dimethyl disulphide

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

STUDIES ON THE INHIBITION OF THE MITOCHONDRIAL ATP-ase BY REDUCTION OF THE RESPIRATORY CHAIN

The effects of inhibition of the electron transport system on the stimulation of the ATP-ase reaction in liver and rat-heart mitochondria by dinitrophenol were studied. Cyanide at concentrations 10−3M and higher effectively reduced the stimulation of ATP-ase by dinitrophenol. A similar but less striking inhibition was observed for rat-heart sarcosomes. This ATP-ase reaction was also inhibited in the presence of 10−4M cyanide with glutamate, β-hydroxybutyrate, DPNH, and succinate as reductants of the respiratory chain. Anaerobiosis also caused a substantial decrease in the ATP-ase reaction. In all instances, complete inhibition of the ATP-ase reaction could not be achieved when the respiratory chain was reduced. The magnesium-stimulated ATP-ase of rat-heart sarcosomes, of aged mitochondria, and of the Keilin–Hartree heart-muscle preparation was insensitive to reduction of the carriers suggesting that this reaction may constitute only part of the total ATP-ase reaction. The compatability of the various mechanisms of oxidative phosphorylation with these results is discussed. </jats:p

OXIDATIVE METABOLISM OF CARBOHYDRATES IN INSECTS: III. HEXOSE MONOPHOSPHATE OXIDATIVE CYCLE IN THE HOUSEFLY, MUSCA DOMESTICA L.

The component reactions of the hexose monophosphate oxidative pathway have been studied in adult houseflies. It has been shown that houseflies possess an active glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconic dehydrogenase, phosphopentose isomerase, phosphoketopentose epimerase, transketolase, and transaldolase.The cycle consists of five main steps: (a) a two-step oxidation of glucose-6-phosphate to ribulose-5-phosphate and CO2; (b) isomerization of ribulose-5-phosphate to ribose-5-phosphate; (c) epimerization of ribulose-5-phosphate to xylulose-5-phosphate; (d) conversion of pentose phosphate to sedoheptulose phosphate and triose phosphate; (e) conversion of sedoheptulose phosphate to hexose phosphate.Balance studies indicate that 2 moles of pentose phosphate are utilized for the formation of 1 mole of heptulose phosphate. However, this stoichiometry is attained after a lag period, suggesting the formation of a transient intermediate. Because the lag was eliminated by the addition of hydroxypyruvate, it is suggested that the lag was due to the time required for the combination of a C2fragment with an appropriate aldehyde acceptor.</jats:p

OXIDATIVE METABOLISM OF CARBOHYDRATES IN INSECTS: III. HEXOSE MONOPHOSPHATE OXIDATIVE CYCLE IN THE HOUSEFLY, MUSCA DOMESTICA L.

The component reactions of the hexose monophosphate oxidative pathway have been studied in adult houseflies. It has been shown that houseflies possess an active glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconic dehydrogenase, phosphopentose isomerase, phosphoketopentose epimerase, transketolase, and transaldolase.The cycle consists of five main steps: (a) a two-step oxidation of glucose-6-phosphate to ribulose-5-phosphate and CO2; (b) isomerization of ribulose-5-phosphate to ribose-5-phosphate; (c) epimerization of ribulose-5-phosphate to xylulose-5-phosphate; (d) conversion of pentose phosphate to sedoheptulose phosphate and triose phosphate; (e) conversion of sedoheptulose phosphate to hexose phosphate.Balance studies indicate that 2 moles of pentose phosphate are utilized for the formation of 1 mole of heptulose phosphate. However, this stoichiometry is attained after a lag period, suggesting the formation of a transient intermediate. Because the lag was eliminated by the addition of hydroxypyruvate, it is suggested that the lag was due to the time required for the combination of a C2fragment with an appropriate aldehyde acceptor.</jats:p

STUDIES ON THE INHIBITION OF THE MITOCHONDRIAL ATP-ase BY REDUCTION OF THE RESPIRATORY CHAIN

The effects of inhibition of the electron transport system on the stimulation of the ATP-ase reaction in liver and rat-heart mitochondria by dinitrophenol were studied. Cyanide at concentrations 10−3M and higher effectively reduced the stimulation of ATP-ase by dinitrophenol. A similar but less striking inhibition was observed for rat-heart sarcosomes. This ATP-ase reaction was also inhibited in the presence of 10−4M cyanide with glutamate, β-hydroxybutyrate, DPNH, and succinate as reductants of the respiratory chain. Anaerobiosis also caused a substantial decrease in the ATP-ase reaction. In all instances, complete inhibition of the ATP-ase reaction could not be achieved when the respiratory chain was reduced. The magnesium-stimulated ATP-ase of rat-heart sarcosomes, of aged mitochondria, and of the Keilin–Hartree heart-muscle preparation was insensitive to reduction of the carriers suggesting that this reaction may constitute only part of the total ATP-ase reaction. The compatability of the various mechanisms of oxidative phosphorylation with these results is discussed. </jats:p

ON THE NATURE OF THE LIPID SUBSTANCES INHIBITING THE DINITROPHENOL-INDUCED ATPase ACTIVITY DURING MITOCHONDRIAL AGING

Null </jats:p