Phosphine Resistance in the Rust Red Flour Beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): Inheritance, Gene Interactions and Fitness Costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12–20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes

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

Insect Pest Management in Stored Grain

Once cereal grain is harvested and put into storage, it provides a resource for a range of insect pests of stored grain. With few exceptions, these insects rarely attack grain in the field before harvest, but once the grain is in storage there is a degree of inevitability that insect infestation will occur. This means that methods are needed to either disinfest grain or to protect it from infestation during storage. The aim of this chapter is to review recent advances in insect pest management in stored grain, ranging from methods that are well established to those that are still being evaluated. This topic has been the subject of considerable laboratory and field research as evidenced by the large and growing body of published studies. Resistance to phosphine resistance and various insecticides, as well as the phase-out of methyl bromide as an ozone-depleting substance, continue to be major drivers for research on management of insects in stored products. Other research has focussed on improving the basic understanding of various methods or ways of improving methods currently in use. Despite extensive research on a wide variety of chemical and non-chemical treatments, very few have been commercialised. Two examples are spinosad, which has been registered as a grain protectant, and sulfuryl fluoride, which is now available as a grain fumigant. The interest in non-chemical treatments, especially aeration cooling, is encouraging. In general, integrated pest management is seen as the goal of entomologists, requiring the strategic integration of multiple methods to provide maximum effect with minimal health and environmental risks. Some of the chemical and non-chemical treatments reviewed in this chapter have great potential to be used as part of an integrated approach

Insecticide Resistance

Insecticides, including contact chemicals and fumigants, are essential components of the majority of stored product protection systems. Their use enables the implementation of effective quarantine systems, ensures food security and facilitates domestic and international trade. Insecticides have many advantages. They can be integrated easily into grain handling logistics; they reliably provide the freedom from insect infestation demanded by many markets; and they are relatively inexpensive to apply. Despite their central importance, however, there are a surprisingly small number of chemicals used in the protection of stored products. Chemical residue levels are tightly regulated as stored products are usually foods. In addition, because of the often large volumes of commodity involved and convenience of application, fumigants are frequently the preferred treatments, rather than liquid insecticides. However, fumigant use requires strict workplace health and safety precautions and must comply with stringent environmental constraints. These factors, coupled with toxicological considerations, limit the range of materials available for application to grain and make them costly to develop. For these reasons, loss of any one chemical treatment will have a significant impact on pest management. Consequently, the development of resistance in stored product pests to any registered insecticide is a particularly significant problem that requires urgent solutions

A co-fumigation strategy utilizing reduced rates of phosphine (PH3) and sulfuryl fluoride (SF) to control strongly resistant rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae)

BACKGROUND Managing resistance to phosphine (PH3) in rusty grain beetle, Cryptolestes ferrugineus, is challenging, as strongly resistant insects of this species require very high concentrations over lengthy exposure periods (>10 days). Recently, approaches that enhance the efficacy of PH3 have gained momentum to control this pest, especially co-fumigations. In this study, efficacy of co-fumigating PH3 with another commercially available fumigant, sulfuryl fluoride (SF), has been evaluated against adults and eggs of two PH3-resistant strains of C. ferrugineus. Concentrations of the mixture, representing lower than current application rates of both fumigants, were tested towards its field use. RESULTS Co-fumigation of PH3 with SF was achieved in two patterns: over a continuous exposure period of 168 h simultaneously and sequentially over two periods of 78 h, in which insects were exposed to SF first followed by PH3 with 12 h aeration in-between. Results of simultaneous fumigations identified two effective co-fumigation rates, SF 185 + PH3 168 g hm−3 and SF 370 + PH3 84 g hm−3 that yielded complete control of adults and eggs. These two rates also were equally effective when they were applied sequentially and produced consistent results. Irrespective of application methods, concentrations of both PH3 and SF failed individually in achieving complete mortality of either adults or eggs or both. CONCLUSION Our results confirmed that a co-fumigation strategy involving half the current standard rate of PH3 (84 g hm−3) with one-fourth of the current maximal registered rate of SF (370 g hm−3) can provide effective control of strongly PH3-resistant C. ferrugineus

Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha Dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. Dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. Dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation