Co-fumigation with phosphine and sulfuryl fluoride: Potential for managing strongly phosphine-resistant rusty grain beetle, Cryptolestes ferrugineus (Stephens): Presentation

Populations of rusty grain beetle, Cryptolestes ferrugineus, have developed a very high level of resistance (1300×) to the fumigant phosphine (PH3) in Australia. Resistant insects triggered control failures, threatening the country’s annual grain market worth AU$8 billion. Although PH3 protocols were amended to manage this new resistance, fumigation requires lengthy exposure periods which has practical difficulties. While there is no suitable replacement for PH3, the current study explores potential approaches to enhance the efficacy of this fumigant. One possibility is co-fumigation of PH3 with another complementary fumigant, sulfuryl fluoride (SO2F2 or SF), with the dual goals: enhanced efficacy and minimise use of both fumigants. A cohort of mixed age eggs and adults of PH3-resistant C. ferrugineus was fumigated with PH3 and SF individually, as well as in combination inside desiccators at 25°C and 60$8 billion. Although PH3 protocols were amended to manage this new resistance, fumigation requires lengthy exposure periods which has practical difficulties. While there is no suitable replacement for PH3, the current study explores potential approaches to enhance the efficacy of this fumigant. One possibility is co-fumigation of PH3 with another complementary fumigant, sulfuryl fluoride (SO2F2 or SF), with the dual goals: enhanced efficacy and minimise use of both fumigants. A cohort of mixed age eggs and adults of PH3-resistant C. ferrugineus was fumigated with PH3 and SF individually, as well as in combination inside desiccators at 25°C and 60%RH for 168 h. Two doses below the maximal registered rates for SF (8.9 mg L- 1, equivalent to 1500 g hm-3) and PH3 (1.0 mg L-1) were tested. Co-fumigation was performed simultaneously for 168 h. Our results revealed that, the mixture of 1.1 mg L-1 or 2.2 mg L-1 of SF and 0.5 mg L-1 of PH3 over 168 h achieved complete control against resistant C. ferrugineus eggs and adults, whereas each of the tested doses failed individually. Our study confirms that SF and PH3 enhance the efficacy of each other when used in combination, which holds great potential for managing resistant C. ferrugineus

Relative tolerance and expression of resistance to phosphine in life stages of the rusty grain beetle, Cryptolestes ferrugineus

Cryptolestes ferrugineus is a serious cosmopolitan pest of stored products. Frequent and indiscriminate usage of phosphine has caused the development of high levels of resistance to this fumigant. As there are few alternatives, it is imperative that resistance to phosphine is managed. Effective management requires knowledge of key factors driving the rate of selection. One of the most important factors is the response of each resistance genotype to phosphine, especially heterozygotes. Moreover, it is important to understand the expression of resistance in all life stages as all stages are subjected to selection during fumigation. We determined the relative tolerance and resistance levels to phosphine in all life stages of homozygous parental strains (susceptible and resistant) and their F1 progeny (heterozygous) and estimated relative dominance of resistance within life stages over 48 h. In susceptible insects, relative tolerance was highest in eggs followed by pupae, then adults which had about the same tolerance as larvae. In homozygous resistant insects, the order of tolerance was adult = egg > pupae > larvae and in heterozygotes larvae > eggs > pupae > adults. All life stages expressed resistance with resistance ratios highest in adults > pupae > larvae > eggs. At LC50, resistance was incompletely recessive in eggs, pupae and adults and incompletely dominant in larvae. Eggs and adults were also incompletely recessive at LC95, but larvae were completely dominant and pupae were incompletely dominant. Our data showed that a proportion of heterozygotes in all life stages, the major carriers of resistance in the field, will survive at very high concentrations, particularly in the egg stage, forming a nucleus for reinfestation or dispersal of resistance

Gene introgression in assessing fitness costs associated with phosphine resistance in the rusty grain beetle

The current study investigates the fitness cost associated with phosphine resistance in the rusty grain beetle, Cryptolestes ferrugineus (Stephens), a problematic pest in the stored commodities that has developed strong resistance to fumigant phosphine. Three characterised insect strains: the susceptible (Ref-S), the strongly resistant (Ref-R), the introgressed resistant (Intro-R) and a segregating population (F25) derived from crossing the Ref-S and Ref-R strains were used in this study. Intro-R was developed by introgressing two phosphine resistance genes, cf_rph1 and cf_rph2 into Ref-S, aimed to reduce the influence of background genetic factors. Intro-R exhibited 592 × resistance to phosphine and homozygous for strong resistance allele, cf_rph2 (L73N). Two key fitness cost criteria, developmental time and fecundity, were assessed under optimal and suboptimal conditions (less favourable diet and low temperature). There was no significant difference in developmental time and fecundity between Ref-S and either Intro-R strain or F25 under optimal conditions. When challenged with a less favourable diet, cracked wheat + cracked sorghum (CW + CS), or exposed to a low temperature (22 °C), both Intro-R and Ref-S had similar developmental time and total numbers of F1 progeny, confirming the absence of significant fitness effects expressed in these conditions. Therefore, we conclude that strongly phosphine resistant C. ferrugineus are unlikely to incur potential fitness costs. This finding will have implications towards developing strategies to manage this pest

Potential for using pheromone trapping and molecular screening in phosphine resistance research: Presentation

Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia.Phosphine resistance monitoring typically involves bioassays of beetles from population samples collected from grain storage facilities. Insects are classified into susceptible or resistant phenotypes based on mortality or survival at one or more discriminating doses. Although valuable, phenotype testing has several drawbacks. First, phenotype testing needs live insects, and considerable effort is required to collect and maintain them before testing. Second, population samples may contain multiple genotypes expressing different levels of resistance that may not be distinguishable using discriminating dose bioassays. Third, collections are likely to be focussed around grain storages to maximise sampling success. Recent research shows that several key pests are actively dispersing through flight. The availability of commercial pheromone lures and recent advances in molecular screening provide an opportunity to provide information on resistance gene frequencies more broadly across the landscape. This approach is proving to be a valuable adjunct to traditional resistance testing in Australia

Utility of biotechnology based decision making tools in postharvest grain pest management: an Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfectant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker

The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica

Utility of biotechnology based decision making tools in postharvest grain pest management: An Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area.A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfestant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

Utility of biotechnology based decision making tools in postharvest grain pest management: an Australian case study

A major concern for the Australian grain industry in recent years is the constant threat of resistance to the key disinfectant phosphine in a range of stored grain pests. The need to maintain the usefulness of phosphine and to contain the development of resistance are critical to international market access for Australian grain. Strong levels of resistance have already been established in major pests including the lesser grain borer, Rhyzopertha dominica (F.), the red flour beetle, Tribolium castaneum (Herbst), and most recently in the rusty grain beetle Cryptolestes ferrugineus (Stephens). As a proactive integrated resistance management strategy, new fumigation protocols are being developed in the laboratory and verified in large-scale field trials in collaboration with industry partners. To aid this development, we have deployed advanced molecular diagnostic tools to accurately determine the strength and frequency of key phosphine resistant insect pests and their movement within a typical Australian grain value chain. For example, two major bulk storage facilities based at Brookstead and Millmerran in southeast Queensland, Australia, were selected as main nodes and several farms and feed mills located in and around these two sites at a scale of 25 to 100 km radius were selected and surveyed. We determined the type, pattern, frequency as well as the distribution of resistance alleles accurately for two major pests, R. dominica and T. castaneum. Overall, this information along with the phenotypic data, provide a basis for designing key intervention strategies in managing resistance problems in the study area

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

Molecular characterization of phosphine (fumigant) resistance in grain insect pests using the rust red flour beetle, Tribolium castaneum (Herbst) as a model organism

Insect resistance to pesticides is an issue of increasing global importance and a vital one for our future food security. The problem of phosphine resistance is particularly acute in tropical and sub-tropical countries like Australia, where it is used to protect 80% of the AU$8 billion annual grain harvest from insect pests of storage. The major reliance of the grains industries in these countries on phosphine are because alternative control measures are generally either expensive, leave residues on the grain or otherwise reduce the quality of the product. When markets increasingly demand insect-free and residue-free products, the resistance problem threatens the export competitiveness of the nation’s grain industry, as similar resistance problems do not exist in other wheat exporting countries, especially those in temperate regions where pest population growth is inhibited over the winter months. In the present study we used the genetic model and the Tenebrionid grain pest, rust red flour beetle, Tribolium castaneum, as a model organism to understand the genetic and molecular basis of evolution of resistance to phosphine in grain insect pests. The genetic dissection of weakly (~3.2×) and strongly (~431×) resistant strains of T. castaneum revealed that a single major gene (named tc_rph1) is responsible for weak resistance to phosphine whereas two major genes (tc_rph1 and tc_rph2) confer strong resistance, one of which is shared with the weakly resistant strain. Neither weak nor strong resistance genes were sex linked but both were inherited as an incompletely recessive autosomal trait. Genetic interaction and complementation analyses between crosses of weak and strongly resistant strains revealed the synergistic interaction of the tc_rph1 and tc_rph2 loci. The genetic confirmation that two genes conferred high-level resistance to phosphine in T. castaneum prompted me to focus on identifying these genes within the T. castaneum genome. Next-generation sequencing (Illumina- GAII) methods allowed us to resequence the whole genome of selected and unselected F4 and F19 T. castaneum populations of a single pair intercross, Susceptible X Strong-Resistant, that segregated for genes of interest in a whole-genome bulk segregant-type analysis. Subsequent differential detection of SNP homozygosity between selected and unselected datasets identified two regions, on Chr8 and Chr9 that are responsible for strong resistance. The linkage of these regions to resistance loci was confirmed by conversion of identified SNPS to polymorphic CAPS markers and fine scale mapping revealed the closest markers for the two resistance loci, tc_rph1 and tc_rph2 on Chr8 and Chr9,respectively, are approximately 0.08 cm (~30 kb) and 0.37cm (~140 kb) away from their respective most likely candidate genes, Na+/K+ ATPase and dihydrolipoamide dehydrogenase (DLD). Genotyping analysis of F4 selected resistant individuals, using the CAPS markers tcc8-597m and tccU7-138.2k, tightly linked to tc_rph1 and tc_rph2 respectively, confirmed the strongly synergistic interaction of tc_rph1 and tc_rph2 resistance alleles. These markers were also used for genotypic fitness analysis by determining the change in allelic frequency of tc_rph1 and tc_rph2 resistance loci over nineteen generations in a cross segregating for strong resistance alleles without phosphine exposure. The results showed a significant decrease in homozygous resistant tc_rph2 allelic frequency in unselected beetles at multiple generations and revealed the existence of a significant fitness cost associated with that resistance allele only, clearly demonstrating that strong resistance to phosphine in T. castaneum carries a significant fitness disadvantage, which may have significant impact on designing resistance management strategies. Phosphine also induces some significant changes in gene expression at high concentrations in strong resistant T. castaneum. A set of genes associated with amino acid metabolism, redox signalling and defence response were found differentially expressed after phosphine exposure. These genes may provide information as to some of the effects of phosphine toxicity and how organisms can react to such a chemical challenge. Interestingly, none of our candidate genes were found to significantly change expression, suggesting that resistance is possibly conferred by point mutations within those genes rather than an alteration of expression