Search

The rusty grain beetle, Cryptolestes ferrugineus, a major pest of stored commodities, has developed very high levels (>1000×) of resistance to the fumigant phosphine. Resistance in this species is remarkably stronger than reported in any other stored product pests demanding the need to understand the molecular basis of this trait. Previous genetic studies in other grain insect pests identified specific variants in two major genes, rph1 and rph2 in conferring the strong resistance trait. However, in C. ferrugineus, although the gene, rph1 was identified as cytochrome-b5-fatty acid desaturase, the rph2 gene has not been reported so far. We tested the candidate gene for rph2, dihydrolipoamide dehydrogenase (dld) using the recently published transcriptome of C. ferrugineus and identified three variants, L73N and A355G + D360H, a haplotype, conferring resistance in this species. Our sequence analysis in resistant strain and phosphine selected resistant survivors indicates that these variants occur either alone as a homozygote or a mixture of heterozygotes (i.e complex heterozygotes) both conferring strong resistance. We also found that one of the three variants, possibly L73N expressing “dominant” trait at low frequency in resistant insects. Comparison of dld sequences between Australian and Chinese resistant strain of this species confirmed that the identified variants are highly conserved. Our fitness analysis indicated that resistant insects may not incur significant biological costs in the absence of phosphine selection for 19 generations. Thus, we propose that the observed high levels of resistance in C. ferrugineus could be primarily due to the characteristics of three unique variants, L73N and A355G + D360H within dld.

Although phosphine resistance monitoring and management programs are well advanced in Australia, the tropical northern part of the continent has received little attention. To address this, grain storages were sampled in the Townsville region in the Burdekin river catchment area of northern Queensland, and insect populations were subjected to resistance testing. Sampling of storages including silos, food processing facilities and feedlots was carried out during July to August 2019 and February to March 2020. Of the several species collected, populations of two major pest species, Rhyzopertha dominica and Tribolium castaneum were subjected to phenotypic resistance and molecular screening using discriminating doses and gene specific DNA marker, respectively. Utilising phenotypic assay, of the 17 populations of R. dominica, 12 were diagnosed as weakly resistant and five as strongly resistant; whereas of the 34 populations of T. castaneum, two were found to be susceptible to phosphine, 15 were determined as weakly resistant and 17 as strongly resistant. The molecular diagnostics, however, determined rph2 alleles for strong resistance in some populations each of R. dominica and T. castaneum that were diagnosed as weakly resistant in the phenotypic assay. The most prevalent rph2 alleles were found to be P49S and K142E in R. dominica populations and P45S and G131S in T. castaneum populations. Our results highlight the need of more comprehensive study towards determining the level of pests and resistance risks in this region.

Development of resistance in major grain insect pest species to the key fumigant phosphine (hydrogen phosphide) across the globe has put the viability and sustainability of phosphine in jeopardy. The resistance problem has been aggravated over the past two decades, due mostly to the lack of suitable alternatives matching the major attributes of phosphine, including its low price, ease of application, proven effectiveness against a broad pest spectrum, compatibility with most storage conditions, and international acceptance as a residue-free treatment. In this review, we critically analyze the published literature in the area of phosphine resistance with special emphasis on the methods available for detection of resistance, the genetic basis of resistance development, key management strategies, and research gaps that need to be addressed.

Next-generation sequencing methods enable identification of the genetic basis of traits in species that have no prior genomic information available. The combination of next-generation sequencing, variant analysis, and linkage is a powerful way of identifying candidate genes for a trait of interest. Here, we used a comparative transcriptomics [RNA sequencing (RNAseq)] and genetic linkage analysis approach to identify the rph1 gene. rph1 variants are responsible for resistance to the fumigant phosphine (PH3) that is used to control insect pests of stored grain. In each of the four major species of pest insect of grain we have investigated, there are two major resistance genes, rph1 and rph2, which interact synergistically to produce strongly phosphine-resistant insects. Using RNAseq and genetic linkage analyses, we identified candidate resistance (rph1) genes in phosphine-resistant strains of three species: Rhyzopertha dominica (129 candidates), Sitophilus oryzae (206 candidates), and Cryptolestes ferrugineus (645 candidates). We then compared these candidate genes to 17 candidate resistance genes previously mapped in Tribolium castaneum and found only one orthologous gene, a cytochrome b5 fatty acid desaturase (Cyt-b5-r), to be associated with the rph1 locus in all four species. This gene had either missense amino acid substitutions and/or insertion/deletions/frameshift variants in each of 18 phosphine-resistant strains that were not observed in the susceptible strains of the four species. We propose a model of phosphine action and resistance in which phosphine induces lipid peroxidation through reactive oxygen species generated by dihydrolipoamide dehydrogenase, whereas disruption of Cyt-b5-r in resistant insects decreases the polyunsaturated fatty acid content of membranes, thereby limiting the potential for lipid peroxidation.

Resistance to phosphine (PH3) in key insect pests of stored grain is increasing, with a requirement for maintaining a dose as high as 1 mg l−1 for 14 d for effective fumigation, which is difficult to achieve under most commercial storage conditions. There is no suitable replacement for PH3, as most of the available alternatives suffer from specific weaknesses, creating an urgent need to increase the efficacy of this fumigant. One such possibility is co-fumigation of PH3 with another fumigant, sulfuryl fluoride (SO2F2—SF), with the goal of decreasing the time required for a successful fumigation. In this study, adult of two PH3-resistant strains in each of four key grain insect pests, Rhyzopertha dominica, (F.) (Coleoptera: Bostrichidae) Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae), and Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) were fumigated with PH3 and SF individually as well as in combinations at 25°C over 48 h. Mortality responses in each species were subjected to probit analysis to determine the LC50 and LC99.9 of PH3, SF, and PH3 + SF. Co-fumigation of PH3 with SF resulted in a 50% reduction of the PH3 concentration required to achieve 99.9% mortality in two pest species. For example, the PH3 + SF mixture, reduced the amount of PH3 required from 14.2–14.5 to 5.6–6.36 mg l−1 and from 2.71–5.03 to 0.93–1.2 mg l−1, respectively, for C. ferrugineus and S. oryzae. The overall mortality response to the PH3 + SF mixture followed an “additive model” suggesting that mutual enhancement in toxicity can be achieved with this mixture specifically to control PH3-resistant insects.

Populations of rusty grain beetle, Cryptolestes ferrugineus, a cosmopolitan grain insect pest, have recently developed a very high-level of resistance (1300×) to fumigant phosphine in Australia, threatening the country’s international grain market worth of 8 billion per year. Due to the failure of the registered rates of phosphine, industry urgently needed alternative strategies to manage this new resistance. To address this, an integrated resistance management strategy (IRM) was developed with three core components: 1. regular monitoring and characterization of resistance, 2. developing effective phosphine protocols, and 3. exploring alternative fumigants and treatments. Resistance was monitored across Australia in pest samples collected from farms and centrals storages using a modified FAO method as well as quick knockdown tests. Our results indicated that high-level resistance in C. ferrugineus is prevalent in eastern Australia, particularly in Queensland and New South Wales, particularly in the bulk handing sector. Time to population extinction was assessed in field-derived reference resistant strains using higher dosages (c) and exposure times (t) and the effective c x t regimes were validated in the field for the use by the grain industry. A phosphine concentration of 720 ppm (1 mg L-1) over 18 days was found to be effective in controlling all life stages of phosphine-resistant C. ferrugineus at 25°C. In a parallel research, the suitability of sulfuryl fluoride (ProfumeTM), as an alternative fumigant to phosphine was assessed through laboratory and field efficacy trials. Our results showed that phosphine-resistant insects are highly susceptible to sulfuryl fluoride and a CT of 750 g hm-3 (5.2 mg L-1) over 6 days at 25˚C would be sufficient against all phosphine-resistant pests including C. ferrugineus. While sulfuryl fluoride has promising potential as a “phosphine resistance breaker” the team is currently exploring ways of co-treating phosphine and sulfuryl f

Introduction: Phosphine (PH3), an effective fumigant used to protect stored grains from insect pests. As resistance to PH3 is widespread, effective fumigation requires lengthy exposure periods (>7 days) which has practical difficulties. There is an urgent need to develop alternative approaches that can increase the efficacy of PH3. One possibility is co-fumigation with PH3 and another fumigant such as sulfuryl-fluoride (SO2F2), which has properties complementary to PH3. Here we present the results of PH3+SO2F2co-fumigation of key stored grain insect pests. Methodology: Adult beetles were fumigated with PH3 and SO2F2individually as well as in combination inside air-tight desiccators at 25°C and 65% RH for 48 hours. Mortality after a recovery period of 14 days was recorded and the data was subjected to probit analysis. Experiments were replicated twice. Results: Comparison of estimated effective doses (LC99.9) of PH3, SO2F2 and PH3+SO2F2 for each of the four PH3-resistant grain insect species, Tribolium castaneum, Cryptolestes ferrugineus, Sitophilus oryzae and Rhyzopertha dominca indicated that co-fumigation with SO2F2 at half of its effective dose (0.22-0.82 mg L-1) reduced the amount of PH3 required to one third (0.33 to 5.8 mg L-1). There was no interaction between resistance to PH3 and sensitivity to SO2F2.The analysis on observed and expected mortality response curves of PH3+SO2F2in all the four species confirmed that the interaction was additive. Conclusion: SO2F2 enhances the toxicity of PH3 in an “additive” manner in PH3-resistant insect species, thus treating PH3 jointly with SO2F2 holds great potentials for pest and resistance management.

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will

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.