BACKGROUND:Phosphine (PH3) is a slow-acting fumigant, therefore control of resistant insect pests requires high concentrations of 540-1080 ppm (0.75-1.5 g m-3) over long exposure periods of up to 21 days. This is difficult to achieve under many practical storage conditions; hence, there is a need to enhance the toxicity of PH3, so that resistant insects can be eradicated in a shorter fumigation time. Co-fumigation with PH3, along with other atmospheric gases (carbon dioxide (CO2) and nitrogen (N2)), enhances PH3 toxicity in laboratory-scale experiments; however, this information has not been translated into practical pest management protocols for adoption by the industry. METHODOLOGY:The effectiveness of co-fumigation with PH3 plus two of the atmospheric gases (N2 and CO2) was evaluated in fumigation chambers and commercial-scale rice storage silos. Strongly PH3-resistant insects of mixed life stages of key grain insect pests, Rhyzopertha dominica, Tribolium castaneum, Sitophilus oryzae, and Cryptolestes ferrugineus were placed in cages inside the mini-fumigation chambers or silos containing freshly milled rice grains. Fumigations were performed with currently registered application rates of PH3 (0.75-1.5 g m-3) in combination with selected concentrations of CO2 (5-30%) and N2 (90-98%)/low oxygen (O2, 5-10%) over a short exposure period of 4 days. Test insect cages were retrieved after venting, and the effectiveness of the hypoxic fumigation was evaluated in terms of per cent mortalities of adults and progeny. RESULTS AND CONCLUSION:Both the pilot scale and field fumigation research confirmed that the triplet mixture, PH3 0.75-1.0 g m-3 + high N2 (90-95%) + CO2 (5-20%) over 4 days was very effective against three strongly PH3-resistant species, R. dominica, T. castaneum, and S. oryzae and caused complete adult and progeny mortality. However, higher concentrations of this triplet mixture, PH3 1.5 g m-3 + high N2 (90-95%) + CO2 (30%) over 4 days was required to achieve complete mortality of adults and progeny of strongly resistant C. ferrugineus. At both the low and high co-fumigation dose rates, the required minimum effective PH3 concentrations (540-1080 ppm) were maintained over the 4-day exposure period, confirming the reliability of these rates for industrial applications. © 2025 Society of Chemical Industry.

Traps baited with aggregation pheromone lures for the lesser grain borer, Rhyzopertha dominica (F.) were deployed at two bulk grain storage facilities in southern Queensland, Australia from January 2018 to March 2019. This trapping provided data on flight activity in this species as well as sample specimens for screening for the strong phosphine resistance allele (K142E) of the rph2 gene. This species is a major pest in Australia where phosphine is the commonly used means of disinfesting stored cereal grains. Therefore, information on infestation pressure from flying beetles and the frequency of resistance genes in these beetles may provide a basis for improved pest and resistance management. Rhyzopertha dominica beetles were trapped at both depots throughout the study, with totals of 3699 and 6683 trapped at the two depots, respectively. Trap catch tended to fluctuate in synchrony at the two depots, with the lowest number caught during winter. There was a total of 15 trapping events during the study and the rph2 allele (K142E) was detected in 14 of them. The overall resistance allele frequency was estimated to be 7.4%, and this variant was present in 12.6% of screened beetles from each site, with most of them being heterozygotes (rph2rs). This study shows that there was a great deal of similarity between the two sites, in relation to flight activity and frequency of rph2 alleles (K142E). It also shows the potential for using pheromone trapping as a passive means of collecting R. dominica beetles for monitoring the frequency of phosphine resistance alleles in a typical grain storage environment.

Phosphine is a lethal metabolic toxin with a mode of action involving oxidative stress that depends on aerobic respiration. Phosphine gas is used as a fumigant to protect food commodities from insect pests. In countries with poorly regulated fumigation practices, recurring sublethal exposure causes serious headache and other symptoms that usually resolve without long term consequences. Industrial accidents can result in serious acute exposure that can cause long term neurological or pulmonary damage. Suicide attempts involving ingestion of phosphine tablets causes severe organ damage. Suicide attempts are usually successful due to the lack of an effective antidote.

Phosphine is the most widely used fumigant for stored grains due to a lack of better alternatives, all of which have serious shortcomings that restrict their use. The extensive use of phosphine has led to the development of resistance among insect pests of grain, which threatens its status as a reliable fumigant. Understanding the mode of action of phosphine as well as its resistance mechanisms provides insight that may lead to improved phosphine efficacy and pest control strategies. The mechanisms of action in phosphine vary from disrupting metabolism and oxidative stress to neurotoxicity. Phosphine resistance is genetically inherited and is mediated by the mitochondrial dihydrolipoamide dehydrogenase complex. In this regard, laboratory studies have revealed treatments that synergistically enhance phosphine toxicity that may be used to suppress resistance development and enhance efficacy. Here, we discuss the reported phosphine modes of action, mechanisms of resistance and interactions with other treatments.

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.

Gut microbes play an important role in the biology and evolution of insects. Australian native dung beetles (Scarabaeinae) present an opportunity to study gut microbiota in an evolutionary context as they come from two distinct phylogenetic lineages and some species in each lineage have secondarily adapted to alternative or broader diets. In this study, we characterised the hindgut bacterial communities found in 21 species of dung beetles across two lineages using 16S rRNA sequencing. We found that gut microbial diversity was more dependent on host phylogeny and gut morphology than specific dietary preferences or environment. In particular, gut microbial diversity was highest in the endemic, flightless genus Cephalodesmius that feeds on a broad range of composted organic matter. The hindgut of Cephalodesmius harbours a highly conserved core set of bacteria suggesting that the bacteria are symbiotic. Symbiosis is supported by the persistence of the core microbiota across isolated beetle populations and between species in the genus. A co-evolutionary relationship is supported by the expansion of the hindgut to form a fermentation chamber and the fermentative nature of the core microbes. In contrast, Australian species of the widespread dung beetle genus Onthophagus, specialise on a single food resource such as dung or fungus, exhibit minimal food processing behaviour, have a short, narrow hindgut and a variable gut microbiota with relatively few core bacterial taxa. A conserved, complex gut microbiota is hypothesised to be unnecessary for this highly mobile genus.IMPORTANCE Dung beetles are a very important part of an ecosystem because of their role in the removal and decomposition of vertebrate dung. It has been suspected that symbiotic gut bacteria facilitate this role, a hypothesis that we have explored with high throughput barcoding. We found that differences in hindgut morphology had the greatest effect on the bacterial community composition. Species with a hindgut fermentation chamber harboured a distinctly different hindgut community compared to those species with a narrow, undifferentiated hindgut. Diet and phylogeny were also associated with differences in gut community. Further understanding of the relationships between dung beetles and their gut microbes will provide insights into the evolution of their behaviours and how gut communities contribute to their fitness.

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.

A decrease in energy metabolism is associated with Alzheimer's disease (AD), but it is not known whether the observed decrease exacerbates or protects against the disease. The importance of energy metabolism in AD is reinforced by the observation that variants of dihydrolipoamide dehydrogenase (DLD), is genetically linked to late-onset AD. To determine whether DLD is a suitable therapeutic target, we suppressed the dld-1 gene in Caenorhabditis elegans that express human Aβ peptide in either muscles or neurons. Suppression of the dld-1 gene resulted in significant restoration of vitality and function that had been degraded by Aβ pathology. This included protection of neurons and muscles cells. The observed decrease in proteotoxicity was associated with a decrease in the formation of toxic oligomers rather than a decrease in the abundance of the Aβ peptide. The mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which like dld-1 gene expression inhibits ATP synthesis, had no significant effect on Aβ toxicity. Proteomics data analysis revealed that beneficial effects after dld-1 suppression could be due to change in energy metabolism and activation of the pathways associated with proteasomal degradation, improved cell signaling and longevity. Thus, some features unique to dld-1 gene suppression are responsible for the therapeutic benefit. By direct genetic intervention, we have shown that acute inhibition of dld-1 gene function may be therapeutically beneficial. This result supports the hypothesis that lowering energy metabolism protects against Aβ pathogenicity and that DLD warrants further investigation as a therapeutic target.

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.

Strong resistance to phosphine (PH3) in the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Laemophloeidae: Coleoptera) poses a serious risk to stored-grain biosecurity. Resistant populations hold risk of surviving in PH3 fumigation, particularly in storage structure that limits achieving very high concentrations of PH3, demanding the need for alternative fumigation strategies. Cofumigation with PH3 and carbon dioxide (CO2) is one alternative approach that has the potential to be used widely. CO2 fumigation of adults of strongly PH3-resistant reference strain of C. ferrugineus, for 48 h, showed that the effective concentration (LC50) of CO2 was 30.99%. This 30% level of CO2 in combination with PH3 decreased the LC50 of PH3 from 6.7 mg/liter to 0.84 mg/liter, an eightfold increase in PH3 efficacy relative to PH3 fumigation in normal air. The LC 99.9 decreased from 16.2 mg/liter to 5.8 mg/liter, a 2.8-fold increase in PH3 efficacy. Comparison of mortality response data of PH 3 alone and the PH3 + CO2 mixture confirmed that CO2 enhances the toxicity of PH3 synergistically in addition to exerting its own toxicity. These results were validated against three independently field-derived strains of strongly resistant C. ferrugineus that confirmed that observed enhancement in toxicity with the PH3 + CO2 mixture was consistent, irrespective of differences in resistance phenotypes and inherent tolerance levels. Results of the current study provide further opportunities to develop new commercially viable strategy to control strongly PH3-resistant C. ferrugineus.

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.

BACKGROUND Next-generation sequencing can enable genetic surveys of large numbers of individuals. We developed a genotyping-by-sequencing assay for detecting strong phosphine resistance alleles in the dihydrolipoamide dehydrogenase (dld) gene of Rhyzopertha dominica populations. The assay can estimate the distribution and frequency of resistance variants in thousands of individual insects in a single run. RESULTS We analysed 1435 individual insects collected over a 1-year period from 59 grain-storage sites including farms (n = 29) and central storages (n = 30) across eastern Australia. Resistance alleles were detected in 49% of samples, 38% of farms and 60% of central storages. Although multiple alleles were detected, only two resistance variants (P49S and K142E) were widespread and each appeared to have a distinct but overlapping geographical distribution. CONCLUSION The type of structure in which the grain is stored had a strong effect on resistance allele frequency. We observed higher frequencies of resistance alleles in bunker storages at central sites compared with other storage types. This contributed to the higher frequencies of resistance alleles in bulk-handling facilities relative to farms. The discovery of a storage structure that predisposes insects to resistance highlights the utility of our high-throughput assay system for improvement of phosphine resistance management practices. (c) 2018 Society of Chemical Industry

Phosphine gas is an excellent fumigant for disinfesting stored grain of insect pests, but heavy reliance on phosphine has led to resistance in grain pests that threatens its efficacy. Phosphine-resistance was previously reported to be mediated by the enzyme DLD. Here we explore the relationship between phosphine toxicity and genotoxic treatments with the goal of understanding how phosphine works. Specifically, we utilized mutant lines either sensitive or resistant to phosphine, gamma irradiation or UV exposure. The phosphine-resistance mutation in the enzyme of energy metabolism, dihydrolipoamide dehydrogenase exhibited cross-resistance to UV and ionizing radiation. Two radiation-sensitive mutants that are defective in DNA repair as well as a mutant that is defective in the activation of the DAF-16 stress response transcription factor all exhibit sensitivity to phosphine that exceeds the sensitivity of the wild type control. A radiation resistance mutation in cep-1, the p53 orthologue, that is deficient in double strand break repair of DNA and is also deficient in apoptosis causes radiation-resistance results but sensitivity toward phosphine.

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.

Metabolism is one of the attributes of life and supplies energy and building blocks to organisms. Therefore, understanding metabolism is crucial for the understanding of complex biological phenomena. Despite having been in the focus of research for centuries, our picture of metabolism is still incomplete. Metabolomics, the systematic analysis of all small molecules in a biological system, aims to close this gap. In order to facilitate such investigations a blueprint of the metabolic network is required. Recently, several metabolic network reconstructions for the model organism Caenorhabditis elegans have been published, each having unique features. We have established the WormJam Community to merge and reconcile these (and other unpublished models) into a single consensus metabolic reconstruction. In a series of workshops and annotation seminars this model was refined with manual correction of incorrect assignments, metabolite structure and identifier curation as well as addition of new pathways. The WormJam consensus metabolic reconstruction represents a rich data source not only for in silico network-based approaches like flux balance analysis, but also for metabolomics, as it includes a database of metabolites present in C. elegans, which can be used for annotation. Here we present the process of model merging, correction and curation and give a detailed overview of the model. In the future it is intended to expand the model toward different tissues and put special emphasizes on lipid metabolism and secondary metabolism including ascaroside metabolism in accordance to their central role in C. elegans physiology.

Phosphine is the only general use fumigant for the protection of stored grain, though its long-term utility is threatened by the emergence of highly phosphine-resistant pests. Given this precarious situation, it is essential to identify factors, such as stress preconditioning, that interfere with the efficacy of phosphine fumigation. We used Caenorhabditis elegans as a model organism to test the effect of pre-exposure to heat and cold shock, UV and gamma irradiation on phosphine potency. Heat shock significantly increased tolerance to phosphine by 3-fold in wild-type nematodes, a process that was dependent on the master regulator of the heat shock response, HSF-1. Heat shock did not, however, increase the resistance of a strain carrying the phosphine resistance mutation, dld-1(wr4), and cold shock did not alter the response to phosphine of either strain. Pretreatment with the LD50 of UV (18 J cm-2) did not alter phosphine tolerance in wild-type nematodes, but the LD50 (33 J cm-2) of the phosphine resistant strain (dld-1(wr4)) doubled the level of resistance. In addition, exposure to a mild dose of gamma radiation (200 Gy) elevated the phosphine tolerance by ~2-fold in both strains.

Phosphine is the only fumigant approved globally for general use to control insect pests in stored grain. Due to the emergence of resistance among insect pests and the lack of suitable alternative fumigants, we are investigating ways to synergistically enhance phosphine toxicity, by studying the mechanism of action of known synergists, such as oxygen, temperature, and arsenite. Under normoxia, exposure of the model organism Caenorhabditis elegans for 24 h at 20°C to 70 ppm phosphine resulted in 10% mortality, but nearly 100% mortality if the oxygen concentration was increased to 80%. In wild-type C. elegans, toxicity of phosphine was negatively affected by a decrease in temperature to 15°C and positively affected by an increase in temperature to 25°C. The dld-1(wr4) strain of C. elegans is resistant to phosphine due to a mutation in the dihydrolipoamide dehydrogenase gene. It also exhibits increased mortality that is dependent on hyperoxia, when exposed to 70 ppm phosphine at 20°C. As with the wild-type strain, mortality decreased when exposure was carried out at 15°C. At 25°C, however, the strain was completely resistant to the phosphine exposure at all oxygen concentrations. Arsenite is also a synergist of phosphine toxicity, but only in the dld-1(wr4)-mutant strain. Thus, exposure to 4 mM arsenite resulted in 50% mortality, which increased to 89% mortality when 70 ppm phosphine and 4 mM arsenite were combined. In stark contrast, 70 ppm phosphine rendered 4 mM arsenite nontoxic to wild-type C. elegans. These results reveal two synergists with distinct modes of action, one of which targets individuals that carry a phosphine resistance allele in the dihydrolipoamide dehydrogenase gene.

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.