Resistance to phosphine in Sitophilus oryzae in Australia: A national analysis of trends and frequencies over time and geographical spread

In Australia, along with many other parts of the world, fumigation with phosphine is a vital component in controlling stored grain insect pests. However, resistance is a factor that may limit the continued efficacy of this fumigant. While strong resistance to phosphine has been identified and characterised, very little information is available on the causes of its development and spread. Data obtained from a unique national resistance monitoring and management program were analysed, using Bayesian hurdle modelling, to determine which factors may be responsible. Fumigation in unsealed storages, combined with a high frequency of weak resistance, were found to be the main criteria that led to the development of strong resistance in Sitophilus oryzae. Independent development, rather than gene flow via migration, appears to be primarily responsible for the geographic incidence of strong resistance to phosphine in S. oryzae. This information can now be utilised to direct resources and education into those areas at high risk and to refine phosphine resistance management strategies.     

Significant population structure in Australian Cryptolestes ferrugineus and interpreting the potential spread of phosphine resistance

An analysis of the population genetics of Cryptolestes ferrugineus, across all major regions in Australia where grain is grown and stored in bulk, provides an initial step in investigating the movement of these insects and implications for the spread of phosphine resistance. Microsatellite data revealed two levels of genetic structure. Across Australia, two clusters were detected, one in eastern Australia (Queensland, New South Wales, Victoria and Tasmania) and Western Australia (Cluster 1) and one in South Australia (Cluster 2). Intermediate between them, in eastern South Australia, are a couple of populations that are equally admixed for the two clusters. Populations in Western Australia and western SA belong to different clusters suggesting that the extensive and arid Nullarbor Plain restricts the natural dispersal of these beetles. Isolation-by-distance and the detection of clustering among local populations suggests there is considerable gene flow at a local scale in C. ferrugineus, and we infer this is by flight. Limited natural gene flow between eastern and western Australia might, in part, explain why extremely high resistance has not been detected in Western Australia.     

Significant population genetic structuring in Rhyzopertha dominica across Turkey: Biogeographic and practical implications

Rhyzopertha dominica is a key pest of stored grain. Understanding the movement of this beetle on broad geographic scales is crucial, particularly when developing strategies to prevent the spread of phosphine resistance. We assessed population genetic structuring in this pest across Turkey, using a combination of mitochondrial (cytochrome oxidase I) and microsatellite markers. In addition, we screened samples for Wolbachia, as this endosymbiont has previously been suggested to be associated with low mitochondrial genetic diversity in this beetle. Mitochondrial genetic diversity was low, with only six haplotypes identified. The genetic diversity was, however, substantially higher than that previously found in Australia or India, suggesting that R. dominica may have originated in the Middle East. Wolbachia were detected only at a single site, indicating they are not impacting the mitochondrial genetic diversity of R. dominica across Turkey. Microsatellite markers indicated there is significant geographic genetic structuring across Turkey, even among sites less than 100 km apart, suggesting there is little movement of beetles across regions within the country. This contrasts with the significantly higher levels of gene-flow found in Australia and the United States. We suggest that the limited movement of beetles across Turkey may be due to a combination of the historically localised agricultural practices (which limits anthropogenic movement among regions), and the mountainous landscape (which limits active flight among regions). Our results demonstrate that the movement of stored product pests may differ significantly across studies conducted in different countries. As a consequence, phosphine resistance management strategies must incorporate region specific information on the extent of beetle movement.     

A rapid assay for the detection of resistance to phosphine in the lesser grain borer,Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Resistance to the fumigant phosphine in stored product insect pests is a global problem. Diagnosis of resistance relies on a bioassay developed by the FAO that involves a mortality assessment after 20-h fumigation of a pest population at a discriminating concentration of gas, followed by a 14-day post fumigation assessment. This bioassay is impractical for monitoring and early detection of phosphine resistance in routine pest management. We utilized the procedure of a commercial resistance detection test kit for rapid detection in field populations of lesser grain borer, Rhyzopertha dominica (F.). We established a knockdown effect of either susceptible or resistant insects by exposing them to a high concentration of phosphine. We assessed the relationship between adult knockdown times and the FAO method for 18 beetle populations utilizing knockdown criteria for a single beetle in a chamber, or for 50% or 100% knockdown times for groups of beetles, exposed to 3000 ppm of phosphine. We also determined the most effective concentrations that would elicit the quickest knockdown while estimating the recovery times from exposure. Results suggest that a KT₁₀₀ test was better than the KT₅₀ and the KT_single tests. Based on the responses of susceptible populations, we established that a KT₁₀₀ of approximately 18 min can be used as a viable knockdown time to distinguish a susceptible from a resistant populations. Higher concentrations of phosphine significantly elicited a quicker recovery in strongly resistant populations compared to susceptible populations. These findings have potential for developing a robust commercial kit for practical phosphine resistance detection in populations of R. dominica by commercial fumigators, and could be incorporated in a resistance management program.     

First report of strong phosphine resistance in stored grain insects in a far northern tropical region of Australia,combining conventional and genetic diagnostics

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.     

Effect of diet on phosphine toxicity,rate of development and reproduction of the rice weevil Sitophilus oryzae (Linnaeus)

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.     

Phosphine resistance in saw-toothed grain beetle,Oryzaephilus surinamensis in the United States

Phosphine (PH₃) fumigation resulting in sub-lethal exposure has led to the development of phosphine resistance in many stored-product insect species worldwide and is a major challenge to the continued effective use of phosphine. In 2016 phosphine resistance was found in Tribolium castaneum (Herbst) and Plodia interpunctella (Hübner) collected from California dried fruit and nut processing facilities. Although Oryzaephilus surinamensis (L.) infests grain, dried fruit, and nuts in storage and processing facilities, phosphine resistance in this species has not been studied in the United States. In this study, the discriminating dose of phosphine for O. surinamensis eggs was estimated using a laboratory susceptible strain; it was found to be 28.4 ppm over a 72-h fumigation period (1 mg/L of phosphine = 714.18 ppm or 1 ppm = 0.0014 mg/L). Discriminating dose bioassays were used to determine phosphine resistance in both eggs and adults of 14 different populations collected from California and Oklahoma. Resistance to phosphine was detected in four out of 14 populations in adults and nine out of 14 populations in eggs and ranged from 2 to 100%. Phosphine percent survival values in both adults and eggs of three populations, namely, Box BR, Box BF, and OKWat were >90%. Lethal concentration values required to kill 99% of individuals in samples for adults of these three populations were predicted as 320.5, 290.7, and 263 ppm, respectively, and those for eggs were 1030.7, 1055.9, and 564.5 ppm, respectively, over a 72-h fumigation period. This study confirms that phosphine resistance is present in O. surinamensis in the United States.     

Detection of phosphine resistance in major stored-product insects in Greece and evaluation of a field resistance test kit

The use of phosphine has been effective against a wide range of stored-product pests in different types of commodities and facilities. However, its continuous and improper use has led to resistance development in -several major insect species. Although phosphine resistance has been reported from many countries across the globe, reports from Europe have been very limited. In the present study, we determined phosphine resistance in insect populations that had been collected from a range of storages across Greece, using two different diagnostic protocols. Apart from the traditional Food and Agriculture Organization (FAO) protocol, a field test kit (known as the Detia Degesch Tolerance Test Kit, DDTTPK) was utilized, for “same day” determination of the resistance status of field collected insects. In total, 53 populations belonging to Rhyzopertha dominica, Sitophilus oryzae, Sitophilus granarius, Cryptolestes ferrugineus, Tribolium confusum, Tribolium castaneum and Oryzaephilus surinamensis were tested. For the majority of the species and populations tested, both FAO and DDPTTK provided similar results, for the susceptibility to phosphine and thus, the quick test could be used with success for an initial same day screening of phosphine resistance. Among the tested species, the populations recorded with the most frequent survival at the FAO testing dose of phosphine was that of R. dominica. The dissimilar evaluation and characterization of resistance to phosphine between diagnostic protocols is particularly important, as it poses risks in the over or underestimation of the resistance status of a given population. Our data indicate that the DDPTTK could be used to determine resistance to phosphine in the field, before the initiation of fumigations to disinfest stored commodities.     

Prevalence of resistance to deltamethrin in Rhyzopertha dominica (F.) in eastern Australia

The pyrethroid, deltamethrin, is currently used as a grain protectant to protect stored grain from insect pests in several countries including Australia. We examined the variation in response to wheat treated with deltamethrin among 15 field populations of the lesser grain borer, Rhyzopertha dominica (F.) collected from Queensland and New South Wales in eastern Australia during 2008 and 2009, to establish whether resistance has developed in this species. Based on the results of treated wheat bioassays, using a discriminating dose of 0.5 mg kg⁻¹, we found that 14 of the 15 field populations were resistant to deltamethrin. The distance between the two most geographically separated field populations was 450 km, and both of these populations contained resistant insects. Deltamethrin applied at 1 mg kg⁻¹ alone or with the synergist piperonyl butoxide at 8 mg kg⁻¹ failed to control resistant R. dominica. On the basis of our findings, we conclude that populations with deltamethrin resistant individuals are common in the eastern part of Australia, and control failures are expected when such populations are present in treated grain.     

Phosphine resistance in stored-product insects collected from various grain storage facilities in Morocco

Despite heavy dependence on phosphine (PH₃) for fumigating stored products, the resistance status of insect pests in Morocco has never undergone a thorough investigation. Some control failures with PH₃ were reported in Morocco, and a previous study showed two field populations of Sitophilus oryzae to be highly resistant to phosphine.We surveyed phosphine resistance in field populations of three major insect pests of stored wheat in Morocco. Around 32% of the samples collected at different storage facilities were found to be infested with one or more species of stored-product beetles. First-generation adult beetles, cultured from the field samples, were subjected to a discriminating dose test for phosphine resistance using an FAO method. The results indicated that, with the exception of one population of S. oryzae, all samples tested contained phosphine-resistant individuals. Treatments at up to 1.8 g m⁻³ of phosphine for 20 h, or at 0.18 g m⁻³ for up to 5 days, indicated that a high degree of resistance was already selected in some of the insect populations.Tests using [³²P]-radiolabelled phosphine showed that the mechanism of resistance in the three insect species tested involved a reduced uptake of the fumigant. The study has highlighted an urgent need for reviewing current fumigation practices in Morocco to ensure effective use of phosphine and avoid further selection of resistance.     

Genetic diversity and its geographic structure in Sitophilus oryzae (Coleoptera; Curculionidae) across India – implications for managing phosphine resistance

The rice weevil, Sitophilus oryzae, is a serious global pest of stored grains. Fumigation with phosphine gas is the primary control method for S. oryzae, but the indiscriminate and prolonged use of phosphine gas has led to the development of heritable resistance. Developing and implementing an effective phosphine resistance management strategy for S. oryzae relies on an understanding of its genetic diversity and any structuring of that diversity geographically. We therefore sequenced the mitochondrial cytochrome c oxidase subunit I gene from 143 S. oryzae specimens collected from 37 locations across India, and from that assessed the genetic diversity of the species and its phylogeographic structuring. In addition, we compared the genetic diversity in Indian S. oryzae populations (the hypothesised origin of this beetle) to global populations. Genetic diversity was low in Indian S. oryzae, with only eight haplotypes (including two very common haplotypes) identified. The low level of mitochondrial diversity observed in this species appears typical of stored product pests, perhaps suggesting that low mitochondrial diversity is associated with repeated phosphine fumigations, which may eliminate low frequency haplotypes. The genetic diversity of S. oryzae in India is, however, higher than in many other countries, though comparable levels were identified in China. There was no evidence of population genetic structure across India, with most haplotypes found in three of the broad biogeographic regions. This lack of phylogeographic structuring indicates significant gene-flow across India, most likely through the incidental anthropogenic transport of this relatively poor (or reluctant) flyer. The major practical implication is that phosphine resistance management for S. oryzae needs to be dealt with country wide, as populations are not isolated.     

Fumigant toxicity of allyl isothiocyanate to populations of the red flour beetle Tribolium castaneum

The phasing out of methyl bromide as a fumigant, the phosphine resistance problems in stored product insect-pests, and the ever-growing concerns with human health and environmental safety have been guiding the search of alternative fumigants. Allyl isothiocyanate (AITC) is the main component of mustard oil with reported pesticide activity and potential as a fumigant of stored foodstuffs. The fumigant toxicity of AITC was assessed in adults of 18 populations of the red flour beetle Tribolium castaneum (Coleoptera: Tenebronidae). These populations were all susceptible to AITC with negligible variation among them. Two of these populations were further used to test the AITC susceptibility of eggs, larvae (early and late instars), and pupae of T. castaneum. All of the developmental stages of both populations were similarly susceptible to AITC. No cross-resistance between phosphine and AITC was observed. Despite the significant variation in body mass, respiration rate, and fitness among the populations of T. castaneum, they were not correlated with AITC susceptibility. Larvae and adult malformations were observed when larvae and pupae were exposed to AITC. These results show the potential of AICT as an alternative fumigant against stored product insects.     

Developmental and population growth rates of phosphine-resistant and -susceptible populations of stored-product insect pests

Phosphine resistance positively contributes towards an individual's fitness under phosphine fumigation. However, phosphine resistance may place resistant individuals at a fitness disadvantage in the absence of this fumigant, which can be exploited to halt or slow down the spread of resistance. This study aimed to determine if there is a fitness cost associated with phosphine resistance in populations of the red flour beetle (Tribolium castaneum (Herbst)), the lesser grain borer (Rhyzopertha dominica (F.)) and the sawtoothed grain beetle (Oryzaephilus surinamensis (L.)). The developmental rate and population growth of phosphine-resistant and -susceptible populations of these three species of stored-product insects were therefore determined under phosphine-free environment. The majority of the phosphine-resistant populations exhibited lower developmental and population growth rates than the susceptible populations indicating that phosphine resistance is associated with fitness cost in all three species, which can potentially compromise the fixation and dispersal of the resistant genotypes. Nonetheless, some phosphine-resistant populations did not show a fitness cost. Therefore, resistance management strategies based on suppression of phosphine use aiming at eventual reestablishment of phosphine susceptibility and subsequent reintroduction of this fumigant will be useful only for insect populations exhibiting a fitness cost associated with phosphine resistance. Therefore recognition of the prevailing phosphine-resistant genotypes in a region is important to direct the management tactics to be adopted.     

Modeling post-fumigation desorption of phosphine in bulk stored grain

Phosphine is a dangerous gas commonly used in fumigations of stored grains throughout the world. Grain that has not fully released the phosphine it absorbed during fumigation may continue to desorb phosphine into the headspace of a shipping container or storage. USOSHA standards for handling phosphine state the acceptable Threshold Limit Value (TLV) of 0.3 ppm. In many cases, during grain transport and handling, the level of phosphine in work environments can exceed the 0.3 ppm TLV which resulted from desorption of phosphine from fumigated grains. It is also important to note that desorbing grain, with low concentrations of phosphine, can facilitate insect resistance. Therefore, it is important to understand the process of phosphine venting and desorption in order to ensure safe handling of fumigated grain. In order to achieve this, the venting and release of phosphine was studied on location in a well-sealed grain silo in Lake Grace, Western Australia. The data set served for verification of a 3D finite element ecosystem model and were compared to the predicted results. Results were calculated using two different fumigant desorption models based on previous literature, i.e., a reversed sorption model and an air-grain equilibrium model. Simulations reproduced accurate trends of desorption but did not accurately reproduce the quantity of fumigant, with 55.5% error for the model based on reversed sorption equations, and 86.3% error for the air-grain equilibrium based model. For both models, additional simulations were conducted to compare the effectiveness of existing grain venting regulations at producing grain that is within post-fumigation safe handling limits. Results revealed that current U.S. standards may be insufficient to guarantee safety based on minimum venting times needed. These results highlight the necessity for continued research into phosphine desorption and the importance for establishing realistic post-fumigation venting guidelines.     

Phosphine resistance in Brazilian populations of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Phosphine resistance was assessed in adults of 22 Brazilian populations of Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). The concentration-mortality bioassays for the detection of phosphine resistance followed the FAO standard method. Twenty populations of S. zeamais were resistant to phosphine and the resistance ratios at the LC₅₀ ranged from 1.1- to 86.6-fold. This is the first report of phosphine resistance in populations of S. zeamais in Brazil, where previous surveys did not detect resistance in this species. There was significant variation in respiration rate (CO₂ production) among the populations (P < 0.05). Respiration rate was significantly inversely correlated with phosphine resistance for this species (P < 0.05). The populations with lower respiration rates showed higher levels of phosphine resistance, suggesting that the lower respiration rate is associated with the physiological basis of phosphine resistance due to reduced fumigant uptake.     

Comparison of the mitochondrial proteomes of phosphine-susceptible and -resistant Tribolium castaneum

Various lines of evidence suggest that mitochondria might be the site of constitutive differences between coleopteran pests of stored products with and without resistance to the fumigant phosphine. In this study two-dimensional polyacrylamide gel electrophoresis with Differential In-Gel Electrophoresis (DIGE) was used to compare soluble proteins from the mitochondria of phosphine-susceptible and -resistant Tribolium castaneum. Eighty-five spots were aligned across all nine gels and a further 111 across all but one gel. Each gel displayed the proteome from a susceptible strain, a resistant strain and a standard made by mixing aliquots from all experimental samples. No significant differences were observed between resistant strains and strains susceptible to phosphine. However, proteins of lower abundance and membrane proteins were not detected so important resistance-associated differences might yet be detected by more exhaustive techniques.     

Mobility parameters of Tribolium castaneum and Rhyzopertha dominica populations with different susceptibility to phosphine

Phosphine (PH₃) is the most commonly used fumigant to protect stored products from arthropod infestations worldwide. Our knowledge about the behavioral differences between phosphine-resistant and -susceptible stored product pest populations is limited. This study evaluated differences in mobility and behavior of populations of two major stored product insects, Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.), which have different susceptibility to phosphine. In this regard, laboratory bioassays in Petri dish arenas were designed to determine if phosphine resistance has an impact on the walking and mobility behavior of adult beetles of both species. Results indicated that there were significant differences between resistant and susceptible populations for both species. Regarding velocity, R. dominica susceptible individuals moved faster than resistant ones. However, the resistant population showed reduced activity for several parameters tested compared to the susceptible population. Similar trends were also noted for T. castaneum. Knowledge of these parameters should be further utilized in management tactics, as resistant populations may behave in a different way in key management indicators such as trapping and sampling, as compared with susceptible ones.     

Effect of low temperatures on the rate of respiration and uptake of phosphine in different life stages of the cigarette beetle Lasioderma serricorne (F.)

Studies were undertaken to establish the effects of low temperatures on the rate of respiration in larvae and adults of a susceptible strain of the cigarette beetle Lasioderma serricorne. The results showed that there was a consistent decrease in the rate of respiration in L. serricorne with a decrease in temperature from 25°C to 5°C. The rate of respiration was slightly affected by circadian rhythm in adult beetles, but not in larvae.The levels of phosphine taken up by different life stages of the beetle were also determined using [³²P]-radiolabelled gas. A comparison between susceptible and phosphine resistant strains showed that the mechanism of resistance in L. serricorne involves a reduced uptake of the fumigant.The uptake of [³²P]-phosphine by different life stages of resistant L. serricorne declined with a decrease in temperature from 25°C to 5°C. Similarly, the uptake by susceptible L. serricorne decreased from 25°C to 10°C, but there was a comparatively higher uptake at 5°C and consequently a higher mortality at the end of the exposure. The significance of the findings in relation to control strategies is discussed.     

Allyl isothiocyanate actions on populations of Sitophilus zeamais resistant to phosphine: Toxicity,emergence inhibition and repellency

The risks associated with the use of synthetic insecticides have caused increased interest in the research of essential oils and their main constituents for use in the pest management of stored products. Allyl isothiocyanate (AITC) is the main component of mustard essential oil and has been reported as a potential replacement pesticide for conventional insecticides that control stored product insect pests. Here, we assessed the toxicity (including emergence inhibition) and repellent actions of AITC on Brazilian populations of the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) resistant to conventional insecticides (e.g., phosphine). We also evaluated physiological (e.g., respiration) and behavioral (e.g., walking and flight) traits of AITC-exposed insects. The AITC showed consistent insecticidal activity against the populations resistant to phosphine and other synthetic insecticides, with LC₅₀ values ranging from 1.5 to 2.9 μL L⁻¹. Significant inhibition of the offspring emergence was achieved after the exposure of parental adults to sublethal levels (i.e., LC₁ and LC₅) of AITC. Reductions in respiration rates were also registered in all the populations sublethally exposed to AITC. In all five populations, a high number of insects avoided AITC-treated (1.5 μL L⁻¹) grain masses, and although individuals of a phosphine-susceptible (i.e., Abre Campo) population increased walking and reduced flight activities, individuals of another phosphine-susceptible (i.e., Tunápolis) population exhibited higher flight activity under AITC exposure. Thus, our findings suggest that AITC is a potential tool that may be integrated into the control strategies of maize weevils where resistance to phosphine and other conventional insecticides is a problem.