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.     

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.     

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.     

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.     

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.     

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.     

TcYellow-h基因过表达参与介导赤拟谷盗对磷化氢的抗性形成

以赤拟谷盗为研究对象,旨在明确表皮相关基因TcYellow-h在害虫磷化氢抗性形成过程中的作用。序列分析结果表明TcYellow-h编码468个氨基酸,其拥有一段保守的MRJP(major royal jelly protein)特征序列;RT-qPCR结果显示,TcYellow-h在赤拟谷盗外周组织器官(头、胸、腹的表皮、翅和足)中表达水平较高,在内部器官(脂肪体、肠道、马氏管)中的表达量相对较低;此外,在不同地理种群中,TcYellow-h的表达量随磷化氢抗性水平的增加呈上升趋势;注射dsRNA能有效抑制TcYellow-h基因的表达,沉默效率为73.0%;干扰抗性种群(GD)TcYellow-h基因的表达后再用LC₃₀的磷化氢处理试虫,赤拟谷盗的死亡率显著上升至64.4%。本研究显示赤拟谷盗表皮相关基因Yellow-h的过表达与磷化氢的抗性形成相关。     

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.     

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.     

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.     

Using immobilization as a quick diagnostic indicator for resistance to phosphine

In the present study, we evaluated a quick diagnostic test for resistance to phosphine in stored-product beetle species. We collected different populations of thirteen species, obtained from different laboratories in different counties, i.e., USA, Greece, Australia, Germany and Spain. There were also tested populations that have been sampled from different facilities (field populations). We used the Detia Degesch Phosphine Tolerance Test Kit (DDPTTK), which is based on the exposure of the insects on a high concentration of phosphine for shorter exposure periods. The tested concentrations to phosphine were 1000 and 3000 ppm. Briefly, 20 adults of the tested populations were placed in a 100 ml plastic syringe. The observations were taken every 2 min and the exposed adults were classified as either walking normally or being immobilized (knocked down), i.e., not walking normally. In light of our findings, the time to reach knockdown of all adults was notably reduced at 3000 ppm in comparison with 1000 ppm. For the majority of the species and laboratory populations tested, at 3000 ppm, the time required for knockdown ranged between 8 and 14 min. In contrast, for some of the field populations, knockdown did not reach 100% even after 300 min of exposure, at either 1000 or 3000 ppm. Based on the results of the present study, we recommend that the DDPTTK can be operated at 3000 ppm, and we provide the critical threshold times per species for the characterization of tolerance/resistance.     

Prevalence of clinically relevant antibiotic resistance genes in surface water samples collected from Germany and australia

The prevalence and proliferation of antibiotic resistant bacteria is profoundly important to human health, but the extent to which aquatic environments contribute toward the dissemination of antibiotic resistant genes (ARGs) is poorly understood. The prevalence of 24 ARGs active against eight antibiotic classes (β-lactams, aminoglycosides, glycopeptides, chloramphenicols, tetracycline, macrolides, trimethoprim, and sulfonamides) was evaluated in surface water samples collected from Germany and Australia with culture independent methods. The ARGs most frequently detected both in Germany and Australia were sulI, sulII (77-100%), and dfrA1 (43-55%) which code for resistance to sulfonamide and trimethoprim. Macrolides resistance gene ermB was relatively more prevalent in the surface water from Germany (68%) than Australia (18%). In contrast, the chloramphenicol resistance gene catII was more frequently detected in Australia (64%) than Germany (9%). Similarly, β-lactams resistance gene ampC was more prevalent in the samples from Australia (36%) than Germany (19%). This study highlights wide distribution of ARGs for sulfonamide, trimethoprim, macroline, β-lactams and chloramphenicol in the aquatic ecosystems. Aquatic ecosystems can therefore be reservoirs of ARGs genes which could potentially be transferred from commensal microorganisms to human pathogens.     

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.     

VOCs,pesticides, nitrate,and their mixtures in groundwater used for drinking water in the United States

Samples of untreated groundwater from 1255 domestic drinking-water wells and 242 public supply wells were analyzed as part of the National Water-Quality Assessment Program of the U.S. Geological Survey between 1992 and 1999. Wells were sampled to define the regional quality of the groundwater resource and, thus, were distributed geographically across large aquifers, primarily in rural areas. For each sample, as many as 60 volatile organic compounds (VOCs), 83 pesticides, and nitrate were analyzed. On the basis of previous studies, nitrate concentrations as nitrogen > or = 3 mg/L were considered to have an anthropogenic origin. VOCs were detected more frequently (44%) than pesticides (38%) or anthropogenic nitrate (28%). Seventy percent of the samples contained at least one VOC, pesticide, or anthropogenic nitrate; 47% contained at least two compounds; and 33% contained at least three compounds. The combined concentrations of VOCs and pesticides ranged from about 0.001 to 100 microg/L, with a median of 0.02 microg/L. Water from about 12% of the wells contained one or more compounds that exceeded U.S. Environmental Protection Agency drinking-water standards or human health criteria, primarily because of nitrate concentrations exceeding the maximum contaminant level in domestic wells. A mixture is defined as a unique combination of two or more particular compounds, regardless of the presence of other compounds that may occur in the same sample. There were 100 mixtures (significantly associated with agricultural land use) that had a detection frequency between 2% and 19%. There were 302 mixtures (significantly associated with urban land use) that had a detection frequency between 1% and <2%. Only 14 compounds (seven VOCs, six pesticides, and nitrate) contributed over 95% of the detections in these 402 mixtures; however, most samples with these mixtures also contain a variety of other compounds.     

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.     

Proteomic assessment of resistance to the fumigant phosphine in the lesser grain borer, Rhyzopertha dominica (F.)

A previous publication compared a strain of the stored-grain pest beetle, Rhyzopertha dominica, resistant to the fumigant phosphine, with a susceptible strain using two-dimensional polyacrylamide gel electrophoresis. Certain proteins appeared to differ and the authors proposed that arginine kinase in particular might be used as a marker for the rapid monitoring of resistance in this species and other pests of stored grain. Here an expanded set of susceptible and resistant strains is surveyed using the two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) system. Of hundreds of spots only two showed a significant difference between the resistant and susceptible strains and the magnitudes of those differences were less than three-fold. Arginine kinase spots were identified but they did not differ significantly. The proposal that certain abundant proteins of R. dominica might be used as markers of phosphine resistance was not supported by the present study.     

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.     

Active exclusion of phosphine as a mechanism of resistance in Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

A strain of Rhyzopertha dominica which is resistant to the fumigant phosphine, was found to absorb less radioactive phosphine than a susceptible strain. The non-absorption of phosphine was enhanced by increased temperature and carbon dioxide. Living resistant insects absorbed less toxin than dead insects, and metabolic detoxication did not appear to contribute to resistance. Since respiratory activity in the presence of the gas is known to be unaffected in the resistant insects, a resistance mechanism based on exclusion of toxic gas is proposed.     

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.