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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Behavioral and physiological responses induced by ozone in five Brazilian populations of Rhyzopertha dominica

This study evaluated locomotory and respiratory responses induced by ozone at a concentration of 500 ppm in five Brazilian populations of Rhyzopertha dominica (Coleoptera: Bostrichidae). Toxicity and body mass were also assessed to establish their relationship with behavioral patterns. The results indicated that none of the evaluated populations of Rhyzopertha dominica showed resistance to ozone. No significant correlations were observed between ozone toxicity and locomotory behavioral patterns. Moreover, no significant correlations were found between ozone toxicity and the respiratory rate of Rhyzopertha dominica. Ozone is a potential alternative for phosphine resistance management, and its rapid degradation constitutes an advantage for the environment.     

Field validation of phosphine efficacy on the first recorded resistant strains of Sitophilus granarius and Tribolium castaneum from the Czech Republic

This study first estimated the current state of phosphine sensitivity (using a knock-down/KT₁₀₀/Degesch kit) in Sitophilus granarius (23 strains) and Tribolium castaneum (8 strains) in Czech Republic grain stores. The resistance of S. granarius (21.7% resistant strains; coefficient of resistance KT₁₀₀ ranged from 0.5 to 2.3 among strains) was substantially lower and less frequent than that of T. castaneum (87.5% resistant strains; coefficient of resistance KT₁₀₀ ranged from 0.9 to 52.5 among strains). The phosphine efficacy of the laboratory and field (i.e., resistant) pest strains was validated during commercial fumigation when suboptimal tarpaulin sealing resulted in low-concentration phosphine exposure (Ct products ranged from 5.9 to 7.4 g*hr/m³). Although even low-dose fumigation led to 100% adult mortality of both laboratory and field strains of S. granarius and laboratory strains of T. castaneum, the mortality of the field strain of T. castaneum ranged from 47% to 95%. Larval emergence from the fumigated commodity samples with pest eggs was zero or near zero for laboratory strains, while 1.3–6.0 (S. granarius) and 63.7–80.00 (T. castaneum) field-strain larvae emerged per sample (100 g). This study shows that although a high proportion of the tested pest populations were still sensitive, several T. castaneum populations showed an elevated level of resistance that may decrease field fumigation efficacy, especially under suboptimal phosphine dosage conditions.     

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.     

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.     

The toxicity of phosphine to adults of Tribolium castaneum (Herbst): Phosphine-induced narcosis

The narcotic effect of phosphine on adults of Tribolium castaneum was examined over concentrations ranging from approx 0.2 to 50 mg/l. Times to narcosis decreased with higher concentrations as did the rate of decrease of these times consistent with a decreasing rate of uptake with increasing concentration. Correlations between narcosis times and mortality were examined but were only of limited value for rapidly estimating mortality response. Moreover, from these correlations it was not possible to resolve the question as to whether narcotised insects have a better chance of survival. Narcosis may have practical implications in terms of phosphine resistance.     

Immediate and delayed effects of short exposures to phosphine on adults and larvae of the khapra beetle,Trogoderma granarium

Adults and larvae of the khapra beetle, Trogoderma granarium Everts were exposed to 3000 ppm of phosphine through the Phosphine Tolerance Test. In a first series of bioassays, observations were taken every 2 min and the exposed individuals were classified either as walking normally or as being immobilized (knocked down), i.e., not walking normally. In the second series of bioassays all individuals were exposed for 90 min to phosphine. For both bioassays delayed mortality was noted after a 7 and 14-day post exposure interval. Larvae were found to be more tolerant than adults, as the time required for the individuals to be immobilized was up to 20 min, which was almost twice as long as the time required for the immobilization of the adults. There were high levels of adult mortality 7 days later, and complete (100%) mortality 14 days after the exposure. In contrast, larval mortality was low, for both post-exposures. Adults were 100% immobilized after the termination of the 90-min exposure interval at 3000 ppm of phosphine, while the percentage of the active larvae was extremely low. Regarding the 7 days post exposure interval the percentage of larval immobilization was higher than that of adults, but this was reversed seven days later. Interestingly, development of the larvae was delayed compared to the control, after the 90 min exposure, in contrast to 20 min exposure which did not cause any delayed effect. Our work provides some first data for the evaluation of the influence of short exposures to phosphine on adults and larvae of T. granarium, which may be very useful in creating an effective initial quantification plan for the control of this species.     

Evaluation of dosimeter tubes for monitoring phosphine fumigations

Within integrated pest management options, fumigation of stored products is one method to help control post-harvest insect infestations in our food and agricultural products. Fumigant gas concentration monitoring is important to confirm that the treatment was adequate to achieve the desired insect control, but monitoring can be relatively expensive and labor intensive. This study evaluated how accurately dosimeter tubes could monitor phosphine fumigation treatments. The dosimeter tube is designed to continuously react with phosphine gas during the fumigation period and yields a measurement in terms of concentration 1 time product or CT, which can be interpreted as cumulative exposure. Two models of dosimeter tubes were evaluated (high range and low range). The reference method for these trials were wireless phosphine monitoring sensors, which recorded gas concentrations at hourly intervals during an exposure, and from this a CT product was also calculated. Model LPG-1, high-range dosimeter tube, measured within ± 25% of the phosphine monitoring sensors for CT dosages less the 70,000 ppm1hr. Model LPG-2, low-range tube, tended to significantly over-estimate phosphine CT dosage by 50%–100% of the phosphine monitoring sensor references. Secondly, bioassays of fumigant efficacy were performed using susceptible and resistant adult Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), lesser grain borers, and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), red flour beetle, for estimating insect control at the varied fumigation CT treatments. For the susceptible strains, CT dosages ∼5000 ppm1hr controlled both species. However, the insect control varied from 60% to 100% for resistant adults at CT dosages of ∼20,000 ppm1hr. The dosimeter tubes function in these ranges of dosages where each insect species are controlled and the dosimeter tube model LPG-1 provides reasonable estimates of the fumigation dosage for a given treatment level.