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.     

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.     

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.     

The effectiveness of various insecticides on some resistant beetle pests of stored products from Uganda

Laboratory tests were carried out using populations of Tribolium castaneum (Herbst), Tribolium confusum Jacquelin du Val, Sitophilus zeamais Motschulsky, Callosobruchus maculatus (F.), and Zabrotes subfasciatus Boheman, from Uganda, firstly to determine whether they were resistant to malathion and/or lindane and secondly to measure the effectiveness and stability of pirimiphos-methyl, fenitrothion, etrimfos, permethrin and deltamethrin dilute dusts in protecting cereals and pulses from these insect pests. All the insect populations tested were resistant to malathion or lindane and some were resistant to both. Of the insecticides tested, deltamethrin at 1 ppm was generally the most effective. The organophosphorus compounds were only effective against the Tribolium species and Sitophilus zeamais. Permethrin was the least effective, only controlling Callosobruchus maculatus.     

Stored grain pest prevalence and insecticide resistance in Egyptian populations of the red flour beetle Tribolium castaneum (Herbst) and the rice weevil Sitophilus oryzae (L.)

As a consequence of the widespread use of insecticides against insect pests in grain warehouses and in the food industry, insecticide resistance has greatly increased among these species all over the world. Nonetheless, insecticide resistance in stored grain insects in Egypt was little studied. Therefore, our study aimed to forecast infestation risks and determine the insecticide resistance level in Egyptian populations of the main insect pests of stored grains. To achieve this goal, we surveyed the populations of stored-product insects in wheat in two different sites in the Alexandria governorate (Egypt) between June and August 2017. Moreover, insecticide resistance levels in Egyptian populations of Tribolium castaneum (Herbst) and Sitophilus oryzae (L.) collected from wheat storage facilities (granaries and flour mills) in Alexandria governorate were also determined. Three contact insecticides, namely malathion, pirimiphos-methyl and cypermethrin, were tested. Tarsal contact bioassays on filter paper were carried out on adults following FAO methods and concentration-mortality lines were estimated to determine the resistance ratios for each insecticide and population. The data from the monitoring of insects revealed that T. castaneum and S. oryzae were the most common species infesting wheat in both Alexandria granaries and mills. Populations of S. oryzae were generally more resistant to malathion than the populations of T. castaneum. However, T. castaneum populations were more resistant to pirimiphos-methyl than the S. oryzae populations. Both populations of T. castaneum and S. oryzae were susceptible to cypermethrin. The detected levels of insecticide resistance indicate that this phenomenon takes place and should be a concern in Egyptian populations of T. castaneum and S. oryzae requiring monitoring and design of resistance management practices.     

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.     

Insecticidal effect of contact insecticides against stored product beetle populations with different susceptibility to phosphine

In the present work we evaluated the effect of alpha-cypermethrin, pirimiphos-methyl and spinetoram on field and laboratory strains of Sitophilus oryzae (L.) and Oryzaephilus surinamensis (L.) with different susceptibility levels to phosphine. The field populations were collected from storage facilities in Greece and were characterized as resistant by using the Food and Agriculture Organization (FAO) protocol, based on the same protocol, populations were characterized as susceptible to phosphine. The insecticides were applied at three dose rates (0.1, 1 and 10 ppm) on wheat and adult mortality was measured after 7, 14 and 21 days of exposure, while progeny production was assessed 65 days later. For S. oryzae populations, complete control was noted at the highest dose on pirimiphos-methyl and spinetoram, while mortality caused by alpha-cypermethrin was 62 and 100% for the field and laboratory populations, respectively. For O. surinamensis, complete control was recorded at the highest dose only on alpha-cypermethrin for the laboratory population, in contrast with the field population, where mortality was only 32% after 21 days of exposure. In general, the variations among populations were negligible for spinetoram, probably due to the fact that the populations tested were not previously exposed to this active ingredient. In contrast, the lowest susceptibility of the field populations to the other two insecticides can be attributed to the fact that these populations might have been exposed to these active ingredients, while any hypothesis for cross-resistance with phosphine has to be examined more thoroughly.     

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.     

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.     

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.     

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.     

Prediction of insect mortality in cooled stored grain

Grain aeration with ambient air is the primary method used in France to prevent and control insect infestations. French grain storage operators consider that complete total mortality of insects can be achieved by maintaining grain temperature at 5 °C for 3 months. A predictive model on insect survival at low temperature was developed, using data from literature to test this hypothesis on Sitophilus oryzae, Sitophilus granarius, Rhyzopertha dominica, Cryptolestes ferrugineus, Oryzaephilus surinamensis, Tribolium castaneum and Tribolium confusum. A Cox proportional hazard regression, belonging to survival analysis, was associated to a Receiver Operator Analysis to evaluate the model and to predict durations of cold exposure required to obtain a total mortality for each species at different developmental stages and temperatures. The model described well the data since occurrence or not of a total mortality was correctly described in 77.2% of all cases. Computed predictions highlighted the facts that species cold-tolerance ranking varied depending on the temperature levels and that difference of cold tolerance between adult and immature stages depended greatly on species. A temperature of 0 °C is sufficient to kill all insect populations within 3 months, except Cryptolestes ferrugineus ones. However, a temperature of 5 °C is highly insufficient to achieve a total mortality. Practical implementations of this work are discussed.     

The repellent effect of plants and their active substances against the beetle storage pests

This review compares research techniques, plant species and forms of their application in worldwide studies on the repellent activity of plant material against storage pests, such as Sitophilus oryzae, Sitophilus granarius, Tribolium castaneum, Rhyzopertha dominica and Oryzaephilus surinamensis. Over 300 plant extracts, essential oils and powders were tested against these five insect species with various methods. The intensity of repellency of the examined plant products against each considered pest species was presented. Evaluation of the repellency potential of particular plant extracts, oils and powders showed that insect repulsion increased with their concentration. Duration of exposure was also an important factor affecting repellent activity.     

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.     

Ozone as a management alternative against phosphine-resistant insect pests of stored products

This study was carried out to assess ozone toxicity to 16 populations of Tribolium castaneum (Herbst), 11 populations of Rhyzopertha dominica (F.) and nine populations of Oryzaephilus surinamensis (L.) collected from six regions of Brazil. These populations were also used to test if there is cross-resistance to ozone and phosphine. The relationship between susceptibility to ozone and respiration rate, and associated fitness costs were also evaluated. The instantaneous population growth rate (r_i), CO₂ production and mean insect body mass of each population were measured. Ozone toxicity was determined using time-response bioassays at the dosage rate of 150 ppm ozone in a continuous flow of 2 L min⁻¹. All of the populations were susceptible to ozone and there was no cross-resistance to ozone and phosphine. The populations of each species differed in respiration rate and body mass, but there was no significant association between respiration rate or body mass and susceptibility to ozone, as was also the case for the instantaneous rate of population increase (r_i). As none of the populations showed resistance to ozone, regardless of their susceptibility to phosphine, ozone is a potential alternative for phosphine resistance management in the insect species evaluated in this study.     

6种储粮害虫对不同颜色的趋性研究

在实验室内利用校正趋色法,研究我国6种储粮害虫对红色等6种颜色的趋性行为反应。结果表明,6种储粮害虫对不同颜色的趋性情况大多随时间的变化而变化,在特定的时间段内,赤拟谷盗、杂拟谷盗、米象、玉米象对不同颜色的行为反应存在显著性差异(P≤0.05),其中,红色、橙色、黑色对赤拟谷盗均有明显的引诱作用,诱集率最高分别为76.23%、39.55%、49.57%;红色、黑色对玉米象有明显的引诱作用,诱集率最高分别为38.48%、38.67%。嗜卷书虱和无色书虱对所测试6种颜色则大多表现出驱避性,其中,红色和紫色分别对嗜卷书虱、无色书虱有明显的驱避作用,驱避率最高分别为70.29%、54.46%。研究结果可为研发相应颜色诱捕器对储粮害虫进行诱捕监测提供参考。     

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.     

Efficacy of phosphine and insect penetration ability in ZeroFly® bags

The deltamethrin incorporated woven polypropylene ZeroFly® storage bag is a promising novel technology for grain storage. However, if grain stored in ZeroFly bags gets infested and has to be fumigated using phosphine (PH₃), data on the effectiveness of such treatments are needed. Additionally, obtaining field data on ability of stored-product insect pests to breach ZeroFly bags would facilitate insect management. Therefore, efficacy of PH₃ in immature and adult Sitophilus zeamais (Motschulsky), Prostephanus truncatus (Horn), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) in experimental cages in maize stored in 100-kg polypropylene (PP), jute and ZeroFly bags was investigated. Post-fumigation mortality of adults was recorded after 7 d, and after 7 wk for immatures. The ability of either S. zeamais or P. truncatus to penetrate fabric of PP, jute and ZeroFly bags was assessed. Phosphine efficacy was good in all the three types of bags and resulted in complete mortality of adults and immatures of the four species tested. Sitophilus zeamais and P. truncatus were more successful in penetrating the PP bag fabric and on average made 84 and 780 holes per bag over a 4 mo-period, respectively; this was followed by jute with 37 and 614 holes. The ZeroFly bag was harder to breach and ≤3 holes per bag were made for both species. This study shows that PH₃ is highly efficacious in insects that infest maize stored in ZeroFly bags, and that these bags are not easily penetrated by stored product insect pests. Hitherto, ZeroFly bags are a good technology for storing grain that is not infested, and fumigation using PH₃ can be effectively conducted if infestation occurs. Therefore, ZeroFly bags can be incorporated in integrated stored product insect management (IPM) programs for bagged grains.     

Radiation sensitivity of successively irradiated generations of Tribolium castaneum (Herbst) (Coleoptera,Tenebrionidae), and Sitophilus oryzae (L.) (Coleoptera,Curculionidae)

Four populations of Tribolium castaneum (Herbst) and of Sitophilus oryzae (L.) were treated with 0 (control), 0·5, 1 or 2 krad of gamma radiation for 7 and 5 successive generations, respectively, and the adults from the subsequent unirradiated generation of each population were checked for fecundity. Groups of 6th generation T. castaneum and S. oryzae adults from each population were tested for resistance to acute radiation by exposure to doses of 16 and 50 krad followed by a check for mortality.No evidence of increased resistance to acute radiation exposures was found. Irradiated T. castaneum populations did not show significant decreases in mean survival time, but some significant decreases in life-span of irradiated S. oryzae populations were found.Fecundity of the 8th generation 0·5 krad T. castaneum was greater than that of the control population, but 1- and 2-krad populations did not show any difference in fecundity. Fecundity of the 6th generation S. oryzae population was not significantly different from that of the control, but 1- and 2-krad populations had significantly reduced fecundities. The test results indicated that possible accidental exposure of some insects to substerilizing doses during commercial radiation disinfestation is unlikely to bring about the development of radiation-resistant insect populations.     

Prevalence and diversity of Wolbachia bacteria infecting insect pests of stored products

Wolbachia are common bacterial symbionts of insects. Because infections of these bacteria can alter the reproduction and biology of the host, there is interest in the potential use of Wolbachia to control populations of pest species. To advance this research, we screened 38 species (88 populations from 16 countries) of stored-product insect pests for Wolbachia infections. Infections were detected in nine species of Coleoptera (Anobiidae – Lasioderma serricorne (Fabricius), Stegobium paniceum (Linnaeus); Curculionidae – Sitophilus oryzae (Motschulsky), Sitophilus zeamais Motschulsky; Dermestidae – Attagenus unicolor (Brahm), Dermestes lardarius Linnaeus; Silvanidae – Oryzaephilus mercator (Fauvel), Oryzaephilus surinamensis (Linnaeus); Tenebrionidae – Tribolium confusum Jaquelin Du Val), one species of Hymenoptera (Trichogrammatidae – Trichogramma deion Pinto & Oatman), and one species of Lepidoptera (Pyralidae – Ephestia kuehniella Zeller). Phylogenetic analyses based on wsp gene sequences identified all Wolbachia isolates as members of Supergroups A or B. Additional sequences for the genes gatB, coxA, hcpA, fbpA and ftsZ were obtained for use in multilocus sequence typing (MLST) to identify the sequence types to which the isolates belonged. Our results identify five new Wolbachia-insect associations and support previous findings that infections of Wolbachia in stored-product insect pests are members of Supergroups A or B.