Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of Nitrogen on Stored-Product Insect Control at Industrial Scale.

In this study, we evaluated the insecticidal effect of nitrogen against Sitophilus granarius (L.), Sitophilus oryzae (L.), Rhyzopertha dominica (F.), Prostephanus truncatus (Horn), Tribolium confusum Jacquelin du Val, and Oryzaephilus surinamensis (L.). Four trials were conducted in chambers containing flour in bags or sacks with >99% nitrogen level. Adults of all the above species, as well as immature life stages (eggs, larvae, and pupae) of T. confusum were used in the trials. Our results showed that nitrogen caused high mortality for all species and life stages tested. Some survival was recorded for R. dominica and T. confusum pupae. Low progeny production was recorded for S. granarius, S. oryzae, and R. dominica. In conclusion, our trials indicated that a high nitrogen environment can provide satisfactory control of various primary and secondary stored-product insect species.

Quick knockdown results in high mortality: is this theory correct? A case study with phosphine and the red flour beetle.

BACKGROUND: The fumigant phosphine is used all over the world for disinfestation of stored grains and commodities. Adults of 23 different populations of Tribolium castaneum from 10 different countries were evaluated for phosphine resistance using a modification of the Detia Degesch Phosphine Tolerance Test Kit (DDPTTK). Adults were exposed to 3000 ppm and recorded for 5-270 min for their mobility. RESULTS: Among the tested populations, high levels of phosphine resistance were recorded in populations from Brazil, Serbia, and Spain. No survivals were recorded after 7 days post exposure for eight of 23 in a tested population. CONCLUSIONS: Our work revealed four scenarios: 1, quick knockdown-low (or no) recovery; 2, Slow knockdown-high recovery; 3, Quick knockdown-high recovery; and 4, Slow knockdown-low recovery. Our data indicate that post exposure period is critical for the evaluation and characterization of phosphine resistance. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Population growth of phosphine resistant and susceptible populations of Lasioderma serricorne (F.) (Coleoptera:Anobiidae) exposed to different temperatures and commodities.

The aim of this work was to investigate the population growth of Lasioderma serricorne (F.) with two populations with different susceptibility to phosphine (one resistant and one susceptible). Population growth was recorded on different days (35 days, 50 days, 65 days, 80 days, 95 days, and 110 days) in two different commodities: (a) mixed food consisted of wheat flour (10 parts) + cornmeal (10 parts) + brewers' yeast (1.5 parts) and (b) wheat flour. Our results clearly indicate that both populations preferred mixed food compared to wheat flour for all combinations tested. Moreover, the increase in temperature from 25 to 30 °C showed a positive effect in some combinations in the population growth of both populations. In general, we found some differences in the production of offspring between the susceptible and the resistant population. Based on the results of the present study, population growth may provide critical information for the fitness advantages or disadvantages of each population.

Residual efficacy of selected larvicides against Culex pipiens pipiens (Diptera: Culicidae) under laboratory and semi-field conditions.

Mosquitoes are a threat worldwide since they are vectors of important pathogens and parasites such as malaria, dengue, yellow fever, and West Nile. The residual toxicity of several commercial mosquito larvicides was evaluated for the control of Culex pipiens pipiens under controlled laboratory and semi-field conditions during late spring and summer of 2013. The evaluation included six different active ingredient formulations, i.e., diflubenzuron Du-Dim), Bacillus thuringiensis var. israelensis (Bti) (Vectobac), spinosad (Mozkill), S-methoprene (Biopren), temephos (Abate), and polydimethylsiloxane (PDMS) (Aquatain), that are currently registered of and had been registered in the past for mosquito control. Under controlled laboratory conditions, the residual activity ranged from 1 week (S-methoprene) up to 2 months (spinosad, PDMS). Exposure of larvicides under semi-field conditions resulted in noticeable differences regarding their efficacy as compared to the laboratory bioassays. Exposure of S-methoprene, Bti, and spinosad, for up to 3 days, resulted in similar adult emergence to the controls. On the other hand, the residual efficacy of diflubenzuron, temephos, and PDMS ranged from 14 to 28 days, depending on the season of exposure. Longevity and fecundity of adults that had emerged from surviving larvae, in most of the cases tested, did not differ significantly from that of the controls. The results of the present study demonstrate the necessity of both field and laboratory studies to draw safe conclusions regarding the efficacy of larvicides against mosquitoes and the selection of the proper formulation for each application scenario. In addition, defining the seasonal variation in the residual toxicity of the tested formulations could be useful for improving mosquitos' management programs.

Efficacy of Spinetoram for the Control of Bean Weevil, Acanthoscelides obtectus (Say.) (Coleoptera: Chrysomelidae) on Different Surfaces.

In this study, the contact toxicity of spinetoram on three different surfaces, concrete, ceramic floor tile and laminate flooring, against Acanthocelides obtectus (Say.) (Coleoptera: Chrysomelidae) was evaluated in laboratory bioassays. Different concentrations were evaluated ranging from 0.0025 to 0.05 mg AI/cm2, against adults of A. obtectus. Adult mortality was measured after 1-, 3-, 5- and 7-day exposure. After 1-day exposure, the mortality was low on all surfaces, ranging from 0 to 27.2%. After 5- and 7-day exposure, spinetoram at concentrations of 0.01 mg/cm2 and above achieved 100% or close mortality on concrete and laminate flooring surface, whereas low concentrations (0.0025, 0.005 and 0.0075 mg AI/cm2) resulted in significantly lower mortality levels, ranging from 1.6 to 30.8%, than high concentrations. In the case of ceramic floor tile surface, spinetoram treatments at all tested concentrations did not result in 100% mortality. Significant differences were recorded among the surfaces, depending on concentrations and exposure intervals. After 3-, 5- and 7-day exposure, mortality levels on ceramic floor tile surface were generally higher at low concentrations than those on the concrete and laminate flooring surfaces, whereas those on concrete and laminate flooring surfaces were significantly higher at high concentrations than ceramic floor tile surface. These results indicate that spinetoram at 0.025 and 0.05 mg AI/cm2 achieve satisfactory control at relatively short exposures on common types of surfaces and thus can be used as an effective insecticide against A. obtectus.

Population-Mediated Responses of Lasioderma serricorne (Coleoptera: Anobiidae) to Different Diagnostic Protocols for Phosphine Efficacy.

We evaluated different monitoring protocols for phosphine resistance in adults of three populations of the cigarette beetle, Lasioderma serricorne (F.), termed LB, 2KT, and E1. The protocols evaluated were as follows: 1) the Food and Agriculture Organization (FAO) bioassay, i.e., the exposure at 30 ppm of phosphine for 20 h, 2) the dose response, based on the exposure at 50-1,000 ppm of phosphine for 3 d, 3) the Cooperation Center for Scientific Research Relative to Tobacco (CORESTA) bioassay, which is based on exposures of 4 d at 200 ppm, and, if there are surviving individuals, of 10 d at 700 ppm, and 4) Detia Degesch Phosphine Tolerance Test Kit (DDPTTK), based on the evaluation of the exposed insects for short intervals of exposure (in our case 90 min) at 3,000 ppm. From the populations tested, E1 indicated the highest survival in the FAO bioassay. Moreover, adults of this population were able to survive at 200 ppm, in the dose response, in contrast with the other populations. However, both 2KT and E1 gave similar results in DDPTTK, but only E1 had survivors after 7 d postexposure. Finally, only adults of E1 survived the CORESTA bioassay for 200 ppm and 4 d, but not for 700 ppm and 10 d. Our results show that, at least in the range of the populations tested here, there are some trends that are comparable among the different diagnostics for L. serricorne, which can be utilized further in designing a widely adopted standardized protocol.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

Effect of Nitrogen on Phosphine-Susceptible and -Resistant Populations of Stored Product Insects.

In this study, we evaluated nitrogen treatment on phosphine-resistant field and -susceptible laboratory populations of different stored product beetles. Nine trials were conducted in commercial nitrogen chambers with the O2 level set at 1.0%. Two different temperatures-i.e., 28 and 40 °C-and three exposure intervals-i.e., 2.5, 3 and 9 d-were used in our tests. Adults of the sawtoothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae); the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae); and the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) were used in the trials. The insects were placed in vials with different commodities per species and population, and their mortality was measured after the termination of each trial. Then, the vials were kept in incubator chambers at 25 °C and 65% relative humidity for 65 d to measure progeny production. Complete parental mortality was observed in all cases for O. surinamensis and S. oryzae, but there was some survival for T. castaneum at 28 °C and 3 d of exposure. In general, progeny production was completely (100%) suppressed, with some exceptions for all species and populations. The results indicate that low oxygen is effective for all species tested, regardless of their resistance status to phosphine, and can be further adopted as an alternative method to mitigate resistance in stored product beetles.