Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes

Phosphine (PH₃) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.     

Influence of phosphine resistance genes on flight propensity and resource location in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): the landscape for selection

Phosphine resistance in Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) has evolved through changes to enzymes involved in basic metabolic pathways. These changes impose metabolic stress and could affect energy‐demanding behaviours. We therefore tested whether phosphine resistance alleles impact the movement of these insects in their quest for new resources. We measured walking and flight parameters of four T. castaneum genotypes: (1) a field‐derived population, (2) a laboratory cultured, phosphine‐susceptible reference strain, (3) a laboratory cultured, phosphine‐resistant reference strain, and (4) a resistant introgressed strain that is almost identical genetically to the susceptible population. The temporal pattern of flight was identical across all populations, but resistant beetles took flight significantly less, walked more slowly, and located resources less successfully than did susceptible beetles. Also, the field‐derived beetles (proved not to be carrying resistance genes) walked significantly faster and more directly towards food resources, and had a higher propensity for flight when compared to the susceptible laboratory beetles. These negative effects suggest survival of beetles with the resistance alleles will be compromised should they leave phosphine application sites. The field for selection therefore extends beyond the site at which phosphine fumigant imposed its effect, and other mutations are also likely to be affected in this way.     

Inheritance pattern and expression of resistance to phosphine in larval stage of Tribolium castaneum (Coleoptera: Tenebrionidae)

Resistance developed by Tribolium castaneum (Herbst), a major pest of stored grain and grain products, to phosphine is a serious concern. Treatment of phosphine is given to all the stages (egg, larva, pupa and adult) of insects simultaneously in the infected commodity. Effective resistance management tactics need a proper understanding of expression of resistance in relation to dosage in immature stages including larvae. So, this study was undertaken to understand the genetic basis of phosphine resistance in larval stage of T. castaneum by crossing the resistant and susceptible parents to obtain the F₁, F₂ and the back-cross progenies. The resistant parent had a resistance ratio of 37.75 when compared to the susceptible parent. The susceptibility levels of the reciprocal F₁ hybrids did not show any significant difference indicating autosomal mode of inheritance of resistance. Level of dominance for the reciprocal F₁ hybrids was estimated (-0.70 and ?0.68) which showed that the resistance is expressed as incompletely recessive in larval stage of T. castaneum. χ² analysis showed that the observed and expected mortalities differed significantly (P < 0.05) in most of the concentrations indicating that more than one gene is responsible for phosphine resistance. Two plateaus at ～ 40 % and ～ 90 % mortality levels for the back-cross progeny to the resistant parent further confirmed the involvement of two or more genes in imparting resistance to phosphine. Our results will aid in enhancing the knowledge on the development of resistance to phosphine in the field and thus facilitate in designing suitable resistance management tactics.     

Response of Tribolium castaneum and Rhyzopertha dominica to various resources,near and far from grain storage

Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) are common cosmopolitan pests of stored grain and grain products. We evaluated the relative attraction of T. castaneum and R. dominica to wheat, sorghum and cotton seeds in the field, near grain storage facilities and well away from storages in southern and central Queensland using multiple trapping techniques. The results show that T. castaneum is more strongly attracted to linted cotton seed relative to wheat, whereas R. dominica did not respond to cotton seed at all and was attracted only to wheat. Significantly more adults of T. castaneum (10–15 times) were attracted to traps placed on the ground, near grain storage, than to equivalent traps that were suspended (1.5 m above the ground) nearby. These results suggest that Tribolium beetles detect and respond to resources towards the end of their dispersal flight, after which they localize resources while walking. By contrast R. dominica was captured only in suspended traps, which suggests they fly directly onto resources as they localize them. The ability of both species to colonize and reproduce in isolated resource patches within the relatively short time of 1 month is illustrated by the returns from the traps deployed in the field (at least 1 km from the nearest stored grain) even though they caught only a few beetles. The results presented here provide novel insights about the resource location behaviours of both T. castaneum and R. dominica. In particular, the relationship of T. castaneum with non‐cereal resources that are not conventionally associated with this species suggests an emphasis on these other resources in investigating the resource location behaviour of these beetles. This new perspective on the ecology of T. castaneum highlights the potential role of non‐cereal resources (such as the lint on cotton seed) in the spread of grain pest infestations.     

Effect of five diatomaceous earth formulations against Tribofium castaneum （Coleoptera： Tenebrionidae）, Oryzaephflus surinamensis （Coleoptera： Silvanidae） and Rhyzopertha dominica （Coleoptera： Bostrychidae）

Laboratory bioassays were conducted to determine the effect of food source on the survival of Tribolium castaneum Herbst, Oryzaephilus surinamensis L. and Rhyzopertha dominica F., after exposure to five diatomaceous earth （DE） formulations： Protect-It, Insecto, Perma-GuardTM, Dryacide and SilicoSec. Adults of these species were exposed to DEs at the rate of 0.5 mg/cm^2 for 1 day on filter paper inside plastic Petri dishes. After exposure, the initial mortality was counted and live individuals of the three species were held for a week in glass vials containing 50 mg wheat flour, rice and whole wheat, respectively. In the second experiment, after 1 day exposure to DEs, beetles were transferred to Petri dishes without food and held for a week to determine if the presence of food source would decrease the mortality of beetles. Experiments were carried out at 27℃ and 55% RH in the dark. The initial mortality in both of the experiments reached 100% for the three species exposed to Protect-It and in the case ofR. dominica and O. surinamensis exposed to Dryacide. In contrast, low level of mortality （〈 10%） was observed for T. castaneum exposed to Perma-GuardTM and Insecto. The mortality after the post-treatment period on food was decreased for the three species exposed to Perma-GuardTM and in the case of T. castaneum and R. dominica exposed to Insecto and SilicoSec. Adults of O. surinamensis were the most susceptible followed by R. dominica and 100% adult mortality was obtained, whereas T. castaneum were the least susceptible beetles to DEs. Protect-It and Dryacide were the most efficient DE formulations and can be used effectively in a stored grain integrated pest management program.     

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae (L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated So_rph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in S. oryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.     

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.     

Distribution of Pyrethroid Resistant Populations of Triatoma infestans in the Southern Cone of South America

BackgroundA number of studies published during the last 15 years showed the occurrence of insecticide resistance in Triatoma infestans populations. The different toxicological profiles and mechanisms of resistance to insecticides is due to a genetic base and environmental factors, being the insecticide selective pressure the best studied among the last factors. The studies on insecticide resistance on T. infestans did not consider the effect of environmental factors that may influence the distribution of resistance to pyrethroid insecticides. To fill this knowledge gap, the present study aims at studying the association between the spatial distribution of pyrethroid resistant populations of T. infestans and environmental variables.Conclusions/SignificanceThe occurrence of these two groups concentrated over a particular region that coincides with the area where populations of the intermediate cytogenetic group were found might reflect the spatial heterogeneity of the genetic variability of T. infestans, that seems to be the cause of the insecticide resistance in the area, even on sylvatic populations of T. infestans, never before exposed to pyrethroid insecticides, representing natural and wild toxicological phenotypes. The strong linear relationship found between LD₅₀ and RR₅₀ suggest RR₅₀ might not be the best indicator of insecticide resistance in triatomines.     

Predator‐parasitoid‐host interaction: biological control of Rhyzopertha dominica and Sitophilus oryzae by a combination of Xylocoris flavipes and Theocolax elegans in stored cereals

Post‐harvest management constraints are economically significant in Africa for a broad range of commodities, resulting in a substantial negative impact on food security and livelihoods. Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae) coexists with Sitophilus oryzae (L.) (Coleoptera: Dryophthoridae) – they infest stored cereal grains, especially in sub‐Saharan Africa, and cause significant losses in storage. Laboratory studies were carried out to determine the efficacy of Xylocoris flavipes (Reuter) (Hemiptera: Anthocoridae) and Theocolax elegans (Westwood) (Hymenoptera: Pteromalidae), alone or in combination, in suppressing populations of R. dominica and S. oryzae in stored rice and wheat grains. The weight loss and moisture of the infested grains were assessed, for the various combinations of the two pest insects and the two natural enemies. In rice there was a synergistic effect of the combined release of T. elegans and X. flavipes against both R. dominica and S. oryzae compared to the effect of one natural enemy alone. With the combined release, only 2% F₁ progeny of the pest beetles emerged compared to the untreated control (100%), whereas 3.5 and 6% emerged when X. flavipes and T. elegans, respectively, were released alone. When the two natural enemies were released in combination, 0.4% R. dominica F₁ progeny emerged, compared to 1.6% with X. flavipes or T. elegans alone. In the combined release only 0.3% S. oryzae F₁ progeny emerged, whereas 65% emerged with X. flavipes alone and 41% with T. elegans alone. No synergistic effect of the combined release of the two natural enemies was detected in wheat. In stored rice, the lowest percentage of weight loss (44%) was found when both natural enemies and both beetle species were present. We conclude that the combination of the predator and the parasitoid should be recommended when R. domininca and S. oryzae coexist and feed on stored cereals, especially rice.     

Repellency of Palizin® (Coconut Soap) with three laboratory techniques against five stored-product insect pests

The heavy economic damages are the major concern in storage that was caused by coleopteran pests. The conventional control that is accomplished by synthetic pesticides created toxicity to non-target organisms, development of resistance and residue’s problems. Nowadays, with changes in legislation and consumer demand, alternatives to synthetic pesticide to manage the store product pest are increasingly needed. The repellent compounds could be considered as one of the mentioned methods in storage. In this study, five widespread species of stored product pest including Sitophilus oryzae Linnaeus, Tribolium castaneum Herbst, Rhizopertha dominica Fabricius, Oryzaephilus surinamensis Linnaeus and Lasioderma serricorne Fabricius were conducted to assay the percentage repellency (PR) of Palizin^® (Coconut Soap 65%). Percentage repellency was achieved with beetles which were exposed to 0, 0.5, 1, 5 and 10% concentration using three techniques: filter papers, Y-shape tube and cup bioassays. In each of these methods, the means of PR reacted to increasing concentration except T. castaneum and O. surinamensis after 72 and 48 h. The maximum PR of Palizin^® belonged to a concentration of 10% (except R. dominica at 5% and 48 h). Among the methods, except filter paper, degradation process resulted to a decreasing trend of PR. In the present experiment, Palizin^® can be recommended as a limiting factor of all beetles and the data which were exported by Cub bioassay were closely adapted to reality condition of storage.     

Effects of heat shock on ovary development and hsp83 expression in Tribolium castaneum (Coleoptera: Tenebrionidae)

Heat shock affects reproductive performance in insects including Tribolium castaneum. In this study, the effects of heat shock on ovary development and hsp83 expression in T. castaneum were investigated. Two lines of T. castaneum, H line and C line, from the same base population were established and maintained for five successive generations. In each generation, the newly hatched beetles (within 3 h after eclosion) in the H line were treated with a heat shock at 40°C for 1 h, and those in the C line were raised at normal temperature (30°C) as control treatment. Four traits related to ovary development were measured for the beetles of the fifth generation: days from eclosion to laying the first eggs (T_o), days from eclosion to laying the first hatchable eggs (T_h), ovariole size on the third day after eclosion, and pupal mass of their offspring. The results showed that the beetles of the H line had a significantly longer pre‐oviposition period (0.6 more days) and smaller ovariole size than those of the C line. No significant difference in pupal mass was observed. Applying heat shock to the offspring of the fifth generation of both lines led to significantly higher hsp83 expression in offspring of the C line than in offspring of the H line. Within each line, the hsp83 expression level in offspring with heat shock was significantly higher than that of offspring without heat shock, but the difference in the C line was much larger than that in the H line. We infer from these results that a tradeoff between heat resistance, registered as hsp83 expression, and ovarian development operates under heat stress in T. castaneum. 2009 Wiley Periodicals, Inc.     

Fine mapping of the Rrs1 resistance locus against scald in two large populations derived from Spanish barley landraces

Key message

In two Spanish barley landraces with outstanding resistance to scald, the Rrs1 _Rh4 locus was fine mapped including all known markers used in previous studies and closely linked markers were developed.

Abstract

Scald, caused by Rhynchosporium commune, is one of the most prevalent barley diseases worldwide. A search for new resistance sources revealed that Spanish landrace-derived lines SBCC145 and SBCC154 showed outstanding resistance to scald. They were crossed to susceptible cultivar Beatrix to create large DH-mapping populations of 522 and 416 DH lines that were scored for disease resistance in the greenhouse using two R. commune isolates. To ascertain the pattern of resistance, parents and reference barley lines with known scald resistance were phenotyped with a panel of differential R. commune isolates. Subpopulations were genotyped with the Illumina GoldenGate 1,536 SNP Assay and a large QTL in the centromeric region of chromosome 3H, known to harbour several scald resistance genes and/or alleles, was found in both populations. Five SNP markers closest to the QTL were converted into CAPS markers. These CAPS markers, together with informative SSR markers used in other scald studies, confirmed the presence of the Rrs1 locus. The panel of differential scald isolates indicated that the allele carried by both donors was Rrs1 _Rh4 . The genetic distance between Rrs1 and its flanking markers was 1.2 cM (11_0010) proximally and 0.9 cM (11_0823) distally, which corresponds to a distance of just below 9 Mbp. The number and nature of scald resistance genes on chromosome 3H are discussed. The effective Rrs1 allele found and the closely linked markers developed are already useful tools for molecular breeding programs and provide a good step towards the identification of candidate genes.     

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l⁻¹ and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1^R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1^R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1^V or the duplicated ace-1^D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.     

Potential of the neonicotinoid imidacloprid and the oxadiazine indoxacarb for controlling five coleopteran pests of stored grain

Abstract The potential for using imidacloprid (a neonicotinoid) and indoxacarb (an oxadiazine) as grain protectants was investigated in bioassays against resistant strains of five stored grain beetles. The species investigated were Rhyzopertha dominica (F.) (the lesser grain borer), Sitophilus oryzae (L.) (the rice weevil), Tribolium castaneum (Herbst) (the rust‐red flour beetle), Oryzaephilus surinamensis (L.) (the saw tooth flour beetle), and Cryptolestes ferrugineus (Stephens) (the flat grain beetle). Each of these species has developed resistance to one or more protectants, including organophosphorus insecticides, synthetic pyrethroids and the juvenile hormone analogue methoprene. Mortality and reproduction after a 2‐week exposure of adults to treated wheat depended on species, dose and insecticide. Imidacloprid had no effect on S. oryzae at any dose, but none of the other species produced any live progeny at 10 mg/kg. Indoxacarb had no effect on T. castaneum at any dose, but none of the other species produced any live progeny at 5 mg/kg. The results show that although both imidacloprid and indoxacarb can control at least four of the five key pests tested at doses comparable to those used for organophosphorus protectants, more potent neonicotinoid or oxadiazine insecticides would be needed than either of these to provide broad spectrum protection of stored grain.     

Flight directionality of Tribolium castaneum soon after take‐off under glasshouse and field conditions

Flight directionality of the rust‐red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), was investigated under glasshouse and field conditions using sticky traps placed around dense experimental infestations of T. castaneum derived from field‐collected samples. Although beetles of this species are known to fly quite readily, information on flight of beetles away from grain resources is limited. Under still glasshouse conditions, T. castaneum does not demonstrate strong horizontal or vertical trajectories in their initial flight behaviour. Flight was significantly directional in half of the replicates, but trapped beetles were only weakly concentrated around the mean direction of flight. In the field, by contrast, emigration of T. castaneum was strongly directional soon after flight initiation. The mean vector lengths were generally >0.5 which indicates that trapped beetles were strongly concentrated around the calculated mean flight direction. A circular‐circular regression of mean flight vs. mean downwind direction suggested that flight direction was generally correlated with downwind direction. The mean height at which T. castaneum individuals initially flew was 115.4 ± 7.0 cm, with 58.3% of beetles caught no more than 1 m above the ground. The height at which beetles were trapped did not correlate with wind speed at the time of sampling, but the data do indicate that wind speed significantly affected T. castaneum flight initiation, because no beetles (or very few; no more than three) were trapped in the field when the mean wind speed was above 3 m s^?1. This study thus demonstrates that wind speed and direction are both important aspects of flight behaviour of T. castaneum, and therefore of the spatio‐temporal dynamics of this species.     

Bioactivity of powders and essential oils of three Asteraceae plants as post-harvest grain protectants against three major coleopteran pests

Powders and essential oils were obtained from Achillea biebersteinii, A. fragrantissima and Ageratum conyzoides and tested for their insecticidal activity against Sitophilus oryzae, Rhyzopertha dominica and Tribolium castaneum. Composition of the oils was identified by gas chromatography (GC) and GC/mass spectrometry (MS). The tested plants showed adulticidal activity, where toxicity varied with dosage of the plant product, the insect tested and the exposure period. When mixed with grains as ground powders, a dosage of 20 g/kg grains killed 88.2, 73.6 and 64.4% of S. oryzae after 12 days of exposure with 79.6, 63.3 and 48% reduction in progeny for powders of A. biebersteinii, A. conyzoides and A. fragrantissima, respectively. Under the same assay conditions, A. biebersteinii and A. conyzoides powders killed 100% of R. dominica. T. castaneum was less susceptible to the plant powders. The LC₅₀ values for powders were 22.8, 31.6 and 39.7 mg/g grains for A. biebersteinii, 27.1, 35.4 and 47.8 mg/g grains for A. conyzoides and 36.9, 48.3 and 78.6 mg/g grains for A. fragrantissima against R. dominica, S. oryzae and T. castaneum, respectively. Upon fumigation, a dose of 60 μl/L air of A. biebersteinii and A. conyzoides oils was sufficient to kill 100% of R. dominica after 12 days of adult exposure, while 91.3% adult mortality was recorded in the case of A. fragrantissima oil. S. oryzae and T. castaneum showed a great susceptibility to the plant oils. Powders exhibited a moderate to strong residual activity where A. conyzoides showed the greatest grain protecting activity.     

Toxic and repellent properties of Xylopia aethiopica (Dunal) A. Richard on Tribolium castaneum Herbst infesting stored millets,Pennisetum glaucum (L.) R. Br.

The toxicity and repellency of Xylopia aethiopica seed extract was investigated in the laboratory against Tribolium castaneum Herbst. Concentration and days after treatment (DAT) caused a significant increase in T. castaneum adult mortality with an interaction effect of both on mortality when filter paper was impregnated with X. aethiopica extract. At 0.2 ml/60 cm² extract, significant mortality was observed at three–seven DAT when compared with one DAT. At 0.4 ml/60cm², 100% mortality was recorded at the lowest exposure period of one DAT. When 0.2 ml extract was applied to 5 g millet seeds, mortality at five–seven DAT was significantly higher than mortality observed in the control. Although repellency was dose-dependent, the percentage of T. castaneum that were repelled from treated filter paper was not significant. At 0.4 ml/60 cm², Class II repellency (26.7%) was observed. The results suggest that X. aethiopica can only effectively control T. castaneum populations that have infested millet but do not prevent cross-infestation via repellency.