Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma

Phosphine gas, or hydrogen phosphide (PH3), is the most common insecticide applied to durable stored products worldwide and is routinely used in the United States for treatment of bulk-stored cereal grains and other durable stored products. Research from the late 1980s revealed low frequencies of resistance to various residual grain protectant insecticides and to phosphine in grain insect species collected in Oklahoma. The present work, which used the same previously established discriminating dose bioassays for phosphine toxicity as in the earlier study, evaluated adults of nine different populations of red flour beetle, Tribolium castaneum (Herbst), and five populations of lesser grain borer, Rhyzopertha dominica (F.) collected from different geographic locations in Oklahoma. One additional population for each species was a laboratory susceptible strain. Discriminating dose assays determined eight out of the nine T. castaneum populations, and all five populations of R. dominica, contained phosphine-resistant individuals, and highest resistance frequencies were 94 and 98%, respectively. Dose-response bioassays and logit analyses determined that LC99 values were approximately 3 ppm for susceptible and 377 ppm for resistant T. castaneum, and approximately 2 ppm for susceptible and 3,430 ppm for resistant R. dominica. The most resistant T. castaneum population was 119-fold more resistant than the susceptible strain and the most resistant R. dominica population was over 1,500-fold more resistant. Results suggest a substantial increase in phosphine resistance in these major stored-wheat pests in the past 21 yr, and these levels of resistance to phosphine approach those reported for other stored-grain pest species in other countries.     

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.     

赤拟谷盗的磷化氢敏感和抗性品系体内羧酸酯酶的活性比较

本文比较测定了赤拟谷盗Tribolium castaneum(Herbst)的磷化氢抗性(Rf=327)和敏感品系害虫的羧酸酯酶活性,研究了该害虫同一品系不同个体间羧酸酯酶的活性差异,比较了两品系害虫在系列磷化氢浓度下熏蒸24h和6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的羧酸酯酶活性。主要结果为:未熏蒸的抗性害虫幼虫和蛹体内的羧酸酯酶活性分别高于敏感品系的1.37和1.16倍;敏感和抗性害虫同品系内不同个体间羧酸酯酶活性分布频率都存在明显差异,抗性害虫中酶活性大的个体数量占的比例较大;磷化氢浓度分别为0.69×10-2、2.78×10-2、5.56×10-2、8.33×10-2和11.11×10-2mg/L时都可导致敏感害虫羧酸酯酶的活性降低,但活性受抑制的程度不因浓度高低呈相应的增减。浓度分别为5.56×10-2、11.11×10-2、13.89×10-2、20.83×10-2和27.78×10-2mg/L的熏蒸中抗性害虫体内酶活性增加,且活性增高的程度与浓度增减也不呈对应变化。在6.94×10-2mg/L磷化氢浓度下熏蒸不同时间的结果中,敏感害虫的酶活性随时间延长而下降,抗性害虫的活性则随时间延长而增大。研究表明赤拟谷盗对磷化氢的抗性可能与羧酸酯酶的活性增加有关。     

The rph2 gene is responsible for high level resistance to phosphine in independent field strains of Rhyzopertha dominica

The lesser grain borer Rhyzopertha dominica (F.) is one of the most destructive insect pests of stored grain. This pest has been controlled successfully by fumigation with phosphine for the last several decades, though strong resistance to phosphine in many countries has raised concern about the long term usefulness of this control method. Previous genetic analysis of strongly resistant (SR) R. dominica from three widely geographically dispersed regions of Australia, Queensland (SR(QLD)), New South Wales (SR(NSW)) and South Australia (SR(SA)), revealed a resistance allele in the rph1 gene in all three strains. The present study confirms that the rph1 gene contributes to resistance in a fourth strongly resistant strain, SR2(QLD), also from Queensland. The previously described rph2 gene, which interacts synergistically with rph1 gene, confers strong resistance on SR(QLD) and SR(NSW). We now provide strong circumstantial evidence that weak alleles of rph2, together with rph1, contribute to the strong resistance phenotypes of SR(SA) and SR2(QLD). To test the notion that rph1 and rph2 are solely responsible for the strong resistance phenotype of all resistant R. dominica, we created a strain derived by hybridising the four strongly resistant lines. Following repeated selection for survival at extreme rates of phosphine exposure, we found only slightly enhanced resistance. This suggests that a single sequence of genetic changes was responsible for the development of resistance in these insects.     

Fitness consequences of malathion-specific resistance in red flour beetle (Coleoptera: Tenebrionidae) and selection for resistance in the absence of malathion

Malathion resistance in the red flour beetle, Tribolium castaneum (Herbst), is a worldwide problem and is very stable once it becomes widespread in natural populations. In the absence of insecticide the proportion of resistant phenotypes may rapidly decline but the development of resistance does not always involve reduced fitness. Malathion-specific resistance in T. castaneum seems not to involve any loss of fitness in laboratory or field conditions. Susceptible beetles were in competition with resistant beetles at different initial frequencies and modifications of susceptible gene frequency were estimated in these laboratory populations over 10 generations. A significant decrease in susceptible gene frequency was observed in Tribolium populations over time. The selection coefficient of the susceptible allele was estimated and the fitness of susceptible alleles in all tests was observed to range from 0.89 to 0.93 compared with the fitness of resistant genotypes, which was assumed to be 1. Data provided evidence that the resistant strains exhibited fitness advantages in the absence of malathion. We also compared the biotic potential (fecundity and developmental time) of the susceptible strain, the homozygous malathion-specific resistant strain, and their hybrids. Malathion-specific resistant strains showed an 8 -23% increase in biotic potential relative to the susceptible strain. These findings are consistent with those of malathion-specific resistance in T. castaneum; the fitness of the insects seems independent of the genetic background and the fitness of the resistant insects is not affected by this resistance mechanism.     

Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae)

The inheritance of resistance to phosphine was studied in two strains of the lesser grain borer, Rhyzopertha dominica (F.), labeled 'Weak-R' and 'Strong-R'. These strains were purified versions of field-selected populations collected in Queensland, Australia. Weak-R and Strong-R were, respectively, 23.4 times (20-h exposure) and 600 times (48-h exposure) resistant to phosphine compared with a reference susceptible strain (S-strain). Each -R strain was crossed with the S-strain and the response to phosphine was measured in their respective F1, F2, and F1-backcross (F1-BC) progenies. Data from testing of reciprocal F1 progeny indicated that resistance in Weak-R was autosomal and incompletely recessive with a degree of dominance -0.96. Modified chi-square analysis and contingency analysis of the observed response to phosphine of F1-BC and F2 progenies rejected the hypothesis of single gene inheritance of resistance. Analysis of the response of the F1, F2, and F1-BC progeny from the Strong-R x S-strain cross also rejected the null hypothesis for single gene resistance. Resistance in the Strong-R strain was autosomal and incompletely recessive with a degree of dominance of -0.64. The Weak-R and Strong-R strains were then crossed. Analysis ofthe F1 and F2 progenies of this reciprocal cross revealed that the strong resistance phenotype was coded by a combination of the genes already present in the Weak-R genotype plus an extra major, incompletely recessive gene. There was also evidence of a minor dominant gene present in approximately 5% of Strong-R individuals.     

Susceptibility of laboratory and field strains of four stored-product insect species to spinosad

Two field strains of the Indianmeal moth, Plodia interpunctella (Hübner); red flour beetle, Tribolium castaneum (Herbst); and lesser grain borer, Rhyzopertha dominica (F.), and one field strain of the rusty grain beetle, Cryptolestes ferrugineus (Stephens), were collected from hard red winter wheat stored on farms in northeastern Kansas. Fifty eggs of P. interpunctella and 25 beetle adults of each species were exposed to 100 g of untreated wheat or wheat treated with various rates of spinosad, to determine susceptibility of the field and corresponding insecticide-susceptible laboratory strains. Mortality of beetle adults and P. interpunctella larvae was assessed after 7 and 21 d postinfestation, respectively. Field strains of P. interpunctella, C. ferrugineus, and T. castaneum were less susceptible to spinosad than the corresponding laboratory strains. The LD50 and LD95 values for P. interpunctella and C. ferrugineus field strains were 1.7-2.5 times greater than values for corresponding laboratory strains. Adults of both laboratory and field strains of T. castaneum were tolerant to spinosad, resulting in <88% mortality at 8 mg/kg. The LD50 and LD95 values for the field strains of T. castaneum were 2.0-7.5 times greater compared with similar values for the laboratory strain. The field and laboratory strains of R. dominica were highly susceptible to spinosad, and one of the field strains was relatively less susceptible to spinosad than the laboratory strain. Our results confirm a range of biological variability in field populations, which is consistent with findings for other compounds, and underscores the need to adopt resistance management programs with stored grain insect pests. The baseline data generated on the susceptibility of the four insect species to spinosad will be useful for monitoring resistance development and for setting field rates.     

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.     

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.     

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.     

Carboxyesterase degradation of malathion in vitro by susceptible and resistant strains of Tribolium castaneum (Herbst) (Coleoptera, Tenebrionidae)

Malathion monoacid was found to be the major metabolite of malathion produced in vitro by homogenates of susceptible and resistant Tribolium castaneum. Slightly increased production of this metabolite was detected in a malathion specific resistant strain. In all strains, monoacid formation was inhibited by triphenyl phosphate. Partial purification of the enzymes responsible failed to indicate a significant increase in enzyme levels in the resistant strain.     

Enhanced esterase gene expression and activity in a malathion-resistant strain of the tarnished plant bug, Lygus lineolaris

Extensive use of insecticides on cotton in the mid-South has prompted resistance development in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). A field population of tarnished plant bugs in Mississippi with 11-fold higher resistance to malathion was used to examine how gene regulation conferred resistance to this organophosphate insecticide. In laboratory bioassays, synergism by the esterase inhibitors S,S,S,-tributylphosphorotrithioate (DEF) and triphenylphosphate (TPP) effectively abolished resistance and increased malathion toxicity by more than 80%. Esterase activities were compared in vitro between malathion susceptible and resistant (selected) strains. More than 6-, 3- and 10-fold higher activities were obtained with the resistant strain using alpha-naphthyl acetate, beta-naphthyl acetate, and p-nitrophenyl acetate, respectively. Up to 95% and 89% of the esterase activity in the susceptible and resistant strains, respectively, was inhibited by 1 mM DEF. Inhibition of esterase activity up to 75% and 85% in the susceptible and resistant strains, respectively, was obtained with 0.03 mM TPP. Esterase activities in field populations increased by up to 5.4-fold during the fall season. The increase was synchronized with movement of the insect into cotton where exposure to pesticides occurred. Esterase cDNA was cloned and sequenced from both malathion susceptible and resistant strains. The 1818-nucleotide cDNA contained a 1710-bp open reading frame coding a 570 amino acid protein which was similar to many insect esterases conferring organophosphate resistance. No amino acid substitution was observed between susceptible and resistant strains, indicating that esterase gene mutation was not involved in resistance development in the resistant strain in Mississippi. Further examination of esterase gene expression levels using quantitative RT-PCR revealed that the resistant strain had a 5.1-fold higher level of esterase mRNA than the susceptible strain. The results of this study indicated that up-regulation of the esterase gene appeared to be related to the development of resistance in the tarnished plant bug.     

棉铃虫对氰戊菊酯抗性遗传分析

本文采用剂量对数-死亡机率值回归线(LD-P线)分析法, 利用室内选育的棉铃虫 Heliccverpa armitera(Hubner)对氰戊菊酯抗性晶系和敏感品系,研究了棉铃虫对氰戊菊酯的抗性遗传规律。结果表明,棉铃虫对氰戊菊酯的抗性为多基因遗传,其主要基因为不完全显性,抗性不属性连锁遗传。利用增效剂对抗性机理进行的研究表明,多功能氧化酶(MFO)是棉铃虫对氰戊菊酯产生抗药性的主要因子,因此,可以推断多功能氧化酶基因为不完全显性。     

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.     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Cross-resistance between dieldrin and fipronil in German cockroach (Dictyoptera: Blattellidae)

The toxicity of fipronil and dieldrin was determined in one susceptible laboratory strain and seven insecticide-resistant field-collected strains of Blattella germanica (L). The Zo960302 and Ga021001 strains were 1,270- and 2,030-fold resistant to dieldrin and 15- and 14-fold resistant to fipronil. The Su960304 and Od010803 strains were 15- and 13-fold resistant to dieldrin and two- and four-fold resistant to fipronil. Three strains showed no or a low level of resistance to dieldrin and fipronil. Crosses were performed between the susceptible strain Danish Pest Infestation Laboratory (DPIL)-SUS and the resistant strains Zo960302 and Su960304 and resistance to dieldrin and fipronil were intermediate compared with the susceptible and the resistant strains. Backcrosses to both of the parental strains showed cosegregation of dieldrin and fipronil resistance. The toxicity of dieldrin and fipronil was correlated when compared at LD50, and 93% of the observed variation in LD50 of fipronil can be ascribed to variation among predictions based on the value of LD50 of dieldrin. The frequency of the A302S substitution in the resistance to dieldrin (Rdl) gene in the highly dieldrin- and fipronil-resistant strains Zo960302 and Ga021001 and the moderately resistant Su960304 was 0.97, 1.0, and 0.38, respectively. We consider the connection between the frequency of the Rdl mutation and dieldrin and fipronil resistance a causal connection and not merely a coincidence.     

Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.     

Multiple Origins of Cyclodiene Insecticide Resistance in Tribolium castaneum (Coleoptera: Tenebrionidae)

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a γ-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5–6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution. Received: 14 October 1998 / Accepted: 4 January 1999