Phosphine resistance in the rust red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): inheritance, gene interactions and fitness costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.     

Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.     

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.     

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.     

Effective strategy for pyramiding three bacterial blight resistance genes into fine grain rice cultivar, Samba Mahsuri, using sequence tagged site markers

Bacterial leaf blight (BB) of rice is a major disease limiting rice production in several rice growing regions of the world. The pathogen, Xanthomonas oryzae pv oryzae, causing the disease is highly virulent to rice crops and is capable of evolving new races. Breeding efforts to incorporate single BB resistant gene often leads to resistance breakdown within a short period. To overcome such breakdown of resistance and develop germplasm with durable disease resistance, we have introgressed three bacterial blight resistance genes, xa5, xa13, and Xa21 into a fine grain rice variety, Samba Mahsuri, using sequence tagged site (STS) markers linked to these genes. Since the efficiency of the STS markers linked to recessive genes to detect homozygotes is less than 100%, we adopted four different pyramiding schemes to minimize loss of recessive resistance genes in advanced backcross generations. Pyramiding scheme A in which a two-gene Samba Mahsuri pyramid line containing Xa21 and xa5 genes was crossed with the Samba Mahsuri line having xa13 gene alone was found to be most effective in preventing the loss of an important recessive gene xa13. We further demonstrated that there was no yield penalty due to pyramiding of multiple genes into the elite indica rice variety.     

Sublethal Exposure to Phosphine Decreases Offspring Production in Strongly Phosphine Resistant Female Red Flour Beetles,Tribolium castaneum (Herbst)

The red flour beetle is a cosmopolitan pest of stored grain and stored grain products. The pest has developed resistance to phosphine, the primary chemical used for its control. The reproductive output of survivors from a phosphine treatment is an important element of resistance development but experimental data are lacking. We exposed mated resistant female beetles to 0.135 mg/L of phosphine for 48 h at 25°C. Following one week of recovery we provided two non-exposed males to half of the phosphine exposed females and to half of the non-exposed control females. Females that had been exposed produced significantly fewer offspring than non-exposed females. Females that remained isolated produced significantly fewer offspring than both exposed females with access to males and non-exposed controls (P<0.05). Some females were permanently damaged from exposure to phosphine and did not reproduce even when given access to males. We also examined the additional effects of starvation prior to phosphine exposure on offspring production. Non-exposed starved females experienced a small reduction in mean offspring production in the week following starvation, followed by a recovery in the second week. Females that were starved and exposed to phosphine demonstrated a very significant reduction in offspring production in the first week following exposure which remained significantly lower than that of starved non-exposed females (P<0.05). These results demonstrate a clear sublethal effect of phosphine acting on the female reproductive system and in some individuals this can lead to permanent reproductive damage. Pest population rebound after a fumigation may be slower than expected which may reduce the rate of phosphine resistance development. The results presented strongly suggest that phosphine resistance models should include sublethal effects.     

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F₁ reciprocal crosses and backcrosses between F₁ larvae and resistant (P_R) and susceptible (P_S) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F₁ crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F₁ larvae was slightly higher than that for P_S, suggesting that resistance is partially recessive. The responses of both backcross progeny (F₁ × P_R, F₁ × P_S) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F₁ × P_R backcross but decreased the correspondence with the F₁ × P_S backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

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.     

Somaclonal variant UC-T3: the expression of Fusarium wilt resistance in progeny arrays of celery,Apium graveolens L.

Summary First generation (S₁) progeny, second generation (S₂) progeny, and backcross (BC) progeny of a celery (Apium graveolens L. var. dulce) somaclonal variant, UC-T3, were evaluated for resistance to the fungus Fusarium oxysporum f. sp. apii, race 2 (FOA₂). Chisquare analysis of S₁ progeny showed that the expression of resistance did not fit a single-locus model. S₂ progeny means were similar among families, except in a heavily infested field. The lowest ranking S₂ family in both the lightly infested and heavily infested fields was significantly more resistant to FOA₂ than individuals of the susceptible progenitor line Tall-Utah 527OR; therefore; it was concluded that the trait was heritable. The phenotypic distribution of the backcross progeny was broad, suggesting that the new resistance was conferred by at least two genes whose expression was dominant to susceptibility. The mean scores for disease resistance of the progeny of crosses between UC-T3 and the moderately resistant line, Tall-Utah 527OHK, generally equaled the resistance found among the progeny of the most resistant parent.     

Genetic enhancement for semi-dwarf and bacterial blight resistance with enhanced grain quality characteristics in traditional Basmati rice through marker-assisted selection

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC₂F₂ was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.     

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.     

西花蓟马对多杀菌素的抗性汰选和遗传方式

在室内进行了西花蓟马Frankliniella occidentalis(Pergande)种群对多杀菌素的抗性筛选和抗性遗传方式分析。经过2年多的汰选,抗性水平相比较初始种群提高了30倍,与同时测定的敏感品系相比抗性达到80.8倍。抗性遗传方式分析结果表明,正、反交后代显性度分别为0.51和0.43,二者差异不显著,说明西花蓟马对多杀菌素的抗性为常染色体、不完全显性遗传;剂量对数死亡机率值曲线分析结果显示回交后代在死亡率50%处,自交后代在死亡率25%和75%处未出现明显平坡,回交和自交后代实测值卡方检验进一步证实抗性为多基因控制。     

Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F₁ hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F₂ crosses, backcrosses of F₁ hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F₁ (BH-R × VA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F₁ crosses, backcrosses and F₂ crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 μg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F₂ crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F₂ and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity.     

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.     

Biochemical mechanism of chlorantraniliprole resistance in Spodoptera litura (Fab) (Lepidoptera: Noctuidae)

Spodoptera litura, a polyphagus insect pest of economic importance, having the ability to develop resistance to various classes of insecticides was selected for the study. Leaf dip bioassay studies were done after ten generations of selection pressure reported the development of resistance up to 80 folds by S. litura against chlorantraniliprole compared to lab susceptible strain. Bioassay studies conducted using enzyme inhibitors such as triphenyl phosphate (TPP) followed by diethyl-maleate (DEM) and piperonyl butoxide (PBO) showed good amount of synergism with chlorantraniliprole and improved efficacy against resistant strain. Findings of bioassay studies were supported by in-vitro enzyme inhibition assays. Esterase activity in gut homogenate of resistant strain was significantly inhibited by TPP suggesting esterase mediated biochemical resistance development in S. litura against chlorantraniliprole.     

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.     

Genetic linkage between malathion and dieldrin resistance in Anopheles arabiensis

A strain of Anopheles arabiensis resistant to both malathion and dieldrin was crossed and backcrossed to a susceptible strain. The progeny were tested on each insecticide in turn. Less than 50% mortality in the second insecticide exposure among the backcross progeny indicated linkage between the resistance genes. In a backcross of A. gambiae X A. arabiensis hybrids a recombination rate of 7.5% was observed. A Y-translocation strain of A. arabiensis showed less than 2.8% recombination between the resistance genes. It is impossible to confirm the genotype of apparent recombinants using existing stocks, but the two resistance mechanisms are biochemically distinguishable. If the two genes are very closely linked, linkage disequilibrium could influence the consequences of switching to malathion spraying after dieldrin resistance has evolved.     

Mode of Inheritance of Amitraz Resistance in a Brazilian Strain of the Southern Cattle Tick, Boophilus microplus (Acari: Ixodidae)*

The southern cattle tick, Boophilus  microplus (Canestrini), has developed resistance to amitraz in several countries in recent years. A study was conducted at the USDA Cattle Fever Tick Research Laboratory in Texas to investigate the mode of inheritance of amitraz resistance with cross-mating experiments. The Muñoz strain, a laboratory reared acaricide-susceptible reference strain, was used as the susceptible parent and the Santa Luiza strain, originating in Brazil, was used as the resistant parent. A modified Food and Agriculture Organization Larval Packet Test was used to measure the levels of susceptibility of larvae of the parental strains, F₁, backcross, F₂, and F₃ generations. Results of reciprocal crossing experiments suggested that amitraz resistance was inherited as an incomplete recessive trait. There was a strong maternal effect on larval progeny’s susceptibility to amitraz in both the F₁ and the subsequent generations. The values of the degree of dominance were estimated at ?0.156 and ?0.500 for the F₁ larvae with resistant and susceptible female parents, respectively. Results of bioassays on larval progeny of the F₁ backcrossed with the resistant parent strain and that of the F₂ generations suggested that more than one gene was responsible for amitraz resistance in the Santa Luiza strain. Comparisons of biological parameters (engorged female weight, egg mass weight, and female-to-egg weight conversion efficiency index) indicated significant differences between different genotypes. The differences appeared to be heritable, but not related to amitraz resistance. Results from this study may have significant implications for the management of amitraz resistance.