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Diagnostic molecular markers for phosphine resistance in U.S. populations of Tribolium castaneum and Rhyzopertha dominica.

Chen Z, Schlipalius D, Opit G, Subramanyam B, Phillips TW - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America; Plant Biosecurity Cooperative Research Center, Bruce, Australian Capital Territory, Australia.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Locations for field-collected populations of T. castaneum in Kansas, USA.
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pone.0121343.g001: Locations for field-collected populations of T. castaneum in Kansas, USA.

Mentions: An opportunity presented itself for us to compare the frequency of the resistance gene in T. castaneum with the percentage of resistant insects in seven locally collected populations. T. castaneum were collected in 2011 from seven farms that had stored wheat on site, all within a 100 km radius of each other, in north-central Kansas, USA (Fig 1) and maintained as large laboratory-reared colonies until used [18]. The approximate geographic locations of the collection sites, by population codes for latitude and longitude, respectively, are: AB1, 39°01′ N and 97°12 W; AB2, 38°59′N and 97°19′W; MCP, 38°22′N and 97°39′W; MIT, 39°20′N and 98°28′W; MIN, 39°07′N and 97°42′W; RUS, 38°53′N and 98°51′W; and WAS, 39°34′N and 97°16′W. In order to score both weak and strong phosphine resistance in these populations we carried out a modified FAO (1975) bioassay with 50 individuals/vial and 3 replicates per population. The discriminating dose for weak resistance was 30ppm for 20 hrs, as described above, and the discriminating dose for the strong resistance phenotype was set at 180ppm for 20 hrs [19] in separate experiments. Molecular marker analysis for the strong resistance SNP marker was performed on all seven populations as described above and its frequency was calculated for each population from the individual genotypes inferred by the CAPS markers.


Diagnostic molecular markers for phosphine resistance in U.S. populations of Tribolium castaneum and Rhyzopertha dominica.

Chen Z, Schlipalius D, Opit G, Subramanyam B, Phillips TW - PLoS ONE (2015)

Locations for field-collected populations of T. castaneum in Kansas, USA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380312&req=5

pone.0121343.g001: Locations for field-collected populations of T. castaneum in Kansas, USA.
Mentions: An opportunity presented itself for us to compare the frequency of the resistance gene in T. castaneum with the percentage of resistant insects in seven locally collected populations. T. castaneum were collected in 2011 from seven farms that had stored wheat on site, all within a 100 km radius of each other, in north-central Kansas, USA (Fig 1) and maintained as large laboratory-reared colonies until used [18]. The approximate geographic locations of the collection sites, by population codes for latitude and longitude, respectively, are: AB1, 39°01′ N and 97°12 W; AB2, 38°59′N and 97°19′W; MCP, 38°22′N and 97°39′W; MIT, 39°20′N and 98°28′W; MIN, 39°07′N and 97°42′W; RUS, 38°53′N and 98°51′W; and WAS, 39°34′N and 97°16′W. In order to score both weak and strong phosphine resistance in these populations we carried out a modified FAO (1975) bioassay with 50 individuals/vial and 3 replicates per population. The discriminating dose for weak resistance was 30ppm for 20 hrs, as described above, and the discriminating dose for the strong resistance phenotype was set at 180ppm for 20 hrs [19] in separate experiments. Molecular marker analysis for the strong resistance SNP marker was performed on all seven populations as described above and its frequency was calculated for each population from the individual genotypes inferred by the CAPS markers.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Kansas State University, Manhattan, Kansas, United States of America; Plant Biosecurity Cooperative Research Center, Bruce, Australian Capital Territory, Australia.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus