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Deep sequencing of pyrethroid-resistant bed bugs reveals multiple mechanisms of resistance within a single population.

Adelman ZN, Kilcullen KA, Koganemaru R, Anderson MA, Anderson TD, Miller DM - PLoS ONE (2011)

Bottom Line: Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin.Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance.These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

View Article: PubMed Central - PubMed

Affiliation: Fralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, Virginia, United States of America. zachadel@vt.edu

ABSTRACT
A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

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Relative expression of metabolic genes in Harlan and Richmond strain bed bugs.Real-time quantitative PCR was performed to measure the relative expression levels of (A) P450, (B) GST, and (C) CE transcripts in the pyrethroid-resistant Richmond strain as compared with the susceptible Harlan strain. Each bar represents the mean of two biological replicates, each performed in triplicate. Error bars indicate the standard deviation from the mean. All samples were normalized to the expression of alpha-tubulin. A second control gene [myosin heavy-chain, indicated by (c)] was included to verify plate-to-plate consistency. Harlan (white) and Richmond (shaded) are indicated for each gene, with Harlan set to 1 for all samples. Fold change is indicated above each bar where 2-fold or greater for all statistically significant samples (** = p<0.01).
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pone-0026228-g004: Relative expression of metabolic genes in Harlan and Richmond strain bed bugs.Real-time quantitative PCR was performed to measure the relative expression levels of (A) P450, (B) GST, and (C) CE transcripts in the pyrethroid-resistant Richmond strain as compared with the susceptible Harlan strain. Each bar represents the mean of two biological replicates, each performed in triplicate. Error bars indicate the standard deviation from the mean. All samples were normalized to the expression of alpha-tubulin. A second control gene [myosin heavy-chain, indicated by (c)] was included to verify plate-to-plate consistency. Harlan (white) and Richmond (shaded) are indicated for each gene, with Harlan set to 1 for all samples. Fold change is indicated above each bar where 2-fold or greater for all statistically significant samples (** = p<0.01).

Mentions: In order to determine if any of the identified P450, GST or CE (signal peptide-containing only) genes were transcriptionally upregulated in pyrethroid-resistant bed bugs, we performed quantitative real-time PCR on cDNA generated from Harlan or Richmond strain adult male bed bugs. Analysis of the expression of P450 genes revealed that genes cyp397a1 (>36 fold), cyp6dm2 (>29 fold) and cyp400a1 (>18-fold) were all significantly over-expressed in Richmond, pyrethroid-resistant bed bugs as compared to the Harlan control (Fig. 4A). Extremely high similarity between CYP395 family members, as well as between contigs cyp6dl1 and cyp6dl2 prevented analysis of their expression using SYBR® green-based qPCR assays. More specific assays employing hydrolysis probes would likely be needed in order to ascertain any expression changes in these genes. qPCR amplification of GST-encoding gene transcripts revealed that only GSTs1 was significantly upregulated in the resistant strain (Fig. 4B). Analysis of eight putative secreted esterase-encoding transcripts [seven with identifiable signal-peptide sequences and one putative homolog of juvenile hormone esterase, which is also likely to be secreted (Fig. S1)] identified at least two, contigs CE3959 and CE21331, which were significantly up-regulated in the resistant strain (Fig. 4C).


Deep sequencing of pyrethroid-resistant bed bugs reveals multiple mechanisms of resistance within a single population.

Adelman ZN, Kilcullen KA, Koganemaru R, Anderson MA, Anderson TD, Miller DM - PLoS ONE (2011)

Relative expression of metabolic genes in Harlan and Richmond strain bed bugs.Real-time quantitative PCR was performed to measure the relative expression levels of (A) P450, (B) GST, and (C) CE transcripts in the pyrethroid-resistant Richmond strain as compared with the susceptible Harlan strain. Each bar represents the mean of two biological replicates, each performed in triplicate. Error bars indicate the standard deviation from the mean. All samples were normalized to the expression of alpha-tubulin. A second control gene [myosin heavy-chain, indicated by (c)] was included to verify plate-to-plate consistency. Harlan (white) and Richmond (shaded) are indicated for each gene, with Harlan set to 1 for all samples. Fold change is indicated above each bar where 2-fold or greater for all statistically significant samples (** = p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198472&req=5

pone-0026228-g004: Relative expression of metabolic genes in Harlan and Richmond strain bed bugs.Real-time quantitative PCR was performed to measure the relative expression levels of (A) P450, (B) GST, and (C) CE transcripts in the pyrethroid-resistant Richmond strain as compared with the susceptible Harlan strain. Each bar represents the mean of two biological replicates, each performed in triplicate. Error bars indicate the standard deviation from the mean. All samples were normalized to the expression of alpha-tubulin. A second control gene [myosin heavy-chain, indicated by (c)] was included to verify plate-to-plate consistency. Harlan (white) and Richmond (shaded) are indicated for each gene, with Harlan set to 1 for all samples. Fold change is indicated above each bar where 2-fold or greater for all statistically significant samples (** = p<0.01).
Mentions: In order to determine if any of the identified P450, GST or CE (signal peptide-containing only) genes were transcriptionally upregulated in pyrethroid-resistant bed bugs, we performed quantitative real-time PCR on cDNA generated from Harlan or Richmond strain adult male bed bugs. Analysis of the expression of P450 genes revealed that genes cyp397a1 (>36 fold), cyp6dm2 (>29 fold) and cyp400a1 (>18-fold) were all significantly over-expressed in Richmond, pyrethroid-resistant bed bugs as compared to the Harlan control (Fig. 4A). Extremely high similarity between CYP395 family members, as well as between contigs cyp6dl1 and cyp6dl2 prevented analysis of their expression using SYBR® green-based qPCR assays. More specific assays employing hydrolysis probes would likely be needed in order to ascertain any expression changes in these genes. qPCR amplification of GST-encoding gene transcripts revealed that only GSTs1 was significantly upregulated in the resistant strain (Fig. 4B). Analysis of eight putative secreted esterase-encoding transcripts [seven with identifiable signal-peptide sequences and one putative homolog of juvenile hormone esterase, which is also likely to be secreted (Fig. S1)] identified at least two, contigs CE3959 and CE21331, which were significantly up-regulated in the resistant strain (Fig. 4C).

Bottom Line: Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin.Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance.These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

View Article: PubMed Central - PubMed

Affiliation: Fralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, Virginia, United States of America. zachadel@vt.edu

ABSTRACT
A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD(50) bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

Show MeSH
Related in: MedlinePlus