Limits...
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.

Show MeSH

Related in: MedlinePlus

Phylogenetic relationship of bed bug P450s.Cytochrome P450 genes of the bed bug, Cimex lectularius. Predicted protein sequences were aligned using ClustalW; tree construction was performed using the Neighbor-joining method in MEGA 4.0. Bootstrap support is indicated where greater than 70%. Clades assigned to the CYP2, CYP3, CYP4 and mito clans are indicated. Family assignments and the naming of new families were performed by David Nelson as described [18].
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198472&req=5

pone-0026228-g002: Phylogenetic relationship of bed bug P450s.Cytochrome P450 genes of the bed bug, Cimex lectularius. Predicted protein sequences were aligned using ClustalW; tree construction was performed using the Neighbor-joining method in MEGA 4.0. Bootstrap support is indicated where greater than 70%. Clades assigned to the CYP2, CYP3, CYP4 and mito clans are indicated. Family assignments and the naming of new families were performed by David Nelson as described [18].

Mentions: At the time this project was initiated, little information was available for the sequences of bed bug genes which may play a role in insecticide detoxification. To obtain gene sequences for all abundantly expressed bed bug metabolic genes, we performed two 454 titanium runs using Harlan or Richmond strain cDNA, obtaining more than 2.5 million reads in total (Table 2). Harlan and Richmond strain reads were assembled together, yielding more than 14,000 contigs greater than 500 bp, with an N50 contig size greater than 1,200 bp (Table 2). From these contigs, we identified at least 34 members of the cytochrome P450 family. Most of these sequences were full length, as determined by the presence of start and termination codons spanning an ORF typical of P450 genes (avg ORF size was 487 a.a., median was 507). Only five sequences [cyp18a1 (282 a.a.), cyp400a1 (287 a.a.), cyp301a1 (296 a.a.), cyp6dm2 (358 a.a.), cyp6dl2 (406 a.a.)] were relatively incomplete, with all other predicted protein sequences greater than 470 amino acids. The relative completeness of P450 coding regions allowed us to assign each P450 gene into a clade and family according to the Committee on Standardized Cytochrome P450 Nomenclature [18]. Of the 34 assembled P450 contigs, 22 derived from the CYP3 clan (Fig. 2). This included at least 8 new members of the CYP6 family, as well as new families CYP395 (at least 9 contigs), CYP 396 (1), CYP 397 (1), CYP 398 (1), CYP399 (1), and CYP 400 (1). We also identified members of clans CYP4 (7 contigs), CYP2 (2 contigs), and MITO (3 contigs) (Fig. 2).


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)

Phylogenetic relationship of bed bug P450s.Cytochrome P450 genes of the bed bug, Cimex lectularius. Predicted protein sequences were aligned using ClustalW; tree construction was performed using the Neighbor-joining method in MEGA 4.0. Bootstrap support is indicated where greater than 70%. Clades assigned to the CYP2, CYP3, CYP4 and mito clans are indicated. Family assignments and the naming of new families were performed by David Nelson as described [18].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198472&req=5

pone-0026228-g002: Phylogenetic relationship of bed bug P450s.Cytochrome P450 genes of the bed bug, Cimex lectularius. Predicted protein sequences were aligned using ClustalW; tree construction was performed using the Neighbor-joining method in MEGA 4.0. Bootstrap support is indicated where greater than 70%. Clades assigned to the CYP2, CYP3, CYP4 and mito clans are indicated. Family assignments and the naming of new families were performed by David Nelson as described [18].
Mentions: At the time this project was initiated, little information was available for the sequences of bed bug genes which may play a role in insecticide detoxification. To obtain gene sequences for all abundantly expressed bed bug metabolic genes, we performed two 454 titanium runs using Harlan or Richmond strain cDNA, obtaining more than 2.5 million reads in total (Table 2). Harlan and Richmond strain reads were assembled together, yielding more than 14,000 contigs greater than 500 bp, with an N50 contig size greater than 1,200 bp (Table 2). From these contigs, we identified at least 34 members of the cytochrome P450 family. Most of these sequences were full length, as determined by the presence of start and termination codons spanning an ORF typical of P450 genes (avg ORF size was 487 a.a., median was 507). Only five sequences [cyp18a1 (282 a.a.), cyp400a1 (287 a.a.), cyp301a1 (296 a.a.), cyp6dm2 (358 a.a.), cyp6dl2 (406 a.a.)] were relatively incomplete, with all other predicted protein sequences greater than 470 amino acids. The relative completeness of P450 coding regions allowed us to assign each P450 gene into a clade and family according to the Committee on Standardized Cytochrome P450 Nomenclature [18]. Of the 34 assembled P450 contigs, 22 derived from the CYP3 clan (Fig. 2). This included at least 8 new members of the CYP6 family, as well as new families CYP395 (at least 9 contigs), CYP 396 (1), CYP 397 (1), CYP 398 (1), CYP399 (1), and CYP 400 (1). We also identified members of clans CYP4 (7 contigs), CYP2 (2 contigs), and MITO (3 contigs) (Fig. 2).

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