Resistant population selection and residual bioassays

A bed bug population from Cincinnati, CIN-1 NS (the CIN-1 strain before selection) collected in 2007 showed >10,000-fold deltamethrin resistance16 but the resistance levels decreased after rearing them for multiple generations. To enhance the pyrethroid resistance level of this population, CIN-1 S (the selected strain) was selected with commercial insecticide, Temprid® (a commercial insecticide that includes β-cyfluthrin and imidacloprid as active ingredients). Bioassays showed dramatic differences in the susceptibility to deltamethrin between the susceptible strain, LA-1 and resistant strains, NY-1, CIN-1 NS and CIN-1 S (Fig. 1A). LA-1 was susceptible to deltamethrin (n = 240, slope = 0.355, LC 50 = 0.003 mg/cm2). However, NY-1 did not show higher percentage of mortality when the concentration of deltamethrin increased from 0.001 mg/cm2 to 1 mg/cm2 (n = 280). Before selection, the resistance ratio of CIN-1 NS relative to LA-1 was 51 (n = 240, slope = 0.275, LC 50 = 0.153 mg/cm2). After selection, the resistance ratio of CIN-1 S compared with LA-1 increased drastically (n = 300, slope = 0.135, LC 50 = 98,100 mg/cm2).

Figure 1 Identification of target genes associated with insecticide resistance. (A) Dose-response curves (log dose versus mortality on a probit scale) for C. lectularius female adults exposed to deltamethrin. LA-1 ( ), a susceptible strain; NY-1 ( ), a deltamethrin resistant strain; CIN-1 NS (x), a deltamethrin resistant strain without selection; and CIN-1 S ( ), a deltamethrin resistant strain after selection were exposed to serially diluted deltamethrin and the mortality was recorded and graphed. (B) Cytochrome P450 genes in C. lectularius. Totally 42 Cytochrome P450 genes (P450s) were identified through assembling of Cimex transcriptome and named by the P450 nomenclature committee. These genes fall into 4 clans, Mito CYP clan, CYP4 clan, CYP3 clan and CYP2 clan. The number of P450s in each clan was labeled on the top of the column. (C) mRNA levels of 42 C. lectularius P450s in LA-1, CIN-1 NS and NY-1. mRNA levels were shown as mean fold relative to their levels in LA-1. P450s highlighted in red showed the significant increase in CIN-1 NS and/or NY-1 compared to their levels in LA-1 (Student t-test, P < 0.05). (D) Relative mRNA levels of cuticular protein genes. Total RNAs were extracted from one week-old female adults were used in qRT-PCR to quantify relative mRNA levels in susceptible LA-1 as compared with the pyrethroid-resistant CIN-1 S. The data shown are mean + SEM (n = 3). Genes highlighted in red showed significant difference between LA-1 and CIN-1 S (Student's t test. * P < 0.05, ** P < 0.01). (E) Same as B except the mRNA levels of Abc transporter genes were quantified. Full size image

Transcriptome analysis and resistance marker selection

RNA isolated from CIN-1 NS strain was sequenced by 454 GS FLX pyrosequencing. The sequences obtained from this strain and other sequences available in the NCBI database (SRX028107, SRX013985, SRX013984 and 7131 ESTs) were assembled by Roche de novo Assembler program (Newbler) (Fig. S1). These data resulted in a total of 2,197,566 aligned reads constituting 756,568,733 bases producing 129,294 ESTs (25,935 contigs and 103,359 singletons). The length of the contigs varied from 100 bp to 8,249 bp with an average length of 850 bp (Fig. S2A). The length of singletons ranged from 50 bp to 511 bp with an average length of 261 bp (Fig. S2B). Gene Ontology (GO) analysis was performed based on BLAST matches to proteins with known functions. Each GO category, the molecular function (ontology level 5), biological process (ontology level 2) and cellular components (ontology level 5) are shown in the Figs. S3A, B, C, respectively. Based on our previous studies21,22 and recent publications18,20, several categories of genes were identified for the potential association with pyrethroid resistance in the bed bug.

Metabolic enzymes

Increased metabolic detoxification by cytochrome P450s, esterases, and/or glutathione S-transferases (GSTs)23,24,25 is one of the major mechanisms involved in pyrethroid resistance. Our previous studies suggested that P450-mediated metabolic detoxification may serve as one of the resistant mechanisms in bed bugs21,22. Typically, each insect genome contains a variable number of P450 genes varying from tens to more than one hundred24. In the current study, 42 cytochrome P450s were annotated from 129,294 ESTs and named by the P450 nomenclature committee (Dr. D. Nelson, personal communication) (Tables S1 and S2). Of these 42 P450 genes, six (CYP15, 18, 303, 305, 306, 307 families) derived from CYP2 clan, six (CYP301, 302, 314, 315, 394 families) belong to Mito clan, 23 (CYP6, 395–400, 404 families) derived from CYP3 clan and seven CYP4 genes belong to CYP4 clan (Tables S1 and S2, Fig. 1B). The relative expression of these 42 P450s were examined among insecticide susceptible strain LA-1 and resistant strains, CIN-1 NS and NY-1, comparing with the expression of the most stable housekeeping gene rpl822 (Fig. 1C). Four P450s, CYP397A1, CP398A1, CYP6DN1, CYP4CM1 were chosen as the target markers on the basis of their significant up-regulation in resistant strain(s) when compared to their expressions in susceptible strains as well as their relative higher expression when compared to that of rpl8 (Fig. 1C). The same criteria were used to select other genes associated with insecticide resistance as described below. As regards to esterases and GSTs, the expression of three genes identified previously18 was compared between LA-1 and CIN-1 S strains. Only esterase, ClCE21331 showed significant increase in the resistant strain (Table S3) and this gene was selected for further validation.

Cuticular proteins

Cuticular proteins are the major components of insect cuticle which serves as the first line of defense to insecticides26. Recent studies reported that cuticle thickening was associated with pyrethroid resistance in Anopheles funestus26. In Colorado potato beetle, the mRNA levels of three putative cuticular proteins were higher in azinphosmethyl resistant beetles than in susceptible beetles27. To determine the role of cuticular proteins in the pyrethroid resistance of bed bugs, the expression of 15 genes coding for putative cuticular proteins was examined in LA-1 and CIN-1 S strains (Fig. 1D, Table S3) and three genes (C2, C10 and C13) that showed increased expression in resistant strains were selected for further studies.

Abc transporters

ATP-binding cassette (Abc) transporters constitute one of the largest classes of transporters that are responsible for the ATP-powered translocation of many substrates across membranes28. These substrates include ions, sugars, amino acids, vitamins, peptides, polysaccharides, hormones, lipids and xenobiotics29. Recent RNA-Seq studies showed that eight out of 27 Abc transporters were up-regulated in insecticide resistant bed bugs when compared to susceptible insects20. We selected 13 contigs annotated as Abc transporters with significant expression levels in adult bed bugs and the mRNA levels of these were quantified (Fig. 1E, Table S3). Genes coding for four Abc transporters, Abc8, Abc9, Abc10, Abc11 showed an increase in expression in resistant strain and selected for further analysis.

Kdr mutations

Pyrethroid insecticides target the sodium channels within the insect nervous system. Point mutations in the sodium channels, termed the kdr mutations, reduce or eliminate the binding affinity of insecticides to sodium channels causing insecticide resistance6. Two mutations, V419L and L925I, in voltage-gated sodium channel α-subunit gene had been identified as very important substitutions responsible for deltamethrin resistance in bed bugs21,30. A causal link between one or both mutations and deltamethrin resistance was reported21. A dual-primer Allele-Specific PCR (dASPCR) approach was developed to identify these two kdr mutations. Two PCR reactions performed with Susceptible Allele-Specific Primer (SASP) and Resistant Allele-Specific Primer (RASP) primers conclusively show status of kdr mutations (Fig. 2A).

Figure 2 Differential expressions of 12 target genes in susceptible and resistant C. lectularius laboratory populations. (A) Gel showing dASPCR results. Lanes 1–5 show 5 independent DNA samples. Two PCRs were performed with different primers, SASP or RASP, under the same reaction conditions. For each DNA sample, there was only one band (highlighted with red star) shown on the gel illustrating this sample either has mutation (band shown on the gel beneath) or has no mutation (band shown on the gel above). (B) Relative levels of resistance to deltamethrin in LA-1, CIN-1 NS and CIN-1 S drawn based on bioassay data. (C) The relative mRNA levels of 12 target genes were quantified by qRT-PCR in susceptible LA-1 and resistant CIN-1 NS as well as CIN-1 S populations and normalized using the mRNA levels of rpl8. The data represent mean + SEM (n = 4 – 12). Statistical significance of the gene expression among samples was calculated using one-way ANOVA followed by Duncan multiple mean separation techniques (SAS v9.4). There was no significant difference among relative expression within samples with the same alphabetic letter. Full size image

Validation of selected markers in resistant and susceptible strains

To validate the relative expression of 12 genes selected as those that play important roles in pyrethroid resistance, the mRNA levels of these genes were quantified in LA-1, CIN-1 NS and CIN-1 S strains that showed different levels of resistance to deltamethirn (Fig. 2B). All of these target genes are significantly overexpressed in resistant strain(s) when compared to their expression in the susceptible population (Fig. 2C). Moreover, most of these genes showed an increase in expression following the selection from CIN-1 NS to CIN-1 S. The causal link between the overexpression of target genes and deltamethrin resistance suggest that these 12 genes might be involved in the pyrethroid resistance of bed bugs and could serve as molecular markers to monitor the pyrethroid resistance in field populations.

Integument affiliated expression of selected genes

The tissue specific expression of a gene normally is related to its function in that tissue31. Bed bug has an extremely flat body prior to blood meal (Fig. 3A). The functional significance of the integument in regards to bed bugs is especially important, as the integument constitutes the major part of their body (Fig. 3A–C). In order to investigate the tissue-specific expression, the mRNA levels of 12 target genes were quantified by qRT-PCR in the head, leg, gut (foregut and midgut), fat body, mesospermalege, ovary and integument (epidermis + cuticle) isolated from one week-old female adult NY-1 (Fig. 3D) and CIN-1 NS (Fig. S4) bed bugs. Interestingly, most of the genes coding for metabolic enzymes, cuticular proteins and Abc transporters are expressed higher in the head, legs and integument dissected from NY-1 bed bugs (Fig. 3D). All these three tissues contain epidermis and cuticle. In addition, higher levels of mRNAs when compared to the levels in other tissues were detected for CYP397A1 in gut, fat body and mesospermalege; CYP398A1 in the mesospermalege; CYP4CM1 in the fat body and mesospermalege; CYP6DN1 in the gut and the esterase ClCE21331 in the gut and mesospermalege (Fig. 3D). Quantification of mRNA levels of genes associated with resistance in CIN-1 NS strain showed similar patter as those in NY-1 strain (Fig. S4).

Figure 3 Spatial expression of resistance associated genes in NY-1 strain. (A) Representative profile of a bed bug. L, leg. (B) Representative anatomic structure of 1 week old female bed bug. H, Head; G, gut including foregut and midgut; F, fat body; M, mesospermalege; O, ovary and I, integument. (C) Average weight of body parts in 1-week-old female bed bugs. The abdomen of bed bug was split to integument and others. The average weights of abdomen integument (0.583 mg/individual), abdomen other parts (0.353 mg/individual), head + thorax (0.430 mg/individual) were calculated from 30 individuals within 3 replicates. (D) The mRNA levels of 12 target genes were quantified by qRT-PCR. The head (H), leg (L), gut (foregut and midgut) (G), fat body (F), mesospermalege (M), ovary (O) and integument (I) were dissected from 1 week-old female adult NY-1 bed bugs. Relative mRNA levels were normalized using the mRNA levels of rpl8. The data shown are mean + SEM (n = 3). There was no significant difference among relative expression within samples with the same alphabetic letter (i.e. a, b and c) (One-way ANOVA followed by Duncan multiple mean separation, SAS v9.4). Full size image

Multiple mechanisms of resistance exist in field collected bed bug populations

In order to inspect mechanisms of insecticide resistance in field populations, we collected 21 bed bug populations from dwellings in four cities located in the Midwestern United States during November 2011 to February 2012 (Table S4, Fig. S5). The relative expression of 12 target genes and the presence of two kdr mutations were investigated by qRT-PCR and dASPCR, respectively. On an average more than three molecular markers showed a difference in each of the 21 field populations tested (Fig. 4A). P450, esterase and cuticular protein mediated resistance were identified in all the field populations except LEX-7. An Abc transporter associated mechanism was also detected in all the populations except LEX-5. One or both kdr mutations are present in 18 populations tested (Fig. 4A). Out of the marker genes studied, ClCE21331 showed the maximum increase (>50 fold) of gene expression in most field populations (76.2%). A majority (57.1%) of the populations tested showed >5 fold overexpression of genes coding for cuticular proteins. The overexpression of P450 and Abc transporter markers in field populations was less than 5-fold in most of the populations tested (Fig. 4A). The geographic distribution analysis of molecular mechanisms showed that seven out of 13 Lexington populations, two Louisville populations, three Cincinnati populations and three Chicago populations showed overexpression of multiple resistance genes (Figs. 4A and 4B). Moreover, among 21 field bed bug populations tested, 100% of populations were associated with at least two molecular mechanisms (not including LA-1 the reference population), implying the prevalent involvement of multiple mechanisms of pyrethroid resistance in the field bed bug populations.

Figure 4 Multiple mechanisms of resistance in field-collected bed bug populations (n = 21) and laboratory strains (LA-1, NY-1 and CIN-1 S). (A) Analysis of transcription profile of 12 molecular target genes and two kdr mutations among 24 field-collected and laboratory populations. The left 12 rectangles are colored on the basis of results of relative mRNA levels compared with that of LA-1 and normalized by the expression of rpl8. The data shown are mean + SEM (n = 3 – 12). The two columns of rectangles on the right represent two kdr mutations identified by dual-primer allele specific PCR. (B) A pie chart showing the percentage of populations with two, three, four or five mechanisms of resistance. Full size image

RNAi studies

To investigate the relative contribution of each mechanism towards overall resistance development, we exploited RNAi to knockdown the expression of insecticide resistance associated genes in CIN-1 S bed bugs and examined the susceptibility of these bed bugs to pyrethroid insecticide. By mimicking traumatic insemination, dsRNAs were injected through the mesospermalege into the body of female bed bugs as described in our recent publication22. Bed bugs injected with dsRNA of malE or target genes suffered <10% mortality within 6 days after injection. In order to investigate comprehensive contribution of each category of target genes to the pyrethroid resistance, dsRNAs synthesized for each target gene from the same category were pooled and injected into one week-old female bed bugs. The target genes tested were successfully suppressed by RNAi (Fig. 5A–C). The expression of closely related non-target P450 genes, CYP399A1, CYP4CN1 and CYP6DM1 were unaffected in the bed bugs injected with dsRNA targeting select P450s (Fig. S6). Expression of esterase ClCE21331 is quite variable in CIN-1 S population, therefore the RNAi analysis was not performed for this gene.

Figure 5 Knockdown in the expression of insecticide resistance associated genes reduced the resistance to pyrethroid insecticide. (A–C) Injection of dsRNA decreases mRNA levels of target genes. Relative expression of 4 target P450s (A), 3 cuticular proteins (B) and 2 Abc transporters (C) in control (malE dsRNA) and dsRNA of target genes injected bed bugs. The relative mRNA levels are shown as a ratio in comparison with the levels of rpl8 mRNA. The data shown are mean + SEM (n = 3). (D) The percent survival of CIN-1 S bed bugs treated with 5 μg β-cyfluthrin on 6th day after injection of dsRNA. Mortality was recorded after 72 h exposure to β-cyfluthrin (3 replicates, 30 individuals for each replicate). Full size image

Six days after injection of dsRNAs, the survivors were exposed to β-cyfluthrin topical application. CIN-1 S bed bugs are highly resistant to deltamethrin, with a high topical dose leading to no mortality. Therefore, we decided to use a more effective synthetic pyrethroid, β-cyfluthrin, to assay insecticide susceptibility of RNAi insects. The P450s or Abc8 and Abc9 knockdown in pyrethroid resistant CIN-1 S bed bugs showed a consistent increase in their susceptibility to β-cyfluthrin compared with control bed bugs injected with malE dsRNA (Fig. 5D). In contrast, there was no significant difference in the susceptibility to β-cyfluthrin between cuticular proteins knockdown and control bed bugs (Fig. 5D).