Bed bugs evolved unique adaptive strategy to resist pyrethroid insecticides

Abstract

Recent advances in genomic and post-genomic technologies have facilitated a genome-wide analysis of the insecticide resistance-associated genes in insects. Through bed bug, Cimex lectularius transcriptome analysis, we identified 14 molecular markers associated with pyrethroid resistance. Our studies revealed that most of the resistance-associated genes functioning in diverse mechanisms are expressed in the epidermal layer of the integument, which could prevent or slow down the toxin from reaching the target sites on nerve cells, where an additional layer of resistance (kdr) is possible. This strategy evolved in bed bugs is based on their unique morphological, physiological and behavioral characteristics and has not been reported in any other insect species. RNA interference-aided knockdown of resistance associated genes showed the relative contribution of each mechanism towards overall resistance development. Understanding the complexity of adaptive strategies employed by bed bugs will help in designing the most effective and sustainable bed bug control methods.

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.

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.

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).

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.

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 6^th day after injection of dsRNA. Mortality was recorded after 72 h exposure to β-cyfluthrin (3 replicates, 30 individuals for each replicate).