Bed bugs (Cimex lectularius) are nocturnal hematophagous ectoparasites that have a long-standing association (∼300 BC) with man [1] and during evolution (when human lived in caves i.e., much earlier) these ectoparasites may have switched hosts [2]. Bed bugs preferentially feed on humans, and 70% of individuals have allergic reactions to bed bug bites and experience inflamed welts and severe itching [3]. Scratching then can cause the welts to become infected due to secondary bacterial agents [4,5]. Anxiety, stress, and insomnia are common responses to bed bug infestations [6]. Although there is no definitive evidence that bed bugs transmit blood-borne infectious diseases; these ectoparasites have become an important public health issue that affects all socioeconomic classes [7].

Bed bugs were extremely common pests in the United States prior to World War II. However, the repetitive use of insecticides with long-lasting residual, such as dichlorodiphenyltrichloroethane (DDT) (an organochlorine), organophosphates, and carbamates, drastically reduced the bed bug population [8]. Since the late 1990s, there has been a worldwide resurgence of bed bugs, particularly in developed countries including Eastern Asia, Europe, Australia, and North America [9–11]. Some of the important factors attributed to the sudden resurgence of bed bugs include increased domestic and international travel (especially from areas where bed bugs remained common); increased commerce involving used furniture and other household items; the shift from broad-spectrum insecticides to narrow control tactics; and the development of insecticide resistance in bed bugs [6,7,9,10,12-17]. Insecticide resistance (DDT and permethrin) has been largely attributed to changes in the bed bugs' gene structure, i.e., point mutations [18] and upregulation of metabolic genes [19].

According to the World Health Organization [20] resistance is defined as “the inherited ability of a strain of some organism to survive doses of a toxicant that would kill the majority of individuals in a normal population of the same species.” During the course of evolution, an insect species attains resistance to overcome the insecticidal effects of synthetic chemicals or natural toxins and hence allow for its survival.

To date, various groups of insects have been shown to develop resistance towards insecticides; these include 56% crop pests; 37% medical and veterinary pests (mosquitoes, ticks, etc.) and 5% beneficial species [21]. Insects develop resistance to various chemicals through four different modes: target site resistance, behavioral resistance, penetration resistance, and metabolic resistance. Target site resistance and behavioral resistance have been well studied for many insects, whereas metabolic resistance and penetration resistance often have been neglected or overlooked. In this review of all four modes of resistance, we focus on the modus operandi of metabolic resistance in bed bugs. We also highlight potential control mechanisms through silencing genes involved in detoxification.

In general, insecticides react with certain target sites (acetylcholinesterases, gamma aminobutyric acid-gated chloride channels, etc.) in the organism thereby inhibiting their enzymatic activity. However, some blood-feeding insects are thought to have evolved resistance (insensitivity) to organophosphate and carbamate insecticides via a mechanism known as target site insensitivity [22]. For example, studies on the mosquitoes, Anopheles albimanus, Culex pipiens pipiens, and Culex quinquefasciatus, revealed alterations in the acetylcholinesterase genes, which in turn reduced the binding efficiency with insecticides thereby overcoming insecticidal effects [23].

Voltage-gated sodium channels play an important role in the rising phase of action potentials in neurons [24]. Voltage-gated sodium channels were first cloned from Drosophila melanogaster and since then several studies have used this information to compare similar sequences among susceptible and resistant insect populations in order to determine possible resistance due to mutations [25–29]. Pyrethroids are well known to exert insecticidal effects by altering the function of voltage-sensitive sodium channels in nerve membranes of many insect species. It has been documented that pyrethroid resistance stems from point mutations in the voltage-gated sodium channels [24]. As a result, this feature has been linked to knockdown resistance, commonly termed kdr. The Kdr phenotype results from point mutations in coding sequences of voltage gated sodium channels (insect nervous system) thereby resulting in resistance to DDT and pyrethroids [30]. Kdr mutations are well documented in various blood-feeding insects including Anopheles gambiae - L1014F; Cu. pipiens - L1014F and L1014S; Pediculus capitis - T929I and L932F; and C. lectularius - V419L and L925I [31–34].

Resistance to DDT and pyrethroids by bed bugs is suspected to result from point mutations in voltage-gated sensitive channels [34]. In a recent study, Zhu et al. [15] further suggested that the widespread distribution of bed bugs across North America can be attributed to target-site sensitivity of the voltage-gated sodium channel α-subunit gene among the populations scanned. However, it will be interesting to decipher whether pyrethroid resistance in bed bugs is attributable solely to mutations in voltage-gated channels, increased detoxification strategies, or a combination of the aforementioned factors.

Behavioral resistance, the ability to survive insecticide applications through behavioral adaptation, is much less common than physiological resistance. Behavioral responses of arthropod crop pests as well as medical, veterinary, and urban pests have been reviewed by numerous authors [35–41]. These reports cover a wide range of taxonomic groups, particularly Acari, Coleoptera, Lepidoptera, Orthoptera, Hemiptera, and Diptera.

Behavioral resistance is well documented in some blood-feeding insects [42]. However, research is scant on behavioral resistance in bed bugs. In a recent study, it was shown that bed bugs avoided resting on deltamethrin-treated filter paper, a kind of behavioral resistance wherein the insect keeps away from the insecticide [43].

Despite the many studies documenting behavioral responses to insecticides, little is known at the functional level about the genes involved in behavioral traits associated with toxins compared to the genes that metabolize them. However, recent high throughput technologies have allowed the identification of putative candidate genes influencing insect behavior and have simultaneously furthered the understanding of the physiology-driven molecular mechanisms influencing insecticide/allelochemicals resistance [44]. Elucidating the genetic basis of behavioral resistance is crucial to an understanding of insect adaptation to toxins.

Many insects overcome the effects of insecticides through decreased cuticular penetration, which is a well-documented phenomenon [45–47]. In simple terms, the thicker the cuticle (with higher protein and lipid content), the less able it is to absorb a toxin. This mode of resistance could include the development of barriers in resistant insects. Penetration resistance is usually associated with other forms of resistance and thus this trait could intensify the effect of the resistance mechanisms. Interestingly, a high number of cuticular domains were identified in our recent 454 pyrosequencing of bed bugs [19]. Morphological (scanning electron microscopy studies) and biochemical (characterization of proteins involved in detoxification) comparisons of cuticular proteins among resistant and susceptible bed bugs may provide more clues to bed bug resistance to insecticides.

Metabolic resistance is basically the biochemical transformation of a toxin, wherein the toxic compound is transformed into a less toxic form [48]. Biotransformation reactions are classified as Phase I or Phase II based on their mode of action on toxic compounds. The Phase I reactions include oxidation, reduction, and hydrolysis; they are involved in detoxification of xenobiotics. The Phase II reaction is a process of biosynthetic conjugation wherein the conjugative enzymes bind with toxins or primary products of Phase I and convert them into more water soluble derivatives, which are excreted from the body. Cytochrome P450 monoxygenases (P450s) are Phase I metabolic enzymes capable of oxidizing endogenous and exogenous compounds by oxidation or other related reactions [48]. Glutathione S-transferase (GST) is a Phase II metabolizing enzyme that catalyzes the reaction of reduced glutathione to compounds bearing electrophilic sites [49].

The detoxification of synthetic chemicals by insects is mainly associated with cytochrome P450s and with large multigene families such as esterases, oxidases, and transferases [50–55]. Among these detoxifying enzyme systems, cytochrome P450s are involved in the metabolism of pyrethroids; GSTs detoxify organochlorine compounds; and esterases and oxidases act primarily on carbamates [56-58]. The putative insect genes involved in metabolic resistance to various insecticides are summarized in Table 1.

Lessons learned to date clearly indicate the complex nature of bed bugs' resistance to pesticides. As of now, resistance in bed bugs appears to be attributed toward: (i) point mutations in the nervous system genes (e.g., voltage-gated sensitive channels); (ii) differential expression of detoxification genes (e.g., cytochrome P450s and GSTs) and (iii) morphological traits such as thickened cuticle in resistant populations compared to susceptible ones. Future studies aimed at deciphering genome-wide (global) expression patterns among susceptible and resistant bed bug strains and gene knock out experiments may provide insights into the genetic basis for insecticide resistance in bed bugs. Further, comparative metagenomics of bed bugs' microbiota may also unravel the role of these microorganisms in host resistance towards synthetic chemicals.