The biochemical basis of insecticide resistance has been extensively studied in various insects and insect-related species over the last few decades. This effort of unraveling the underlying mechanisms of development of resistance has provide tremendous amount of information that is crucial for pests and vector control programs. These two mechanisms acting alone or together account for most of the cases of rapid development of resistance to insecticides in arthropods (Hemingway 2000).
2.1. Enhanced detoxification:
Insects enhance their detoxification of insecticides both by qualitative and quantitative change in the detoxification enzymes systems. Metabolic resistance is by far the most challenging in the control of insecticide resistance. This mechanism involves alterations of the genome that lead to amplification, upregulation and/or coding sequences change of primary detoxification enzymes. Three enzymes families have been implicated in the enhancement of insecticide degradation, including cytochrome P450 (P450), glutathione-S-transferase (GST), and esterase (EST) enzymes.
P450 enzymes are heme-containing proteins found in various organisms, and involved in the detoxification of wide range of xenobiotics such as insecticides and plants allelochemicals, and also involved in other physiological processes in insects (Feyereisen 1999). They are non specific enzymes that attack functional groups of various insecticides. The basic principal of their mediation is trough insertion of an oxygen molecule into the structure of an insecticide to make it more water soluble for direct elimination or further processing by others enzymes systems. Enhanced P450-mediated detoxification has been found in many insect associated with enhanced metabolic detoxification of various class of insecticides. This mechanism acts principally by alterations in the genomic region encoding a given P450 , and occurs either by mutations in trans-regulatory loci or via indels or mutations in cis-acting elements (Liu, 2012). For instance in housefly Rutgers strain resistant to organophosphate and carbamates, upregulation of two P450 genes, CYP6A1 and CYP6D1 were caused by mutations in negative trans-regulatory loci (loss-of-function mutation) on chromosome 2 (Carino et al., 1994; Liu and Scott 1996). The insertion of the transposable element, Accord in the 5’ end of CYP6g1 lead to the upregulation of this gene in Drosophila (Catania et al., 2004). Another mean of increased detoxification by P450 has been described as being a mutation in the coding sequence that generate more potent enzyme. Amichot (2004), found that mutations (Ar335 to Ser, Leu336 to Val and Val476 to Leu) in CYP6AG2 gene of DDT-resistant strain of Drosophila lead to higher catalytic activity.
Glutathione S-transferases (GSTs) are the second class of enzymes involved in the detoxification of endogenous and xenobiotics compounds. Their primary role accounts for the protection of cells against cytotoxic or genotoxic compounds such as reactive oxygen species In insect they are involved in the detoxification of various class of insecticides (Salinas and Wong, 1999). The detoxification process of these enzymes occurs via o-dealkylation or o-dearylation reactions of insecticides. Insects also raise their levels of insecticide detoxification overexpression or amplification of GST genes. In DDT resistant Aedes aegypti, an elevated GST activity was found by Grant and Hammock (1980). Molecular evidence indicated the presence trans-acting element that upregulated the GST2 gene. The upregulation of GST genes has also been identified in Anopheles gambiae, resistant to pyrethroids as demonstrated by heterologous expression of four GST genes (Ortelli et al., 2003). Western blots using antibodies raised against these GSTs enzymes indicated that the expression of GSTE2-2 is elevated in a DDT-resistant strain of A. gambiae. GST enzymes have been also reported to sequester deltamethrin in the yellow mealworm to provide resistance (Kostaropoulos et al., 2001).
Esterases represent a large group of enzymes hat catalyze t the hydrolysis of aliphatic and aromatic esters. In insects esterase-mediated metabolic resistance of insecticides has been detected in almost all insect pests and vector species, against all classes of insecticides with ester moiety in their structures such as organophosphates, carbamates and pyrethroids. The level of insect esterase highly variable depending on the life stage, sex, tissue, hormones, strain, food , environmental conditions and numerous other factors (Devorshak and Roe,1999). The mechanism of their mediation of resistance occurs principally through gene amplification , upregulation and coding sequence mutations. In the peach-potato aphid, Myzys persicae, Devonshire (1977) noted that homogenates of resistant aphids hydrolysed paraoxon 60 times faster than did those of susceptible aphids, confirmed also by assay with naphtyl acetate. This increase in the rate of metabolism of paraoxon was found to be mediated b amplification of esterase genes. Similarly in organophosphate-resistant Culex quinquefasciatus mosquito, isolation and sequencing of B esterase cDNA confirmed that amplification of esterase genes were responsible for resistance (Vaughan et al., 1995). Esterase mutations have also been documented in various insecticide-resistant species. Oppenoorth and Van Asperen in 1960 proposed the “mutant aliesterase theory” in the housefly resistant to diazinon and parathion. They posit that increased hydrolsysis of these organophosphates by phosphotriesterases and carboxylesterase through qualitative mutations of genes encoding theses esterases. Molecular studies by Newcomb et al. (1997) in the sheep blowfly and housefly provided evidence that mutation in esterase E3 gene that change a glycine to aspartic acid (Gly137Asp) was responsible for the observed resistance to organophosphates.
2.2.Target site insensitivity
This mechanism of resistance involves a structural change in the target site of the insecticide leading to a reduced ability for binding to its active site or to affect its function after binding, thereby reducing its toxicity (Scott, 1990; Feyereisen, 1995). Several point mutations leading to changes in one or more critical amino acid residues in the insecticide receptor protein have been reported as responsible for the development of resistance to various class of insecticide in various insect species. Target sites in which such mutations have been documented involve sodium channel, Gaba-gated chloride channel, acetylcholinesterase and midgut receptors. Although it is possible that reduction in the expression level of these receptor could lead to reduced binding, no such evidence has been published to date.
Sodium channel is the target site of DDT and pyrethroids insecticides. Their binding to this channel alters its gating kinetics, causing the channel to remain open for longer period of time, which allow a large influx of sodium. As a consequence the insect become over-stimulated, ultimately resulting in death (Narahashi, 1988). Mutations in the sodium channel gene result in change of the resulting amino acids sequence, leading to ineffective binding of insecticide ultimately leading the development of resistance. This mechanism of resistance was first discovered in the housefly that survived to DDT without being knocked down, and thus called knock down resistance (kdr) (Farnham 1977). It has been later shown that the kdr phenotype was due to a mutation that changes a leucine to phenyalanine at position 1014 ( Leu1014Phe) on the sodium channel (Ingles et al., 1996). Several of these point mutations are found in the sodium channel in a wide number of agricultural pests and disease vectors.
AChE is an enzyme responsible for the degradation of acetylcholine, once it has carried the message across the synaptic cleft. Organophosphates and carbamates insecticides irreversibly bind to AchE, causing the overstimulation of the nervous system ultimately leading to the death of the insect. Mutations in the AChE gene reduce the binding of insecticides, resulting in the development of resistance. Several studies have reported the existence of an altered acetylcholinesterase gene in many insecticide resistant species. For instance the Colorado potato beetle a serine to gycine mutation of acetylcholinesterase gene was responsible for the development of resistance to azinphosmethyl (Zhu et al., 1996). similar mutations were found in acetylcholinesterase genes of resistants insects in Drosophila (Fournier et al., 1989; Weill et al., 2002), the housefly (Walsh et al., 2001), and Culex pipiens mosquito (Weill et al., 2002). In a study by(Baek et al., 2005) Baek et al. (2005) sequence comparison of ace1 cDNA between prothiofos-susceptible and resistant strains of diamondback moth revealed that a total of three amino acid substitutions were closely associated with the prothiofos-resistant strain. Among them the Gly227Ala mutation along with two other ones on the ace1 genes are likely responsible for the AChE insensitivity in the diamondback moth.
Gaba receptors (chloride channel)
Target site insensitivity was also attributed to mutations on gamma amino butyric acid (GABA) receptor on chloride channel. Normally GABA binds to this receptor and cause rapid closure of chloride channel to prevent the elimination of action potential. Cyclodiennes insecticides such as dieldrin bind to this GABA receptors and prevent GABA from exerting its regulatory function thereby allowing chloride channel to remain constantly opened, this will result in the death of the insect. In Drosophila resistance to dieldrin(Rdl) was due to a point mutation (alanine to serine) of the GABA receptor protein (ffrench-Constant et al., 1993). The same mutation to be responsible of resistance was also found in diamondback moths resistant to fipronil (Li et al., 2006).
Midgut receptors of Bt cry toxins:
Cry toxins are insecticidal proteins produced by the soil bacterium Bacillus thuringiensis (Bt) and used either as spray or expressed in plants to control pest. After ingestion by insects, the toxin is activated by gut proteases and bind to midgut receptors proteins (cadherin, aminopeptidases, maltase) with pores formation in the midgut epithelium resulting in the death of insect by osmotic lysis (Soberon et al., 2009). However as with any insecticide used for pest control, resistance has arisen with Bt Cry toxins, and the primary mechanism of its development is through reduced binding of the toxin to its targets. Mutations within a 12-cadherin domain protein were found to cause Cry1Ac resistance in laboratory selected strains of Heliothis virescens by Gahan et al.( 2001). Likewise, genetic analysis by Xu et al.(2005) demonstrated that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive cadherin gene in Helicoverpa armigera. In the pink bollworm Pectinophora gossypiella, Bt Cry1Ac resistance was found to be caused by an alteration of the cadherin gene BtR-4) (Morin et al., 2003). In the diamondback moth Plutella Xylostella L., selected for resistance to Bt toxins, binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), Bacillus thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). Results showed no difference of binding for Cry1Aa, Cry1Ac, and Cry1Ca between resistant and susceptible strain of the moth. However Cry1Ab showed significantly reduced binding, explaining at least resistance to this toxin (Wright et al., 1997). In Culex pipiens, maltase1 is known as the target molecule of binary toxin from Bacillus sphaericus. In studying the molecular basis of this toxin using immunohistochemical and in situ hybridization, Darboux et al.(2002) noted that the maltase1 protein was not present in the midgut of binary toxin-resistant strain GEO, despite the correct transcription of its gene. Further analysis revealed the presence of six missense mutations and one mutation in the maltase 1 gene, that result in the loss of membrane anchor ultimately leading to premature termination of the translation in the resistant strain.