Genetic and molecular mechanism of organophosphate-based insecticide resistance in Culex pipiens (Northern House Mosquito)

March 23, 2011

Culex pipiens, commonly referred to as the (northern) house mosquito, is the most widely distributed mosquito species, inhabiting every continent except Antarctica. In the northeastern United States, C. pipiens is the most common mosquito pest in both urban and suburban settings, and is an indicator of water being polluted with organic matter. C. pipiens vectors several diseases, including Filiarisis (caused by the parasitic nematode Wucheria bancroftii), Japanese Encephalitis, Meningitis, Urticaria, and is the principle vector of West Nile Virus and Saint Louis Encephalitis in the northeastern United States (Culex pipiens, 2010). Males of the species are herbivorous and feed strictly on plant juices and nectars. Females, however, take blood meals in order to facilitate the development of fertilized eggs. During this process, anticoagulant saliva is injected. This medium, when contaminated, can transmit the aforementioned diseases. Females prefer to feed within enclosed structures such as homes or barns increasing the frequency of their interactions with humans, and also lending them the name house mosquito. Blood meals are primarily taken from birds, but most populations will have individuals that also feed on humans and livestock animals (Culex pipiens, 2010). This greatly magnifies both the mosquitos potential to both contract and transmit pathogens (CBWinfo, 2010). Thus, as a medically and economically important pest, these mosquitoes have been subject to control measures implemented by humans. These measures include chemical pesticides as well as biological controls (sterile male release), and each of these measures have been met with their own limited degree of success. However, in the mid-to-late twentieth century, pressures grew from the scientific community to implement an ecologically friendly solution to the pest problem. Specifically, a particularly effective group of pesticides, organochlorides, were found to be particularly harmful to bird populations in areas with high treatment levels or subject to runoff from such areas. Discontinued organochlorides include

DDT, aldrin, dieldrin and chlordane, though the list is much longer. The use of organochlorides is still widespread in developing nations (Organophosphates, 2004). Thus, organophosphates became the key tool in chemical control of C. pipiens populations. Organophosphates are now the most widely used class of insecticides with agricultural, residential, and veterinary uses. Organophosphates are esters of phosphoric acid; RNA, DNA and many enzyme cofactors are organophosphates. Organophosphate in the context of this paper refers to a group of chemical pesticides, which act as acetylcholinesterase inhibitors, irreversibly binding and deactivating acetylcholinesterase, should an organism be exposed (Corbett, 1974). These compounds, in a mechanism analogous to nerve agents such as sarin or VX gasses, irreversibly phosphorylate acetylcholinesterase. This causes the levels of acetylcholine to rise to fatal levels. Acetylcholine is a neurotransmitter which is essential to the normal neural function of many higher animals, including both insects and humans. The chemical is critical in the function of the peripheral nervous system, activating muscular function. In the central nervous system, acetylcholine is linked to memory as well as sensory perception. In the autonomic nervous system, acetylcholine is one of numerous neurotransmitters, and the only neurotransmitter used in somatic motor responses (Himmelheber et al., 2000). Acetylcholine will slow heart rate as well as cause excitatory muscular twitching (Campbell & Reece, 2002). To terminate neural transmission, acetylcholinesterase, a hydrolytic enzyme, binds to and decomposes acetylcholine into an acetyl group and choline. The choline is then recycled into more acetylcholine for future use in nerve terminals. Acetylcholinesterase has a high catalytic rate, degrading 2.5 * 10^4 molecules of acetylcholine each second. Thus, acetylcholinesterase is only required in low concentrations in nervous tissue (Purves et al., 2008). If acetylcholinesterase is deactivated, such

as by being bound by organophosphates, acetylcholine poisoning will occur. Acetylcholine poisoning is the phenomenon that kills those subjected to weaponized nerve agents. Repeated and homogenous exposure to organophosphates, as with any selection pressure, will spur adaptation. Cases of organophosphate resistance in C. pipiens have been thoroughly documented throughout the world, wherever organophosphate application is heavy (Georghiou & LagunesTejeda, 1991). Studies have been conducted, but the exact mechanism has not been fully described. Hypotheses point toward mutations that differ in resistant individuals from their pesticide-susceptible counterparts: genes which cause an overproduction of detoxifying esterases and the production of insensitive acetylcholinesterase (acetylcholinesterase-1, abbreviated AchE1) which is the molecule targeted by the organophosphates (Raymond et al., 2001) Three genes, at loci Est-2, Est-3 (Pasteur, Iseki, & Georghiou, 1981), and ace-1 (Alout et al., 2007) seem to confer different levels of resistance via different mechanisms: overproduction of esterases which detoxify organophosphates, and production of acetylcholinesterase which is highly insensitive to inactivation by organophosphates (Raymond et al., 1998). At the two Est loci are genes which code for esterases, and together are referred to as the Ester superlocus, as they are located 0.67 units of crossing over apart (Pasteur, Iseki, & Georghiou, 1981).This means that the two genes are not only on the same chromosome, but are so closely linked that when they are replicated, there are only 0.67% of the offspring which are not the parental genotype (that is, both Est-2S and Est-3S or Est-2R and Est-3R, susceptible and resistant respectively) as the result of crossing over events. This linkage corresponds to about a 2kBP separation (Raymond et al., 1998). Esterases are a large group of hydrolytic enzymes that are found in many biochemical pathways. These enzymes break the bond between oxygen and

hydrogen, leaving one protonated acid (left with H+) and an alcohol (left with OH-). Organophosphates, being esters of phosphoric acids, are decomposed into phosphoric acid and the subgroup alcohol. Est-2 codes for B-class esterases, and Est-3 codes for A-class esterases, both of which are carboxylester hydrolases. In organophosphate resistant individuals, both are present in quantities higher than necessary for the normal function within the cell. Six distinct overproduced enzymes have been identified, four B and two A (Raymond et al., 1998). B class carboxylesterases are 67kDa monomers (made up of one subunit, of size 67kDa), while A class carboxylesterases are dimers of identical 60kDa subunits. B class carboxylesterases have been shown to be overproduced by a factor of 500 fold, and A class carboxylesterases have been shown to be overproduced by a factor of 70 fold (Mouches et al., 1987). This overproduction is possible via one or more of the following mechanisms which have been proposed: gene amplification of Est-2 only (Guillemaud et al., 1997), coamplification (or companyamplification) of Est-2 and Est-3 together (Cui et al., 2006a), and/or gene upregulation (Rooker et al., 1996). Another mode of resistance is by modification of the enzyme acetylcholinesterase. Acetylcholinesterase is a hydrolytic enzyme that splits the neurotransmitter acetylcholine into an acetyl group and a choline group, which is then recycled back into neural tissue as a precursor to acetylcholine. If the acetylcholinesterase produced by mosquitoes is not susceptible to inactivation via bonding by organophosphates, the chemical controls will no longer be effective. In the laboratory, mosquitoes were maintained under conditions in which they were routinely exposed to high levels of organophosphates. This selective force resulted in a population of strictly resistant mosquitoes, as the susceptible mosquitoes were killed. The resistant mosquitoes were then homogenized (blended into a pulp), and the acetylcholinesterase was extracted via

high-speed centrifuging and SDS-acrylamide electrophoresis (Cuany et al., 1993). Amino acid sequencing revealed that a single base pair mutation, G119S (that is, a glycine is changed to a serine at the 119 position), within the gene that codes for AcehE1, renders the acetylcholinesterase insensitive to inactivation by organophosphates, but still allows the enzyme to perform its normal functions within cells of the organism. Interestingly, this single-base-pair mutation only renders certain mosquitoes insensitive to organophosphates (and carbamates as well, a different class of pesticides whose biochemical mode of action is similar to organophosphates), but not all. For example, C. pipiens, as well as Anopheles gambiae, the mosquito vector of malaria, have been documented to become resistant to organophosphates via the G119S mutation where the level of exposure is high and constant, while Aedes aegypti, the mosquito vector of yellow and dengue fevers, has never been shown capable of developing resistance via the G119S mutation (Weill et al., 2004). G119S-mutated acetylcholinesterase is not susceptible to organophosphate inactivation when produced by Ae. aegypti, but for some reason this mutation does not occur in natural populations. An investigation of the code for the ace-1 gene in Ae. aegypti shows that the three-base codon for glycine in the 119 position is different than that found in the resistance-capable species of C. pipiens and A. gambiae. The different version of the codon would require two adjacent base mutations a far less likely event, in order to create the glycine-serine switch required of resistance (Weill et al., 2004). 31 of 44 additional mosquito species have the constrained codon while 13 others have the resistance-prone codon. 50% of these resistance-prone species have in fact developed resistance, while the rest are not chemically controlled (Weill et al., 2004). The G119S point mutation confers high levels of resistance to chlorpyrifos, fenitrothion, malathion, and parathion, while remaining susceptible to dichlorvos, trichlorfon and fenthion. This mutation has been observed

in Europe, Asia, Africa and the Americas (C. pipiens entire home range) (Cui et al., 2006b). A separate ace-1 mutation, F290V, is another single-amino acid swap, changing a phenylalanine (abbreviated F) to valine (abbreviated V) at the 290th position, which also confers resistance to deactivation by organophosphate exposure. The amino acid residue in the 290th position lines the active site of the enzyme acetylcholinesterase. While this mutation was identified in 2007, it was identified in a population of mosquitoes maintained from samples collected in 1987 (Alout et al., 2007). Another acetylcholinesterase-encoding gene, Ace-2, is also present in mosquitoes, but seems to play no role in resistance. Deactivation-resistant acetylcholinesterase is a common mode of organophosphate-insecticide resistance, not just in mosquitoes but other arthropods as well (Georghiou & Lagunes-Tejeda, 1991). Sampling of mosquitoes worldwide between 1996 and 2005 was performed to determine the distribution and frequency of resistance-conferring alleles in natural populations of C. pipiens. The same three genes were identified to have been involved in resistance: Est-2, Est-3 and Ace-1. Esterases Ester4, Ester5 and EsterB12 were identified to be products of the Est-2 (Ester B) locus. Esterases EsterA1 and EsterA13 were identified to be products of the Est-3 (Ester A) locus. The G119S mutated acetylcholinesterase was produced by the Ace-1R gene, as well as the Ace-1D (duplicate copies of both the susceptible and resistant genes). The novel F290V point mutation was also identified to be a product of the Ace-1 gene. There was a large heterogeneity of allelic frequencies observed, implying differing application of insecticide in different geographic areas. Comparison between the 1996 and 2005 samples revealed no novel resistanceconferring mutations, but the large range of resistance-conferring genotypes allows for multiple genetic lines of resistance (Cheikh et al., 2009). Gene duplication has been put forth as the mechanism for new gene function, specifically in the ace-1 gene. The ace-1D gene has evolved

at least three independent times, indicating that the previous proposed gene-duplication mechanism was erroneous (it hinged on relatively ancient events, e.g. those which took place hundreds of millions of years ago). This resistance-selective force (widespread use of organophosphate pesticides) has only been an evolutionary factor for half a century. The ace-1D gene consists of two alleles: one coding for an acetylcholinesterase which is sensitive to organophosphate deactivation, one coding for an acetylcholinesterase which is insensitive to deactivation by organophosphates. Labbe et al. suggest the fitness cost associated with duplicating the sensitive and insensitive alleles is reduced due to the resulting advantageous heterozygosity (Labbe et al., 2007). In short, this means that the heterozygous condition (having both the susceptible and resistant alleles) makes the phenotype more fit to survive and reproduce than does either the homozygous dominant (resistant) or homozygous recessive (susceptible) genotype. This is the theory that explains why an individual who is heterozygous for the sicklecell anemia trait has a fitness advantage over his/her homozygous parents: he/she will not develop sickle-cell anemia, but is also highly immune to contracting parasitic malaria. Similarly, the mosquito which has the heterozygous ace-1D gene will produce enough acetylcholinesterase which is resistant to inactivation by organophosphates to survive, but is still producing some susceptible acetylcholinesterase (Labbe et al., 2007). In summary, Culex pipiens, a cosmopolitan mosquito and vector of medically and economically important diseases, has developed major resistance to organophosphate-based insecticides. This represents a serious logistical threat to the main mode of chemical control of C. pipiens. Organophosphate insecticides are esters of phosphoric acid which irreversibly phosphorylate acetylcholinesterase, an enzyme critical in the normal function of neural cells. Resistance to organophosphate (acetylcholine) poisoning is related to one of two factors:

overproduction of esterases or production of mutated acetylcholinesterase. Esterases detoxify the organophosphates (which are esters); mutant acetylcholinesterase is resistant to deactivation by organophosphate. These conditions are met by the products of three mutant genes: Est-2, Est3 and ace-1. The first two code for esterases which are normally present in much lower quantities than those found in susceptible individuals. The ace-1 locus contains mutations which produce the aforementioned resistant acetylcholinesterase.

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