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

March 23, 2011

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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

mosquito’s 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

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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

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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 & Lagunes-

Tejeda, 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

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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 company-

amplification) 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

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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

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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 resistance-

conferring 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

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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 sickle-

cell 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:

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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, Est-

3 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.

Works Cited

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Cui, F., Raymond, M., Berthomieu, A., Alout, H., Weill, M., & Qiao, C.L. (2006b). Recent emergence of insensitive acetylcholinesterase in Chinese populations of the mosquito Culex pipiens. Journal of Medical Entomology, 43, 878-883.

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