Evolution of Insecticide Resistance in the Mosquito - American

Chapter 10

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Evolution of Insecticide Resistance in the Mosquito Culex pipiens: The Migration Hypothesis of Amplified Esterase Genes M . Raymond and N. Pasteur Laboratoire Génétique et Environnement, Institut des Sciences de l'Evolution, Unité de Recherche Associée, Centre National de la Recherche Scientifique 327, Université de Montpellier II (C.C. 065), F-34095 Montpellier, Cedex 05, France Resistance to organophosphorus insecticides has been studied at the gene and the population levels in Culex pipiens in various geographic areas. Only three loci have developed major resistance alleles in this species, including Est-2 (or esterase B), at which resistance occurs through gene amplification. Gene amplification involving a same particular haplotype has been found at the esterase B locus of mosquitoesfromvarious continents. This situation, which has been explained by a unique amplification event followed by migration and selection by OP insecticides, has been sometimes questioned. A clarification of the hypotheses proposed is presented, and how it is possible to prove or disprove them. Recent data on the extent of polymorphism at the esterase B locus in susceptible populations provide a strong support of the migration hypothesis. The wide use of organic insecticides to control medically important pest species has been a powerful agent of selection in natural populations of many insect species which have developed various degrees of resistance (1,2). In a few species, such as the mosquito Culex pipiens, it is possible to identify each gene conferring resistance to organophosphorous insecticides in single individuals. This mosquito, common in temperate and tropical countries, is subjected to insecticide control in many places. World-wide surveys of resistance to organophosphorus insecticides have disclosed that only three loci have developed major resistance alleles (3-6). The first two loci, Est-2 (or esterase B) and Est-3 (or esterase A), code for detoxifying carboxylester hydrolases (EC 3.1.1.1), and resistance alleles correspond to an esterase overproduction (4,7,8). Six distinct electromorphs have been described so far at the Est-2 locus (named B l , B2, B4, B5, B6 and B7) and four at the Est-3 locus ( A l , A2, A4 and A5) (3,4,6,9-11). In the case of esterase B , overproduction corresponds to the amplification of a D N A segment containing the structural gene (4,10,12). The third locus, Ace, codes the acetylcholinesterase 0097-6156/96/0645-0090$15.00/0 © 1996 American Chemical Society

In Molecular Genetics and Evolution of Pesticide Resistance; Brown, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

10. RAYMOND & PASTEUR

Evolution of Insecticide Resistance in Mosquitoes 91

(insecticide target), and insensitive alleles have been reported in various places (e. g. 13-15) but it is not known how many Ace alleles have occurred independently. R


Analysis by Restriction Endonuclase Digestion How resistance to organophosphorus insecticides evolved in natural populations of Culex pipiens could be studied at the molecular level for the esterase B locus, for which molecular tools have been developed for field studies. It is possible to build a restriction map of the D N A area within and around the esterase B structural gene, in susceptible mosquitoes with a single copy of the gene, as well as in mosquitoes with an amplified gene. When such maps are compared, large differences are observed. For example, the map found in S-Lab, a susceptible reference strain from California, and the map from Tem-R, a strain also from California possessing the B l amplification, have only 21% of their restriction sites in common (76). Similar results are found in comparing maps from different susceptible strains, or in comparing strains with distinct overproduced electromorph (4,16,17). However, when strains with the B2 electromorph are compared, restriction maps are strictly identical (16), independently of their geographical origins (Table I). A similar situation is found for B l electromorph, which possesses the same restriction map in mosquitoes from various parts within the Americas and in China (18). How can such similarity be explained ? How to Explain the Similarity of the Restriction Maps? A large part of the polymorphism detected by restriction enzymes around the esterase B structural gene is probably neutral. The identity of the restriction maps of B1 or B2 haplotypes in many geographic areas indicates therefore that these alleles are identical by descent. There are two possibilities: either they were first amplified in a particular place, and have then spread (Figure 1A), or they have first spread and then been independently amplified in various places (Figure IB). The first scenario has been proposed by Raymond et al. (16) and Qiao and Raymond (18), based on the argument that the amount of divergence between distinct restriction maps (such as between S-Lab and Tem-R) could indicate a large amount of polymorphism in natural populations, so that the probability of independently amplifying a same allele is very low. In addition, the selective advantage provided by the amplification itself promotes its spread in places treated with organophosphate insecticides. The multiple and independent amplification of B2 has been favoured by Hemingway et al. (79) and Ketterman et al. (20), based on variation in the kinetics of esterases studied on partially purified enzymes. Only an analysis of the polymorphism of susceptible populations could discriminate between these two possibilities. Under the first scenario, the polymorphism at the esterase B locus in non-treated populations should be extensive, and the probability of sampling a non-amplified allele already amplified elsewhere should be very low. On the other hand, the second scenario predicts that either the non-amplified B l or B2 allele (which are amplified most commonly world-wide) is present at a detectable frequency in susceptible populations.


MOLECULAR GENETICS AND EVOLUTION OF PESTICmE RESISTANCE

92


Table I. Geographic distribution of amplified B esterase identified by restriction fragment length polymorphism analysis from published information. The year of collection of the material studied is indicated. a

References

Country

Year

Allele

RE

North America: USA California USA California U S A California USA Illinois U S A Texas

1974 1974 1986 1986 1986

Bl Bl Bl Bl B2

12 13 1 1 13

(23) (16) (10) (10) (16)

Latin America: French Guiana French Guiana Venezuela Puerto Rico Cuba

1991 1991 1991 1992 1986

Bl B2 Bl Bl B8

1 1 1 13 1

(18) (18) (18) (18) (22)

Europe: France Corsica Cyprus France France Italy

1984 1988 1987 1986 1991 1992

B4 B4 B5 B2 B4 B4

6 1 6 3 1 1

(4) (24) (4) (25) (26) (21)

Africa: Egypt Congo Ivory coast

1987 1988 1986

B2 B2 B2

13 13 13

(16) (16) (16)

Asia: Sri Lanka China China China China China Pakistan

1986 1992 1992 1992 1992 1992 1985

B2 Bl Bl B2 B6 B7 B2

1 13 2 2 2 2 13

(22) (18) (11) (ID (11) (11) (16)

b

a

RE: number of restriction enzymes used Unnamed by Vaughan et al. 1995.



Evolution of Insecticide Resistance in Mosquitoes 93

a> _

1-2

if Downloaded by UNIV MASSACHUSETTS AMHERST on October 8, 2012 | http://pubs.acs.org Publication Date: September 27, 1996 | doi: 10.1021/bk-1996-0645.ch010

A

B

Figure 1. The two possible scenarios for the identity of amplified alleles in various geographical areas. A) amplification occurs once and extensive migration is promoted through organophosphorus resistance. B) migration of the nonamplified allele occurs first, and then is amplified independently in several organophosphorous treated areas. The circle represents an amplification event. Study of Polymorphism at the Esterase B Locus in Susceptible Populations. Culex pipiens susceptible populations still exist in northern France and northern Europe. Three such susceptible populations were sampled and analyzed for esterase electromorph and D N A polymorphism (77). At the protein level, 16 alleles were found for esterase B in one French population (N = 74), and 14 in an English one (N = 50). One electromorph had the same mobility as B2, but it was never associated with A2 (a characteristic of the amplified B2 throughout the world), and it was concluded that this similar migration is probably coincidental. At the D N A level, 24 alleles at the esterase B locus were identified in a sample of 72 mosquitoes from one population, with the use of only one restriction enzyme (Figure 2). Restriction maps of two nonamplified alleles randomly sampled from a single breeding site in Belgium were built with 6 restriction enzymes (Figure 2). 60% of the sites are different among the two maps. In addition, these two maps were not more related than a pair drawn at random from a pool containing other European alleles (Figure 2). The huge polymorphism found in susceptible populations considerably strengthens the hypothesis that amplification of an allele occurs before it is spread. It is still possible that non amplified B2 or B1 alleles exist at very low frequencies in every susceptible populations, but how such a situation would be created and maintained requires specific explanation before further considerations.

Conclusion The unique amplification event prior extensive migration of esterase B haplotype (Figure 1A) seems the most likely hypothesis to explain the existing data. This hypothesis is based on 1) the existence of a large neutral polymorphism around the esterase B structural gene in susceptible mosquitoes, and 2) the presence of the same amplified haplotype in populations from distant geographical areas. The second point



14 (50) 16(74)

Protein

24 (72)

_

DNA

Number of alleles (sample size)

B

i

G

1

1

H

II

HE

II

if

1

1

1 P

1

B

B

1

P

HE

III

ir 1 11 1

III

ir

RHR BGH

III

RHR B H R

E

1

1 ill Mr RHR BRGH

B

BRHR BGH

RHRBGHEPBR

E

1

P

P

E B

11

P

i

I I I

B

1 1

P E

G B

R

1

R

Figure 2. Analysis of susceptible populations in Brittany (France), Bruges (Belgium) and Bristol (England) at the esterase B locus. The French and the English population have been analysed for the number of alleles at the protein and D N A level (bottom left inset). The Belgium population has been studied for two esterase B alleles taken at random, for which a restriction map has been built and compared (top right inset) with restriction maps of B5 and B4 (3rd and 4th from top), and M S E (bottom). Restriction enzymes are abbreviated as: B (BamHl), G (Bgll), E (EcoRl), R (EcoRV), H (Hindffl) and P (Pstl). SOURCE: Adapted from ref.17.

-~\

G

G |

Y

GE

? Tr


P.

en H

i

o

as

o

1

9.

i

3

2


Evolution of Insecticide Resistance in Mosquitoes

95


has been substantially documented (Table I), and the first point is now supported by extensive studies from susceptible populations from northern Europe (77), as well as from small samples studies from Portugal (16), Italy (27) and Venezuela (18). When distinct restriction maps are found for the same overproduced electromorph, as described by Poirie et al. (4) in the Mediterranean region or by Vaughan et al. (22) in Cuba, this indicates that independent amplification of distinct alleles coding co-migrating electromorphs has occurred. This situation does not contradict the migration hypothesis. Acknowledgments We are very grateful to C. Bernard, M . Marquine and G. Pistre for technical help. Research was supported in part by the "Programme Environnement du CNRS" (G.D.R. 1105) and INRA. This is paper no. ISEM 95.090 of the Institut des Sciences de l'Evolution.

Literature Cited 1. Georghiou, G. P.; Mellon, R. B. In Pesticide resistance to pesticides, G. P. Georghiou; T. Saito, Eds; Plenum Press: New York, N.Y., 1983, p. 1-46. 2. Georghiou, G. P.; Lagunes-Tejeda, A. The occurrence of resistance to pesticides in arthropods; Food and Agriculture Organization: Rome, 1991. 3. Pasteur, N.; Iseki, A.; Georghiou, G. P. Biochem. Genet. 1981, 19, 909-919. 4. Poirié, M.; Raymond, M.; Pasteur, M. Biochem. Genet. 1992, 30, 13-26. 5. Wirth, M.; Marquine, M.; Georghiou, G. P.; Pasteur, N. J. Econ. Entomol. 1990, 27, 202-206. 6. Georghiou, G. P. In Insecticides: mechanism of action and resistance, D. Otto; B. Weber, Ed; Intercept: Andover, 1992, p. 407-408. 7. Fournier, D.; Bride, J.-M.; Mouchès, C.; Raymond, M.; Magnin, M.; Bergé, J.-B.; Pasteur, N.; Georghiou, G. P. Pestic. Biochem. Physiol. 1987, 27, 211-217. 8. Mouchès, C.; Magnin, M.; Bergé, J.-B.; De Silvestri, M.; Beyssat, V.; Pasteur, N.; Georghiou, G. P. Proc. Natl. Acad. Sc., USA 1987, 84, 2113-2116. 9. Pasteur, N.; Sinègre, G.; Gabinaud, A. Biochem. Genet. 1981, 19, 499-508. 10. Raymond, M.; Beyssat-Arnaouty, V.; Sivasubramanian, N.; Mouchès, C.; Georghiou, G. P.; Pasteur, N. Biochem. Genet. 1989, 27, 417-423. 11. Xu, J.; Qu, F.; Liu, W. J. Med. Coll. PLA 1994, 9, 20-23. 12. Mouchès, C.; Pasteur, N.; Bergé, J. B.; Hyrien, O.; Raymond, M.; Robert de Saint Vincent, B.; De Silvestri, M.; Georghiou, G. P. Science 1986, 233, 778-780. 13. Bisset, J. A.; Rodriguez, M. M.; Hemingway, J.; Diaz, C.; Small, G. J.; Ortiz, E. Med. Vet. Entomol. 1991, 5, 223-228. 14. Raymond, M.; Fournier, D.; Bride, J.-M.; Cuany, A.; Bergé, J.; Magnin, M.; Pasteur, N. J. Econ. Entomol. 1986, 79, 1452-1458. 15. Bourguet, D.; Capela, R.; Raymond, M. J. Econ. Entomol. 1995, Submitted. 16. Raymond, M.; Callaghan, A.; Fort, P.; Pasteur, N. Nature 1991, 350, 151-153. 17. Raymond, M.; Qiao, C. L.; Callaghan, A. Genet. Res. 1995, 66, in press. 18. Qiao, C.-L.; Raymond, M. Heredity 1995, 74, 339-345.



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MOLECULAR GENETICS AND EVOLUTION OF PESTICIDE RESISTANCE

19. Hemingway, J.; Ketterman, A. J.; Karunaratne, S. H. P. P.; Jayawardena, K. G. I.; Vaughan, A., In First International Conference on Insect Pests in Urban Environment, Willey, K. B.; Robinson W. H., Eds.; BPCC Wheatons Ltd: Exeter, 1993, p. 319-328. 20. Ketterman, A.; Karunaratne, S. H. P. P.; Jayawardena, K. G. I.; Hemingway, J. Pestic. Biochem. Physiol. 1993, 47, 142-148. 21. Severini, C.; Marinucci, M.; Raymond, M. J. Med. Entomol. 1994, 31, 496-499. 22. Vaughan, A.; Rodriguez, M.; Hemingway, J. Biochem. J. 1995, 305, 651-658. 23. Mouchès, C.; Pauplin, Y.; Agawarl, M.; Lemieux, L.; Herzog, M.; Abadon, M.; Beyssat-Arnaouty, V.; Hyrien, O.; Robert de Saint Vincent, B.; Georghiou, G. P.; Pasteur, N. Proc. Natl. Acad. Sc., USA 1990, 87, 2574-2578. 24. Raymond, M.; Marquine, M. J. Evol. Biol. 1994, 7, 315-337. 25. Rivet, Y.; Marquine, M.; Raymond, M. Biol. J. Linn. Soc. 1993, 49, 249-255. 26. Chevillon, C.; Pasteur, N.; Marquine, M.; Heyse, D.; Raymond, M. Evolution 1995, 49, 997-1007.


Evolution of Insecticide Resistance in the Mosquito - American

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